Degree Of Membrane Depolarization Research Articles

Computational diversity in the hippocampus: a matter of components.

Computational diversity in the hippocampus: a matter of components.

Enhanced NMDA receptor-mediated intracellular calcium signaling in magnocellular neurosecretory neurons in heart failure rats

An enhanced glutamate excitatory function within the hypothalamic supraoptic and paraventricluar nuclei is known to contribute to increased neurosecretory and presympathetic neuronal activity, and hence, neurohumoral activation, during heart failure (HF). Still, the precise mechanisms underlying enhanced glutamate-driven neuronal activity in HF remain to be elucidated. Here, we performed simultaneous electrophysiology and fast confocal Ca²⁺ imaging to determine whether altered N-methyl-d-aspartate (NMDA) receptor-mediated changes in intracellular Ca²⁺ levels (NMDA-ΔCa²⁺) occurred in hypothalamic magnocellular neurosecretory cells (MNCs) in HF rats. We found that activation of NMDA receptors resulted in a larger ΔCa²⁺ in MNCs from HF when compared with sham rats. The enhanced NMDA-ΔCa²⁺ was neither dependent on the magnitude of the NMDA-mediated current (voltage clamp) nor on the degree of membrane depolarization or firing activity evoked by NMDA (current clamp). Differently from NMDA receptor activation, firing activity evoked by direct membrane depolarization resulted in similar changes in intracellular Ca²⁺ in sham and HF rats. Taken together, our results support a relatively selective alteration of intracellular Ca²⁺ homeostasis and signaling following activation of NMDA receptors in MNCs during HF. The downstream functional consequences of such altered ΔCa²⁺ signaling during HF are discussed.

Effect of ester to amide or N-methylamide substitution on bacterial membrane depolarization and antibacterial activity of novel cyclic lipopeptides.

Cyclic lipopeptides derived from the fusaricidin/LI-F family of naturally occurring antibiotics represent particularly attractive candidates for the development of new antibacterial agents. In comparison with natural products, these derivatives may offer better stability under physiologically relevant conditions and lower nonspecific toxicity, while preserving their antibacterial activity. In this study we assessed the ability of cyclic lipodepsipeptide 1 and its analogues--amide 2, N-methylamide 3, and linear peptide 4--to interact with the cytoplasmic membranes of selected Gram-positive bacteria. We also investigated their bacteriostatic/bactericidal modes of action and in vivo potency by using a Galleria mellonella model of MRSA infection. Cyclic lipopeptides 1 and 2 depolarize the cytoplasmic membranes of Gram-positive bacteria in a concentration-dependent manner. The degree of membrane depolarization was influenced by the structural and physical properties of 1 and 2, with the more flexible and hydrophobic peptide 1 being most efficient. However, membrane depolarization does not correlate with bacterial cell lethality, suggesting that membrane-targeting activity is not the main mode of action for this class of antibacterial peptides. Conversely, substitution of the depsipeptide bond in 1 with an N-methylamide bond in 3, or its hydrolysis to peptide 4, lead to a complete loss of antibacterial activity and indicate that the conformation of cyclic lipopeptides plays a role in their antibacterial activities. Cyclic lipopeptides 1 and 2 are also capable of improving the survival of G. mellonella larvae infected with MRSA at varying efficiencies, reflecting their in vitro activities. Gaining more insight into the structure-activity relationship and mode of action of these cyclic lipopeptides may enable the development of new antibiotics of this class with improved antibacterial activity.

Computational modelling elucidates the mechanism of ciliary regulation in health and disease

