Biomedical subjects

William D Atchison

Publications and source records attributed to William D Atchison.

21 records · Page 2Linked to original sources

PubMed2002

Effects of methylmercury on human neuronal L-type calcium channels transiently expressed in human embryonic kidney cells (HEK-293).

Methylmercury (MeHg) disrupts the function of native, high voltage-activated neuronal Ca(2+) channels in several types of cells. However, the effects of MeHg on isolated Ca(2+) channel phenotypes have not been examined. The aim of the present study was to examine the action of MeHg on recombinant, neuronal L-type voltage-sensitive Ca(2+) channels. Human embryonic kidney cells (HEK-293) were transfected with human neuronal cDNA clones of the alpha(1C-1) subunit in combination with alpha(2b) and beta(3a) Ca(2+) channel subunits and the reporter jellyfish green fluorescent protein for transient expression. Current from expressed channels (I(Ba)) and their response to MeHg applied acutely were measured using whole-cell voltage-clamp recording techniques and Ba(2+) (5 mM) as charge carrier. Amplitude of I(Ba) in these cells was reduced by the dihydropyridine (DHP), nimodipine, and enhanced by Bay K8644 [S-(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3 pyridine carboxylic acid methyl ester]. MeHg (0.125-5.0 microM) caused a time- and concentration-dependent reduction in amplitude of the peak and sustained current through these channels. However, even at the highest concentration of MeHg tested, reduction of current amplitude by MeHg was incomplete. Washing with MeHg-free solution could not reverse its effects. The steady-state inactivation curve was unaltered by MeHg. Increasing the stimulation frequency or the extracellular Ba(2+) concentration each attenuated slightly the reduction in amplitude of I(Ba) by MeHg. In the presence of MeHg (5.0 microM), Bay K8644 still increased the remaining current, and nimodipine (10 microM) reduced residual current that was resistant to MeHg. Thus, although MeHg reduces the amplitude of recombinant, heterologously expressed L-type channel current, a portion of current is resistant to reduction by MeHg. Furthermore, DHP agonists and antagonists retain their ability to affect L-type Ca(2+) channel current even in the presence of MeHg.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2002

Characteristics of block by Pb2+ of function of human neuronal L-, N-, and R-type Ca2+ channels transiently expressed in human embryonic kidney 293 cells.

Lead (Pb(2+)) is a well-known inhibitor of voltage-dependent Ca(2+) channels in their native environments in several types of cells. However, its effects on discrete Ca(2+) channel phenotypes in isolation have not been well studied. We compared how specific subtypes of human neuronal high-voltage-activated Ca(2+) channels were affected by acute exposure to Pb(2+). Expression cDNA clones of human alpha(1C), alpha(1B), or alpha(1E) subunit genes encoding neuronal L-, N-, and R-subtypes of Ca(2+) channels, respectively, along with a constant alpha(2)delta and beta(3) subunits were transfected into human embryonic kidney 293 cells. Currents through the respective transiently expressed channels were measured using whole-cell recording techniques with Ba(2+) (20 mM) as charge carrier. Extracellular bath applications of Pb(2+) significantly reduced current amplitude through all three types of Ca(2+) channels in a concentration-dependent manner. The order of potency was: alpha(1E) (IC(50) = 0.10 microM), followed by alpha(1C) (IC(50) = 0.38 microM) and alpha(1B) (IC(50) = 1.31 microM). Pb(2+)-induced perturbation of function of alpha(1C) and alpha(1B) containing Ca(2+) channels was more easily reversed than for alpha(1E)-containing Ca(2+) channels after washing with Pb(2+) free solution. The current-voltage relationships were not altered after 3-min exposure to Pb(2+) for any of the three types. However, the steady-state inactivation relationships were shifted to more negative potentials for channels containing alpha(1B) and alpha(1E) subunits, but not for those containing alpha(1C) subunits. Pb(2+) accelerated the inactivation time of current in all three subtypes of Ca(2+) channels in a concentration- and voltage-dependent manner. Therefore, different subtypes of Ca(2+) channels exhibit differential susceptibility to Pb(2+) even when expressed in the same cell type. Current expressed by alpha(1E)-containing channels is more sensitive to Pb(2+) than that expressed by alpha(1C)- or alpha(1B)-containing channels. Several Ca(2+) channel phenotypes are quite sensitive to the inhibitory action of Pb(2+). Furthermore, it seems that Pb(2+) is more likely to combine with Ca(2+) channels in the closed state.

Calcium Channel Blockers↗

PubMed2002

Impairment of synaptic vesicle exocytosis and recycling during neuromuscular weakness produced in mice by 2,4-dithiobiuret.

Chronic treatment of rodents with 2,4-dithiobiuret (DTB) induces a neuromuscular syndrome of flaccid muscle weakness that mimics signs seen in several human neuromuscular disorders such as congenital myasthenic syndromes, botulism, and neuroaxonal dystrophy. DTB-induced muscle weakness results from a reduction of acetylcholine (ACh) release by mechanisms that are not yet clear. The objective of this study was to determine if altered release of ACh during DTB-induced muscle weakness was due to impairments of synaptic vesicle exocytosis, endocytosis, or internal vesicular processing. We examined motor nerve terminals in the triangularis sterni muscles of DTB-treated mice at the onset of muscle weakness. Uptake of FM1-43, a fluorescent marker for endocytosis, was reduced to approximately 60% of normal after either high-frequency nerve stimulation or K(+) depolarization. Terminals ranged from those with nearly normal fluorescence ("bright terminals") to terminals that were poorly labeled ("dim terminals"). Ultrastructurally, the number of synaptic vesicles that were labeled with horseradish peroxidase (HRP) was also reduced by DTB to approximately 60%; labeling among terminals was similarly variable. A subset of DTB-treated terminals having abnormal tubulovesicular profiles in their centers did not respond to stimulation with increased uptake of HRP and may correspond to dim terminals. Two findings suggest that posttetanic "slow endocytosis" remained qualitatively normal: the rate of this type of endocytosis as measured with FM1-43 did not differ from normal, and HRP was observed in organelles associated with this pathway- coated vesicles, cisternae, as well as synaptic vesicles but not in the tubulovesicular profiles. In DTB-treated bright terminals, end-plate potential (EPP) amplitudes were decreased, and synaptic depression in response to 15-Hz stimulation was increased compared with those of untreated mice; in dim terminals, EPPs were not observed during block with D-tubocurarine. Nerve-stimulation-induced unloading of FM1-43 was slower and less complete than normal in bright terminals, did not occur in dim terminals, and was not enhanced by alpha-latrotoxin. Collectively, these results indicate that the size of the recycling vesicle pool is reduced in nerve terminals during DTB-induced muscle weakness. The mechanisms by which this reduction occurs are not certain, but accumulated evidence suggests that they may include defects in either or both exocytosis and internal vesicular processing.

Animals↗