Mark A. Simmons

Amyloid β peptides act directly on single neurons

Abstract A peptide consisting of residues 25–35 of the amyloid β protein was applied to single neurons while monitoring membrane current by whole cell voltage clamp recording. Within minutes of direct exposure of a neuron to the amyloid β peptide, a paroxysmal increase in neuronal membrane conductance was observed. This conductance does not resemble previously described ionic conductances in terms of its time-dependence, voltage-dependence or sensitivity to changes in extracellular or intracellular ionic constituents. The effect of the amyloid β peptide was not mimicked or blocked by substance P nor was it prevented by low intracellular or extracellular Ca. The increased membrane permeability elicited by the peptides may lead to the neuropathology observed in Alzheimers disease.

Muscarinic suppression of the M-current is mediated by a rise in internal Ca2+ concentration

The role of intracellular Ca2+ in the muscarinic suppression of M-current was examined. Intracellular injection of Ca2+ buffer into cells in the intact ganglion reduced the response to muscarinic agonist. In similar experiments on isolated cells, Ca2+ buffer was introduced into the cytoplasm using a perfused recording pipette. Ca2+ buffer (20 mM) with the free Ca2+ concentration set to normal resting levels produced a reversible reduction of the muscarinic response. In a second line of investigation, it was found that pharmacological procedures designed to deplete internal stores of Ca2+ produced a decrease in the muscarinic response. These results, taken together with previous work, support the hypothesis that the muscarinic suppression of M-current is mediated by the release of Ca2+ from intracellular stores.

Myosin light chain kinase occurs in bullfrog sympathetic neurons and may modulate voltage-dependent potassium currents

A polyclonal antibody against myosin light chain kinase (MLCK) of chicken gizzard recognized a 130 kd peptide of bullfrog sympathetic ganglia as MLCK. MLCK immunoreactivity was confined to the neuronal cell body. A synthetic peptide corresponding to an inhibitory domain of MLCK (Ala783-Gly804) was applied intracellularly to isolated sympathetic neurons during whole-cell recordings of ionic currents. The peptide inhibitor reversibly decreased M-type potassium current (IM) while not affecting A-type of delayed rectifier-type potassium currents. Intracellular application of an active fragment of MLCK enhanced IM, whereas application of an inactive MLCK fragment did not. The results suggest that IM can be modulated by MLCK-catalyzed phosphorylation.

Inhibition by wortmannin of M-current in bullfrog sympathetic neurones

1 The actions of wortmannin, an inhibitor of myosin light chain kinase (MLCK), on M‐type potassium current of dissociated bullfrog sympathetic neurones have been examined. 2 The amplitude of M‐current was measured by whole cell recordings from cells pretreated with wortmannin (0.01–10 μm) or the wortmannin vehicle, dimethylsulphoxide (0.0001–0.1 vol%), for 30 min. Internal (recording pipette) solutions having three different pCa values (6, 7 and 8) were used for the measurements. 3 Irrespective of the pCa, M‐current was not detectable when the cells were pretreated with 10 μm wortmannin. Wortmannin, 3 μm, produced 85–95% inhibition of the M‐current. Pretreatment with 10–30 nm wortmannin was without effect on M‐current. 4 The M‐current inhibition by wortmannin at concentrations of 0.1–1 μm depended on the pCa of the internal solution. Inhibition occurred only when the calcium‐rich (pCa = 6) internal solution was used. 5 Pre‐treatment of the cells with wortmannin (10 μm) did not affect rapidly‐inactivating A‐type or delayed rectifier‐type potassium currents nor did it alter inwardly rectifying sodium‐potassium current (IH). 6 These observations show that M‐current inhibition by wortmannin has two pharmacological profiles. One is calcium‐dependent and occurs at lower concentrations (0.1–1 μm), and is attributed to inhibition of MLCK by wortmannin. At higher concentrations (3–10 μm), wortmannin has an additional, calcium‐independent action, inhibiting the M‐current by an unknown mechanism.

The adenosine 2A receptor agonist GW328267C improves lung function after acute lung injury in rats.