BackgroundCiliary dysfunction leads to a number of human pathologies, including primary ciliary dyskinesia, nephronophthisis, situs inversus pathology or infertility. The mechanism of cilia beating regulation is complex and despite extensive experimental characterization remains poorly understood. We develop a detailed systems model for calcium, membrane potential and cyclic nucleotide-dependent ciliary motility regulation.ResultsThe model describes the intimate relationship between calcium and potassium ionic concentrations inside and outside of cilia with membrane voltage and, for the first time, describes a novel type of ciliary excitability which plays the major role in ciliary movement regulation. Our model describes a mechanism that allows ciliary excitation to be robust over a wide physiological range of extracellular ionic concentrations. The model predicts the existence of several dynamic modes of ciliary regulation, such as the generation of intraciliary Ca2+ spike with amplitude proportional to the degree of membrane depolarization, the ability to maintain stable oscillations, monostable multivibrator regimes, all of which are initiated by variability in ionic concentrations that translate into altered membrane voltage.ConclusionsComputational investigation of the model offers several new insights into the underlying molecular mechanisms of ciliary pathologies. According to our analysis, the reported dynamic regulatory modes can be a physiological reaction to alterations in the extracellular environment. However, modification of the dynamic modes, as a result of genetic mutations or environmental conditions, can cause a life threatening pathology.

Voltage-Gated K+ Channel Dysfunction in Myocytes from a Dog Model of Subarachnoid Hemorrhage

Delayed cerebral vasospasm after subarachnoid hemorrhage is primarily due to sustained contraction of arterial smooth muscle cells. Its pathogenesis remains unclear. The degree of arterial constriction is regulated by membrane potential that in turn is determined predominately by K+ conductance (GK). Here, we identified the main voltage-gated K+ (Kv) channels contributing to outward delayed rectifier currents in dog basilar artery smooth muscle as Kv2 class through a combination of electrophysiological and pharmacological methods. Kv2 current density was nearly halved in vasospastic myocytes after subarachnoid hemorrhage (SAH) in dogs, and Kv2.1 and Kv2.2 were downregulated in vasospastic myocytes when examined by quantitative mRNA, Western blotting, and immunohistochemistry. Vasospastic myocytes were depolarized and had a smaller contribution of GK toward maintenance of their membrane potential. Pharmacological block of Kv current in control myocytes mimicked the depolarization observed in vasospastic arteries. The degree of membrane depolarization was found to be compatible with the amount of vasoconstriction observed after SAH. We conclude that Kv2 dysfunction after SAH contributes to the pathogenesis of delayed cerebral vasospasm. This may confer a novel target for treatment of delayed cerebral vasospasm.

Comparison of Relaxations Evoked by Photoactivation of NO-Containing Compounds and Nitrergic Nerve Stimulation in 5-Hydroxytryptamine- and Potassium-Contracted Rat Gastric Fundus

1. The aim of the present study was to further investigate our earlier proposal of liberation of nitric oxide (NO) by photoactivation of molecules containing NO or NO 2, which in turn relaxes gastric smooth muscle, and to determine whether presynaptic- and/or postsynaptic NO-mediated relaxation is affected differently by the degree of membrane depolarization in rat gastric fundus smooth muscle. 2. During contraction of rat gastric fundus with 5-hydroxytryptamine (5-HT, 10 μM), low (K +, 25 mM) and high potassium (K +, 65.4 mM), relaxation responses to nitrergic nerve stimulation, photoactivation of caged NO compounds (streptozotocin [STZ], N ω-nitro- L-arginine-methylacetate [ L-NAME], N ω-nitro- D-arginine-methylacetate [ D-NAME]), and sodium nitroprusside (SNP) were compared. 3. Nitrergic nerve (presynaptic) stimulation and photoactivation (postsynaptic) of all caged NO compounds produced rapid, transient and reversible relaxation of 5-HT and low-K +-contracted tissues. However, when contractions were induced by high K +, the relaxation induced by nerve stimulation was abolished, whereas relaxations induced by photoactivated NO compounds were significantly ( P<0.01) reduced. 4. The relaxation induced by sodium nitroprusside (SNP), but not papaverine, was also diminished in high-K +-contracted tissues. The magnitude of photoactivated NO-induced relaxation was related to the amount of NO release, light intensity and concentration of compounds. 5. The evidence that photoactivated NO-induced relaxation is mediated by cGMP comes from the observation that zaprinast, but not forskolin, potentiated the relaxation. 6. It is concluded that rat gastric smooth muscle relaxes to photoactivation of NO or NO 2-carrying molecules via NO, and it appears that degree of membrane depolarization may be a critical factor in dissociating the response to presynaptic- and postsynaptic NO-mediated relaxation in this muscle.

