Site Selectivity of Competitive Antagonists for the Mouse Adult Muscle Nicotinic Acetylcholine Receptor

Liu, Man; Dilger, James P.

doi:10.1124/mol.108.051060

Cited by 19 publications

(32 citation statements)

References 25 publications

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“…Following procedures established in our laboratory (Durante et al, 1993; Liu et al, 2009; Mendelev et al, 2009), thoracic aorta VSMCs were obtained from male Sprague-Dawley rats (100–125 g) and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and Primocin (100 mg/L; Invivogen) at 37°C in 95% air/5% CO 2 . Cells were split and propagated through passage 6.…”

Section: Methodsmentioning

confidence: 99%

Phosphodiesterases Regulate BAY 41-2272-Induced VASP Phosphorylation in Vascular Smooth Muscle Cells

Adderley¹,

Joshi²,

Martin³

et al. 2012

Front. Pharmacol.

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BAY 41-2272 (BAY), a stimulator of soluble guanylyl cyclase, increases cyclic nucleotides and inhibits proliferation of vascular smooth muscle cells (VSMCs). In this study, we elucidated mechanisms of action of BAY in its regulation of vasodilator-stimulated phosphoprotein (VASP) with an emphasis on VSMC phosphodiesterases (PDEs). BAY alone increased phosphorylation of VASPSer239 and VASPSer157, respective indicators of PKG and PKA signaling. IBMX, a non-selective inhibitor of PDEs, had no effect on BAY-induced phosphorylation at VASPSer239 but inhibited phosphorylation at VASPSer157. Selective inhibitors of PDE3 or PDE4 attenuated BAY-mediated increases at VASPSer239 and VASPSer157, whereas PDE5 inhibition potentiated BAY-mediated increases only at VASPSer157. In comparison, 8Br-cGMP increased phosphorylation at VASPSer239 and VASPSer157 which were not affected by selective PDE inhibitors. In the presence of 8Br-cAMP, inhibition of either PDE4 or PDE5 decreased VASPSer239 phosphorylation and inhibition of PDE3 increased phosphorylation at VASPSer239, while inhibition of PDE3 or PDE4 increased and PDE5 inhibition had no effect on VASPSer157 phosphorylation. These findings demonstrate that BAY operates via cAMP and cGMP along with regulation by PDEs to phosphorylate VASP in VSMCs and that the mechanism of action of BAY in VSMCs is different from that of direct cyclic nucleotide analogs with respect to VASP phosphorylation and the involvement of PDEs. Given a role for VASP as a critical cytoskeletal protein, these findings provide evidence for BAY as a regulator of VSMC growth and a potential therapeutic agent against vasculoproliferative disorders.

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Section: Methodsmentioning

confidence: 99%

Phosphodiesterases Regulate BAY 41-2272-Induced VASP Phosphorylation in Vascular Smooth Muscle Cells

Adderley¹,

Joshi²,

Martin³

et al. 2012

Front. Pharmacol.

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“…and euthanized by pneumothorax and exsanguination. Vascular SMCs (VSMCs) were harvested from the thoracic aorta and isolated by collagenase and elastase digestion as described (Durante et al, 1993; Liu et al, 2009). Cells were cultured serially (passage 1–6) in Dulbecco’s minimum essential media (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM l -glutamine, sodium pyruvate, 5 mM HEPES, and Primocin (100 mg/l) in the presence of 95% air/5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

Control of Vascular Smooth Muscle Cell Growth by Connexin 43

Joshi¹,

Martin²,

Shaver³

et al. 2012

Front. Physio.

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Connexin 43 (Cx43), the principal gap junction protein in vascular smooth muscle cells (VSMCs), regulates movement of ions and other signaling molecules through gap junction intercellular communication (GJIC) and plays important roles in maintaining normal vessel function; however, many of the signaling mechanisms controlling Cx43 in VSMCs are not clearly described. The goal of this study was to investigate mechanisms of Cx43 regulation with respect to VSMC proliferation. Treatment of rat primary VSMCs with the cAMP analog 8Br-cAMP, the soluble guanylate cyclase (sGC) stimulator BAY 41-2272 (BAY), or the Cx inducer diallyl disulfide (DADS) significantly reduced proliferation after 72 h compared with vehicle controls. Bromodeoxyuridine uptake revealed reduction (p < 0.05) in DNA synthesis after 6 h and flow cytometry showed reduced (40%) S-phase cell numbers after 16 h in DADS-treated cells compared with vehicle controls. Cx43 expression significantly increased after 270 min treatment with 8Br-cAMP, 8Br-cGMP, BAY or DADS. Inhibition of PKA, PKG or PKC reversed 8Br-cAMP-stimulated increases in Cx43 expression, whereas only PKG or PKC inhibition reversed 8Br-cGMP- and BAY-stimulated increases in total Cx43. Interestingly, stimulation of Cx43 expression by DADS was not dependent on PKA, PKG or PKC. Using fluorescence recovery after photobleaching, only 8Br-cAMP or DADS increased GJIC with 8Br-cAMP mediated by PKC and DADS mediated by PKG. Further, DADS significantly increased phosphorylation at MAPK-sensitive Serine (Ser)255 and Ser279, the cell cycle regulatory kinase-sensitive Ser262 and PKC-sensitive Ser368 after 30 min while 8Br-cAMP significantly increased phosphorylation only at Ser279 compared with controls. This study demonstrates that 8Br-cAMP- and DADS-enhanced GJIC rather than Cx43 expression and/or phosphorylation plays important roles in the regulation of VSMC proliferation and provides new insights into the growth-regulatory capacities of Cx43 in VSM.