There is a significant unmet need for treatments of patients with acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS). The primary mechanism that leads to resolution of alveolar and pulmonary edema is active vectorial Na(+) and Cl(-) transport across the alveolar epithelium. Several studies have suggested a role for adenosine receptors in regulating this fluid transport in the lung. Furthermore, these studies point to the A(2A) subtype of adenosine receptor (A(2A)R) as playing a role to enhance fluid transport, suggesting that activation of the A(2A)R may enhance alveolar fluid clearance (AFC). The current studies test the potential therapeutic value of the A(2A)R agonist GW328267C to accelerate resolution of alveolar edema and ALI/ARDS in rats. GW328267C, at concentrations of 10(-5) M to 10(-3) M, instilled into the airspaces, increased AFC in control animals. GW328267C did not increase AFC beyond that produced by maximal β-adrenergic stimulation. The effect of GW328267C was inhibited by amiloride but was not affected by cystic fibrosis transmembrane conductance regulator inhibition. The drug was tested in three models of ALI, HCl instillation 1 h, LPS instillation 16 h, and live Escherichia coli instillation 2 h before GW328267C instillation. After either type of injury, GW328267C (10(-4) M) decreased pulmonary edema formation and restored AFC, measured 1 h after GW328267C instillation. These findings show that GW328267C has beneficial effects in experimental models of ALI and may be a useful agent for treating patients with ALI or prophylactically to prevent ALI.

Virtual screening to identify novel antagonists for the G protein-coupled NK3 receptor.

The NK(3) subtype of tachykinin receptor is a G protein-coupled receptor that is a potential therapeutic target for several neurological diseases, including schizophrenia. In this study, we have screened compound databases for novel NK(3) receptor antagonists using a virtual screening protocol of similarity analysis. The lead compound for this study was the potent NK(3) antagonist talnetant. Compounds of the quinoline type found in the virtual screen were additionally evaluated in a comparative molecular field analysis model to predict activity a priori to testing in vitro. Selected members of this latter set were tested for their ability to inhibit ligand binding to the NK(3) receptor as well as to inhibit senktide-induced calcium responses in cells expressing the human NK(3) receptor. Two novel compounds were identified that inhibited NK(3) receptor agonist binding, with potencies in the nM range and antagonized NK(3) receptor-mediated increases in intracellular calcium. These results demonstrate the utility of similarity analysis in identifying novel antagonist ligands for neuropeptide receptors.

Hyperpolarizing shift of the M-current activation curve after washout of muscarine in bullfrog sympathetic neurons

The mechanism underlying the over-recovery of an M-type potassium current following the washout of muscarine (20 microM) has been examined. Whole-cell recordings were made from single neurons dissociated from bullfrog sympathetic ganglia. During over-recovery, the maximum M-conductance decreased by about 2.8 nS while the steady-state M-current activation curve was displaced in the hyperpolarizing direction by about 13 mV. These data suggest that a hyperpolarizing shift in the kinetics of M-current causes over-recovery in amphibian autonomic neurons.

Gamma-aminobutyric acidA receptor function is modulated by cyclic GMP

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the vertebrate nervous system. Modulatory effects of intracellular ATP on the GABA response in isolated bullfrog dorsal root ganglion neurons were examined using whole-cell voltage clamp. Investigation of the plausible mechanisms ATP might utilize to regulate the GABA response led to the discovery that intracellular cyclic GMP may play an important role in modulating inhibitory neurotransmission. This modulatory effect of cyclic GMP is likely to be mediated via a cyclic GMP-dependent protein kinase.

An endogenous 'hypertensive factor' enhances the voltage- dependent calcium current

The effects of an immunoaffinity‐purified putative endogenous hypertensive factor (HF) on voltage‐dependent calcium current in frog cardiac myocytes were assessed. In 9 out of 10 cells, HF reversibly increased the peak amplitude of the calcium current. HF increased peak calcium current density at −5 mV from a control level of 1.8 ± 1.3 pA/pF (mean ± SD) to 4.4 ± 2.0 pA/pF. HF shifted the peak of the calcium current‐voltage relationship in the hyperpolarizing direction. HF shifted the voltage dependence of the inactivation of the calcium current to more negative potentials with prepulses from −80 to 0 mV, but the inactivation was not affected with prepulses more positive than 0 mV. Modulation of the voltage‐dependent calcium current by HF may be the mechanism underlying its pressor effects.