Agonist-Induced Activation of Rat Mesenteric Resistance Vessels

The effects of noradrenaline (NA, 10(-5) M) and [arginine8]vasopressin (AVP, 10(-7) M) on tension in Ca2+-free medium and on membrane potential, and the inhibition of NA- and AVP-induced contractions by isradipine, have been compared in mesenteric resistance vessels (MRVs) from Wistar-Kyoto (WKY) rats. The release of intracellular Ca2+ by AVP contributed significantly less to its tension development than does that by NA. Nonetheless, the concentration-response curves for inhibition by isradipine of NA- and AVP-induced tonic tension were nearly identical. Similarly, these two agonists produced the same degree of membrane depolarization. In addition, both agonists were able to stimulate large contractions in vessels previously depolarized by 80 mM K+. AVP also stimulated 45Ca influx into rat cultured aortic smooth muscle cells. In contrast to the stimulation of 45Ca influx by KCl depolarization, the agonist-stimulated 45Ca influx was insensitive to inhibition by organic Ca2+ antagonists. It is concluded that Ca2+ entry through receptor-operated Ca2+-permeable channels (ROCs) may contribute to agonist-induced activation of rat aortic and MRV smooth muscle.

Agonist-Induced Activation of Rat Mesenteric Resistance Vessels

The effects of noradrenaline (NA, 10−5M) and [arginine8]vasopressin (AVP, 107M) on tension in Ca2+ -free medium and on membrane potential, and the inhibition of NA- and AVP-induced contractions by isradipine, have been compared in mesenteric resistance vessels (MRVs) from Wistar–Kyoto (WKY) rats. The release of intracellular Ca2+ by AVP contributed significantly less to its tension development than does that by NA. Nonetheless, the concentration-response curves for inhibition by isradipine of NA- and AVP-induced tonic tension were nearly identical. Similarly, these two agonists produced the same degree of membrane depolarization. In addition, both agonists were able to stimulate large contractions in vessels previously depolarized by 80 mM K−. AVP also stimulated 45Ca influx into rat cultured aortic smooth muscle cells. In contrast to the stimulation of 45Ca influx by KCI depolarization, the agonist-stimulated 45Ca influx was insensitive to inhibition by organic Ca2+ antagonists. It is concluded that Cap+ entry through receptor-operated Ca2+ -permeable channels (ROCs) may contribute to agonist-induced activation of rat aortic and MRV smooth muscle.

Mechanisms underlying the electrical and mechanical responses of the guinea-pig internal anal sphincter to field stimulation and to drugs.

The electrical membrane characteristics and the response of the circular muscle of the guinea-pig internal anal sphincter (i.a.s.) to field stimulation were studied in vitro using intracellular microelectrodes and conventional mechanical recording techniques. The i.a.s. developed its own tone (3-4 g), following initial stretch (1 g) and spontaneous spike potentials were evident. In the absence of spike potentials, tone declined and disappeared. Tone was not significantly reduced by phentolamine (1 X 10(-6)M). The resting membrane potential, measured between spontaneous spike potentials, was -45 +/- 3.0 mV (n = 224); the space constant (lambda) was 1.13 +/- 0.1 mm (n = 13). Spikes usually overshot by approximately 15 mV. The frequency of spike potential discharge (1-3 Hz) varied with the degree of membrane depolarization, being increased in K+-rich and decreased in K+-deficient solutions or by the presence of Mn2+. It was not significantly affected by C1-withdrawal but was increased in Na+-deficient solutions with or without tetrodotoxin (TTX; 1 X 10(-6)M). Field stimulation (1-20 Hz, 0.5 ms, supramaximal voltage) produced inhibitory junction potentials (i.j.ps) and relaxed tone; at high frequencies (50 Hz or greater), contractions were observed but excitatory junction potentials (e.j.ps) were not. I.j.ps and relaxations were inhibited by apamin (1 X 10(-6)M), TTX (1 X 10(-6)M) but not by atropine (1 X 10(-6)M), phentolamine (1 X 10(-6)M) or hexamethonium (1 X 10(-6)M). I.j.ps were reduced by hyperpolarization and enhanced by depolarization of the membrane by current pulses (15s). The mean equilibrium potential for the i.j.p. was -94 mV (correlation coefficient, gamma = 0.71, n = 5, p less than 0.001). I.j.ps were enhanced in K+-deficient solutions and reduced in K+-rich solutions. Together these results suggest that the i.j.p. is mediated by an increased GK. The absence of [Ca2+]o or the presence of Mn2+ (2 mM) abolished the i.j.p.; in contrast Na+-deficient or C1-free solutions were ineffective in this respect. Tetraethylammonium (5-50mM) abolished the i.j.p.; the accompanying relaxation was reduced by about 80%. The major aspect of the relaxation to nerve stimulation is mediated by membrane hyperpolarization.

