Start Of Depolarization Research Articles

Local Ca2+ coupling between mitochondria and sarcoplasmic reticulum following depolarization in guinea pig urinary bladder smooth muscle cells.

Spatiotemporal changes in cytosolic Ca2+ concentration ([Ca2+]c) trigger a number of physiological functions in smooth muscle cells (SMCs). We previously imaged Ca2+-induced Ca2+ release following membrane depolarization as local Ca2+ transients, Ca2+ hotspots, in subplasmalemmal regions. In this study, the physiological significance of mitochondria on local Ca2+ signaling was examined. Cytosolic and mitochondrial Ca2+ images following depolarization or action potentials were recorded in single SMCs from the guinea pig urinary bladder using a fast-scanning confocal fluorescent microscope. Depolarization- and action potential-induced [Ca2+]c transients occurred at several discrete sites in subplasmalemmal regions, peaked within 30 ms, and then spread throughout the whole-cell. In contrast, Ca2+ concentration in the mitochondria matrix ([Ca2+]m) increased after a delay of ~50 ms from the start of depolarization, and then peaked within 500 ms. Following repolarization, [Ca2+]c returned to the resting level with a half-decay time of ~500 ms, while [Ca2+]m recovered more slowly (∼1.5 s). Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, a mitochondrial uncoupler, abolished depolarization-induced [Ca2+]m elevations and slowed [Ca2+]c changes. Importantly, short depolarization-induced changes in [Ca2+]m and transmembrane potential in mitochondria coupled to Ca2+ hotspots were significantly larger than those in other mitochondria. Total internal reflection fluorescence imaging revealed that a subset of mitochondria closely localized with ryanodine receptors and voltage-dependent Ca2+ channels. These results indicate that particular mitochondria are functionally coupled to ion channels and sarcoplasmic reticulum fragments within the local Ca2+ microdomain, and thus, strongly contribute to [Ca2+]c regulation in SMCs.

Induced polarization and surface electrochemistry

Induced-polarization (IP) responses arising from activation overvoltage are investigated, with the goal of relating established electrochemical properties and processes to IP time constants. In activation overvoltage, an electrical double layer with a well-defined areal capacitance forms at the mineral-electrolyte interface. This study shows that the known capacitance of the interface is compatible with observed initial time constants for the decay of electrode polarization. Furthermore, because the electrical double-layer capacitance is determined largely by the electrolyte and not the mineral, the conclusion is that, although the polarization is caused by the presence of the mineral, the time constants of the decay are determined primarily by the electrolyte and not the mineral. The dependence of the capacitance on the sign and magnitude of the potential implies that the effective decay time constant will be small at the start of depolarization and will progress to larger values as the decay proceeds.

Spectral components of cytosolic [Ca2+] spiking in neurons.

We show here, by means of evolutionary spectral analysis and synthesis of cytosolic Ca2+ ([Ca2+]c) spiking observed at the single cell level using digital imaging fluorescence microscopy of fura-2-loaded mouse cerebellar granule cells in culture, that [Ca2+]c spiking can be resolved into evolutionary spectra of a characteristic set of frequencies. Non-delayed small spikes on top of sustained [Ca2+]c were synthesized by a main component frequency, 0.132+/-0.012 Hz, showing its maximal amplitude in phase with the start of depolarization (25 mM KCI) combined with caffeine (10 mM) application. Delayed complex responses of large [Ca2+]c spiking observed in cells from a different set of cultures were synthesized by a set of frequencies within the range 0.018-0.117 Hz. Differential frequency patterns are suggested as characteristics of the [Ca2+]c spiking responses of neurons under different conditions.

Time course of Ca 2+-dependent K + and Cl − currents in single smooth muscle cells of guinea-pig trachea

The time course of two types of Ca 2+-dependent currents were compared in single smooth muscle cells freshly isolated from guinea-pig trachea. When the pipette solution contained mainly 140 mM KCl, depolarization from −60 mV to 0 mV evoked an initial inward current followed by an outward current which consisted of transient ( I to) and sustained components. In addition, a long-lasting inward tail current ( I tail) was occasionally observed after the repolarization to −60 mV. Although I to often occurred repetitively during depolarization, the first I to reached the peak of ∼50 ms after the start of depolarization and had the largest amplitude in most cells examined. The amplitude of I tail increased with the increase in depolarization period up to about 500 ms. Pharmacological analyses indicate that I to and I tail are Ca 2+-dependent K + and Cl − currents ( I K-Ca and I Cl-Ca), respectively, and suggest that not only Ca 2+-influx through Ca 2+ channels but also subsequent Ca 2+ release from stores contributes to activate these currents. Spontaneous transient outward and inward currents, I K-Ca and I Cl-Ca, respectively, were simultaneously recorded at −40 mV. In over 80% of the spontaneous current events, outward and inward currents coupled one to one and always occurred in this order. Puff-application of 10 mM caffeine also induced I K-Ca and I Cl-Ca in this order at −40 mV. When caffeine was applied twice with various intervals, the current amplitude in the second application depended upon the period of the interval. The recovery of I Cl-Ca during the interval was faster than that of I K-Ca. The results indicate that the activation and decay time courses of I Cl-Ca are slower but its recovery is faster than those of I K-Ca.

