Lymphatic vasomotion

1. Experimental findings in the past decade have greatly advanced present understanding of electrical/mechanical rhythmicities in smooth muscle, including vasomotion. Lymphatic vessels show strong vasomotor activity and have provided a key experimental system to study these processes. 2. Evidence from lymphatic vessels, blood vessels and other smooth muscles indicates that rhythmical contractions arise through a Ca2+ store-controlled pacemaker mechanism, which can function to cause smooth muscle constriction. 3. Such a model fits with observations that vasomotion can be near synchronous over large vessel lengths involving many cells. 4. The alternative interpretation that smooth muscle rhythmicities are generated by a cardiac-like electrical pacemaker mechanism has not been substantiated in any smooth muscle preparation under normal physiological conditions. However, elements of this latter mechanism are likely to be present at least in some smooth muscles, serving to modulate pacemaking.     

Influence of Infrasound Exposure on the Whole L-type Calcium Currents in Rat Ventricular Myocytes

This study was designed to examine the effect of infrasound exposure (5 Hz at 130 dB) on whole-cell L-type Ca²⁺ currents (WLCC) in rat ventricular myocytes and the underlying mechanism(s) involved. Thirty-two adult Sprague-Dawley rats were randomly assigned to infrasound exposure and control groups. [Ca²⁺]_i, WLCC, mRNA expression of the a_1c subunit of L-type Ca²⁺ channels (LCC), and SERCA2 protein were examined on day 1, 7, and 14 after initiation of infrasound exposure. Fluo-3/AM fluorescence and the laser scanning confocal microscope techniques were used to measure [Ca²⁺]_i in freshly isolated ventricular myocytes. The Ca²⁺ fluorescence intensity (FI), denoting [Ca²⁺]_i in cardiomyocytes, was significantly elevated in a time-dependent manner in the exposure groups. There was a significant increase in WLCC in the 1-day group and a further significant increase in the 7- and 14-day groups. LCC mRNA expression measured by RT-PCR revealed a significant rise in the 1-day group and a significant additional rise in the 7- and 14-day groups compared with control group. SERCA2 expression was significantly upregulated in the 1-day group followed by an overt decrease in the 7- and 14-day groups. Prolonged exposure of infrasound altered WLCC in rat cardiomyocytes by shifting the steady-state inactivation curves to the right (more depolarized direction) without altering the slope and biophysical properties of I _Ca,L. Taken together, our data suggest that changes in [Ca²⁺]_I levels as well as expression of LCC and SERCA2 may contribute to the infrasound exposure-elicited cardiac response. Zhaohui Pei and Zhiqiang Zhuang contributed equally to this work.     

The actions of ryanodine on Ca2+-activated conductances in rat cultured DRG neurones; evidence for Ca2+-induced Ca2+ release

The whole-cell recording technique was used to investigate the actions of a calcium release channel ligand, ryanodine, on calcium-activated chloride conductances, and to evaluate ryanodine-sensitive Ca²⁺-induced Ca²⁺ release from intracellular stores in cultured neonatal rat DRG neurones. The aim of the project was to use ryanodine as a pharmacological tool to evaluate calcium-induced calcium release in the cell bodies of cultured DRG neurones. Action potential after-depolarizations were attenuated by extracellular application of the chloride channel blocker, niflumic acid (10 μM), and by ryanodine (10 μM); these actions occurred without concurrent changes in evoked action potentials. Ryanodine and caffeine (10 mM) activated calcium-dependent conductances and the responses to ryanodine were attenuated by depletion of caffeine-sensitive Ca²⁺ stores. The current clamp data were complicated by changes in potassium conductances so studies were carried out under voltage clamp and voltage-activated calcium currents and calcium-activated chloride and non-selective cation currents were isolated pharmacologically. Ryanodine (10 μM) evoked delayed, inward, calcium-activated non-selective cation and chloride currents which reversed close to 0 mV and were attenuated by N-methyl-d-glucamine, niflumic acid and dantrolene. Consistent with actions on action potential after-depolarizations, niflumic acid (10 μM) and ryanodine (10 μM) attenuated calcium-activated chloride currents evoked by calcium entry through voltage-activated calcium channels. Niflumic acid and ryanodine had no effects on voltage-activated calcium currents evoked from a holding potential of –90 mV by voltage step commands to 0 mV. In conclusion calcium-activated chloride conductances appear to be activated in part by calcium released from ryanodine-sensitive stores, and significant calcium-induced calcium release may occur locally in cell bodies of DRG neurones as a result of calcium entry through voltage-activated channels during an action potential. Received: 6 July 1998 / Accepted: 30 November 1998     