A cellular mechanism for myogenic regulation of cat cerebral arteries.

Autoregulation of cerebral blood flow is accomplished through integration of metabolic, neurogenic and myogenic mechanisms. Myogenic mechanisms involve activation of cerebral arterial muscle cells as transmural pressure increases, providing a means through which vessel caliber can be regulated to maintain blood flow constant. The cellular mechanisms involved in this myogenic response may involve changes in the electrical potential across the plasma membrane. When isolated cat middle cerebral arteries are cannulated and prepared in a manner allowing manipulation of transmural pressure, the muscle cell membrane depolarizes as pressure increases. The degree of membrane depolarization in response to an elevated pressure is dependent upon extracellular Ca2+ [( Ca]o), increasing as [Ca]o is elevated and markedly decreasing as [Ca]o is reduced to low levels. When these arterial preparations are maintained at a physiological pressure of around 100 mm Hg, spontaneous action potentials can be recorded which increase in frequency upon further elevation in pressure. Vessels exhibiting such electrical activity can be observed to decrease in diameter as pressure is increased. Such finding suggest a membrane electrical mechanism for myogenic autoregulation of cerebral arteries.

Effects of a 4-aminopyridine in calcium movements and changes of membrane potential in pinched-off nerve terminals from rat cerebral cortex.

Abstract: A study was conducted on the influence of 4‐aminopyridine (4‐AP) on the radiocalcium uptake and membrane potential of rat cortical synaptosomes previously depolarized by biochemical procedures. The initial calcium entry into isolated nerve terminals was substantally enhanced in the presence of 10‐−4M 4‐AP in potassium‐rich media (60 mM). The fast initial phase of potassium‐stimulated calcium entry involves different kinetic characteristics in the presence and the absence of 4‐AP. In the presence of 4‐AP, the fast component of calcium entry reached a peak during the first 15 s immediately following depolarization. The potassium‐stimulated synaptosomal calcium influx at 15 s was 3.5 ± 0.17 times higher in the presence of 4‐AP (10‐−4M) and 2.6 ± 0.11 times higher in the absence of 4‐AP as compared with the respective unstimulated uptake values. In the absence of 4‐AP, however, the calcium entry did not show a peak until 45 s after depolarization. The total amount of calcium accumulated in the synaptosome treated and untreated by 4‐AP is equal at the end of this initial uptake period. The effect of 4‐AP on membrane potential during depolarization of synaptosomes evoked by potassium‐rich medium was also determined by means of a potential sensitive fluorescent dye. It was found that 4‐AP had no effect on the final level of membrane potential, induced by high [K+]o; however, the kinetics showed significant differences. The initial phase of synaptosomal membrane depolarization was slower in the presence of 4‐AP: τ‐4‐AP: 42 ± 4.0 s, τ‐control: 18 ± 2.5 s. The initial calcium entry into the nerve terminals was increased during the 4‐AP treatment in potassium‐rich media—as resulted from our calcium uptake studies—and thus, during this higher calcium influx, the degree of membrane depolarization was decreased. This faster initial calcium influx constitutes a feasible explanation for the unique effect of 4‐AP termed “chemical potentiation” on transmitter release at chemical synapses.