The intracellular pH of frog skeletal muscle: its regulation in hypertonic solutions.

Intracellular pH (pHi) was followed with micro-electrodes in frog semitendinosus muscle, superfused at 22 degrees C with hypertonic solutions (external pH, pHo, 7.35) containing 2.5, 15 or 50 mM-K. Tonicity was doubled by addition of 250 mM-mannitol or, in a few cases, 125 mM-extra NaCl. Tripling of tonicity was accomplished by adding 500 mM-mannitol. Because of the ability of hypertonicity to minimize contracture, the course of pHi could be followed from the start of depolarization. The pHi of fibres after about 40 min in Ringer solution (2.5 mM-K, HEPES buffer) of twice normal tonicity was 7.40 +/- 0.04 (S.E. of mean) (n = 17), about 0.2 higher than at normal tonicity. The membrane potential, Vm, was -87.7 +/- 1.3 mV. When the muscle was depolarized in 50 mM-K to about -30 mV, the pHi rapidly fell by 0.3-0.5 unit (n = 9), and then promptly returned. This recovery was followed by a much slower and progressive rise to above control. Removing Na from the medium did not affect the degree of acidification, but the pHi recovered at a slightly slower rate, did not reach control value and showed no progressive rise. A less pronounced transient acidification was also observed when the muscle was depolarized in 15 mM-K to about -60 mV. When contracture was prevented either by 1-2 mM-tetracaine under isotonic conditions or by raising tonicity 3-fold, 50 mM-K produced no transient acidification. When the pHi of resting fibres in Ringer solution (2.5 mM-K) of twice normal tonicity was reduced by 5% CO2 from 7.40 to 7.12 +/- 0.07 (n = 3), it recovered at a slow rate (0.06 +/- 0.03 delta pHi h-1). Depolarization by 15 or 50 mM-K enhanced recovery rate 4-6-fold. These solutions of twice normal tonicity, as compared to those of normal tonicity, shifted the curve relating pHi recovery rate and membrane potential along the potential axis in the direction of hyperpolarization. This shift may be due to increased ionic shielding of fixed negative charges at the inner membrane surface. At twice normal tonicity, the very slow pHi recovery of resting fibres from CO2-induced acidification, as well as the more rapid recovery in depolarized fibres, could be abolished by 1 mM-amiloride or by removing Na. The application of amiloride during pHi recovery in 50 mM-K was not associated with an observable change in Vm. SITS had no significant effect on recovery.(ABSTRACT TRUNCATED AT 400 WORDS)

Response to light of various retinal cell types during spreading depression

The retina in low Cl − media gives rise to spreading depression (SD) accompanied by a large, receptor side negative potential change (SDP). Changes during SD in the membrane potential and the responsiveness to light of various retinal cell types are studied, using a pair of intra- and extracellular electrodes and applying dim white light flashes to the retina at 5 sec intervals throughout the experiment. The study shows that the amaerine and ganglion cells depolarize strongly during SD to complete loss of response to light, and that the start of depolarization in these cell types precedes a little that of SDP. The other cell types continue to respond to light flashes during SD, though their response amplitude decreases as the cells depolarize due probably to an increase in [K +] 0 accompanying SD. The results suggest that the amaerine and ganglion cells take an initiative role in the SD generation, and that the changes in the other cell types are mostly of passive nature.

The effects of metal ions and caffeine on electro-mechanical coupling in crayfish muscle fibers.

1. The effects of divalent metal ions on electro-mechanical coupling were examined in crayfish muscle fibers, using the voltage clamp method. Metal ions were added by substitution for Na. The relation between tension and membrane potential was directly figured by leading tension and potential to the Y and X axes of the oscilloscope and by depolarizing the membrane very slowly with the linearly rising pulse.2. Replacement of NaCl with equimolar choline chloride shifted the tension-membrane potential relation toward a more negative potential.3. Divalent metal ions elevated the mechanical threshold potential and decreased the tension at the given membrane potential. They not only shifted the curve relating tension and membrane potential along the potential axis but also decreased the slope of the curve. The order of the inhibitory action was Cd>Co≥Mn>Ni>Ca≥Mg>Sr>Ba. Their inhibitory effects appeared fairly rapidly after addition of metal ions and disappeared, fully reversibly, after their removal.4. The time courses of tension rise after the start of depolarization and its fall after the end of depolarization were explained as the sum of two or three exponential components. The time constants were not changed by metal ions.5. Caffeine lowered the mechanical threshold potential and shifted the tension-membrane potential relation toward a more negative potential.6. It is suggested that the inhibitory effect of metal ions on tension results from their stabilizing action on the Ca-permeability of the internal membrane system.