The voltage sensor is responsible for ΔpH dependence in Hv1 channels

The dissipation of acute acid loads by the voltage-gated proton channel (H_v1) relies on regulating the channel’s open probability by the voltage and the ΔpH across the membrane (ΔpH = pH_ex − pH_in). Using monomeric Ciona-H_v1, we asked whether ΔpH-dependent gating is produced during the voltage sensor activation or permeation pathway opening. A leftward shift of the conductance-voltage (G-V) curve was produced at higher ΔpH values in the monomeric channel. Next, we measured the voltage sensor pH dependence in the absence of a functional permeation pathway by recording gating currents in the monomeric nonconducting D160N mutant. Increasing the ΔpH leftward shifted the gating charge-voltage (Q-V) curve, demonstrating that the ΔpH-dependent gating in H_v1 arises by modulating its voltage sensor. We fitted our data to a model that explicitly supposes the H_v1 voltage sensor free energy is a function of both the proton chemical and the electrical potential. The parameters obtained showed that around 60% of the free energy stored in the ΔpH is coupled to the H_v1 voltage sensor activation. Our results suggest that the molecular mechanism underlying the H_v1 ΔpH dependence is produced by protons, which alter the free-energy landscape around the voltage sensor domain. We propose that this alteration is produced by accessibility changes of the protons in the H_v1 voltage sensor during activation.

The voltage-gated proton channel (H_v1) is gated by internal and external pH changes favoring proton extrusion during acute cytosolic acidosis, an essential function for cell physiology and pathophysiology (1). The wide diversity of cell types in which H_v1 is expressed, including different immune cells, sperm, microglia, lung epithelial cells, osteoclasts, cardiac cells, and cancer cells (1), renders this channel as a promising pharmacological target. A selective H_v1 modulation can be achieved by studying the molecular mechanisms of its distinctive pH dependence; although pH modulates other voltage-gated ion channels (2–5), the pH dependence of H_v1 is unique, since the channel opening depends only on the ΔpH (pH_ex − pH_in) established across the membrane (6). Briefly, voltage-gated ion channels have a voltage-sensing domain (made up of transmembrane segments S1 to S4) coupled to a pore domain (S5 to S6) (7). Quite interestingly, H_v1 consists of only four transmembrane segments (from S1 to S4) flanked by intracellular N- and C-terminal domains (8, 9); thus, the permeation pathway, voltage sensor, and pH sensor(s) are contained in these four transmembrane segments. Moreover, the C-terminal domain folds into a coiled-coil structure between two H_v1 subunits to produce a dimer (10, 11), resulting in monomeric voltage-gated proton channels when the N- and C-terminal domains are deleted (10). Although the H_v1 monomer lacks the dimer cooperative opening (12, 13), it maintains its distinctive biophysical properties (10). However, although it has been reported that the monomeric H_v1 gating depends on pH, it is not clear whether the distinctive ΔpH-dependent gating is still present in this channel.The remarkable pH dependence of the H_v1 opening has been observed since the first proton current measurements (14, 15). However, its detailed analysis was only possible when fine precautions to control the pH during the proton current measurement were taken into account (6). These studies showed that conductance-voltage (G-V) curves and activation kinetics shift along the voltage axis as a function of the ΔpH regardless of the internal or external pH (6, 16). Although cloning of the H_v1 gene (8, 9) facilitated the search for mutations affecting the channel’s pH dependence (16–19), its molecular mechanism has remained elusive. In this regard, the H_v1 pH dependence has been challenging to understand, because it has mainly been studied by measuring the dimer’s proton current; in fact, the pH dependence of these currents could be produced by modulating the channel cooperativity, voltage sensor activation, or channel opening. Indeed, there is evidence indicating that pH affects both the voltage sensor movement (20, 21) and the channel’s unitary conductance (22). Consequently, the simultaneous processes that occur during H_v1 gating obscure the source of this ΔpH dependence. To overcome these difficulties, we used a nonconducting mutant of the Ciona intestinalis H_v1 (CiH_v1) monomer to monitor the pH dependence exclusively on the voltage sensor activation. By measuring the voltage sensor movements directly from gating currents in the absence of cooperativity (10, 23) and proton currents (24–26), we asked whether the ΔpH-dependent gating in H_v1 originates during the voltage sensor activation or the permeation pathway opening. We found that the H_v1 voltage sensor movements are modulated by pH. In detail, the voltage sensor activation is coupled to the chemical free energy stored in the ΔpH, resulting in a ΔpH-dependent shift of the gating charge-voltage (Q-V) curves along the voltage axis. Accordingly, the H_v1 ΔpH-dependent gating is a consequence of the voltage sensor modulation. On the other hand, the gating currents kinetics depended on the internal and external pH values, indicating that the pH modulates the voltage sensor movements in a state-dependent manner.     

细胞受体外机械力作用的若干反响

细胞受力后会有很多反响,作者介绍当前对细胞施加机械力的多种试验方法以及受试后的各种反响。其中内容主要包括激活离子通道、对细胞膜上蛋白分子的作用、对内皮细胞ICAM-1表达的影响、对细胞骨架的作用、对内皮细胞ICAM-1表达的影响、剪切力作用原癌基因c-fos和c-myc的表达等。