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“…This EC 50 of 178 nM closely corresponds to the properties of pancuronium at human AChRs. 29,32 Equilibration of the synapse with an antagonist concentration of 237 nM results in 94% occupancy of the receptors and an endplate current (epc, the current produced by release of ACh from all 30 vesicles) whose amplitude is 10% of control. Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Many antagonists are selective between the two binding sites. 28,30–32 A range of values for the high L 1eq and low L 2eq affinity sites were considered (Table 1). “Standard” conditions refer to ℓ −1 =3s −1 , L 1eq =30 nM, L 2eq =300 nM, [C]=7.9×L 1eq =237 nM where ℓ −1 is the antagonist dissociation rate for the high affinity site, L 1eq and L 2eq are the equilibrium constants for antagonists at the high and low affinity sites respectively and [C] is the antagonist concentration.…”

Section: Methodsmentioning

confidence: 99%

Simulation of the Kinetics of Neuromuscular Block

Dilger

2013

Anesthesia &Amp; Analgesia

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Background The onset time for paralysis varies three-fold among nondepolarizing muscle relaxants. Possible explanations include: a) pharmacokinetic differences among drugs and b) buffering of drug molecules by acetylcholine receptors as they diffuse into the neuromuscular junction. Although some pharmacokinetic models consider buffered diffusion, these models do not account for either the high density of receptors or synapse geometry. Here, I used computer simulations to calculate the kinetics of buffered diffusion. The goal was to determine the conditions under which buffered diffusion could account for differences in onset time among nondepolarizing muscle relaxants. Methods Monte Carlo simulation was used along with a realistic 3-dimensional model of the rat neuromuscular junction. Simulations determined the time dependence of the number of drug-bound receptors. A 1000-fold range of drug potency was examined. In some simulations, the drug concentration outside the junction was changed instantaneously. In other simulations, the concentration changed according to predictions of pharmacokinetic models assuming time-dependent changes in plasma drug concentration. The rate constant for equilibration of drug between plasma and muscle, keo, was varied between 0.15 and 0.6 min−1. Twitch amplitude was calculated from receptor occupancy assuming a high safety margin for neuromuscular transmission. Some simulations used a synaptic model with an increased nerve-muscle contact width. Results Simulations with instantaneous changes in drug concentration at the synapse, indicated that the time to 50% twitch depression (onset time) was 0.1–30s and was proportional to drug potency. This corresponds to iontophoretic application of drug to isolated neuromuscular junctions, but is too fast to explain onset times in humans. When pharmacokinetic models were used to calculate the drug concentration outside of the synapse, buffered diffusion increased onset times of potent drugs (drugs for which the effective concentration at 50% twitch height is less than 600 nM). Simulations using keo=0.6 min−1 and a model with a 2–3 fold wider nerve-muscle contact width indicated that buffered diffusion could account for the differences in clinical onset times among the nondepolarizing muscle relaxants. Discussion Monte-Carlo simulation provides a biophysically appropriate way to incorporate buffered diffusion into pharmacokinetic modeling. The simulations indicated that buffered diffusion could account for differences in onset time among drugs. However, a better understanding of the geometry of the human neuromuscular junction is needed before the magnitude of the effect of buffered diffusion can be quantified.

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Site Selectivity of Competitive Antagonists for the Mouse Adult Muscle Nicotinic Acetylcholine Receptor

Cited by 19 publications

References 25 publications

Phosphodiesterases Regulate BAY 41-2272-Induced VASP Phosphorylation in Vascular Smooth Muscle Cells

Phosphodiesterases Regulate BAY 41-2272-Induced VASP Phosphorylation in Vascular Smooth Muscle Cells

Control of Vascular Smooth Muscle Cell Growth by Connexin 43

Simulation of the Kinetics of Neuromuscular Block

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