Mean Time Constant Research Articles

Hyperpolarization-Activated Current (I h) in Vestibular Calyx Terminals: Characterization and Role in Shaping Postsynaptic Events

Calyx afferent terminals engulf the basolateral region of type I vestibular hair cells, and synaptic transmission across the vestibular type I hair cell/calyx is not well understood. Calyces express several ionic conductances, which may shape postsynaptic potentials. These include previously described tetrodotoxin-sensitive inward Na(+) currents, voltage-dependent outward K(+) currents and a K(Ca) current. Here, we characterize an inwardly rectifying conductance in gerbil semicircular canal calyx terminals (postnatal days 3-45), sensitive to voltage and to cyclic nucleotides. Using whole-cell patch clamp, we recorded from isolated calyx terminals still attached to their type I hair cells. A slowly activating, noninactivating current (I(h)) was seen with hyperpolarizing voltage steps negative to the resting potential. External Cs(+) (1-5 mM) and ZD7288 (100 μM) blocked the inward current by 97 and 83 %, respectively, confirming that I(h) was carried by hyperpolarization-activated, cyclic nucleotide gated channels. Mean half-activation voltage of I(h) was -123 mV, which shifted to -114 mV in the presence of cAMP. Activation of I(h) was well described with a third order exponential fit to the current (mean time constant of activation, τ, was 190 ms at -139 mV). Activation speeded up significantly (τ=136 and 127 ms, respectively) when intracellular cAMP and cGMP were present, suggesting that in vivo I(h) could be subject to efferent modulation via cyclic nucleotide-dependent mechanisms. In current clamp, hyperpolarizing current steps produced a time-dependent depolarizing sag followed by either a rebound afterdepolarization or an action potential. Spontaneous excitatory postsynaptic potentials (EPSPs) became larger and wider when I(h) was blocked with ZD7288. In a three-dimensional mathematical model of the calyx terminal based on Hodgkin-Huxley type ionic conductances, removal of I(h) similarly increased the EPSP, whereas cAMP slightly decreased simulated EPSP size and width.

Is tonic vergence protective against strabismus during development?

Young children have increased accommodative demand due to their typically hyperopic refractive error, while they have reduced vergence demand relative to adults, due to their decreased interpupillary distance. The neural coupling of accommodation and vergence implies that young children may experience an apparent cue conflict for stable accommodative and vergence responses (Bharadwaj & Candy, 2009). Some children develop strabismus, potentially resulting from this conflict (Parks, 1958). How most children are able to avoid strabismus is poorly understood. The purpose of this study was to determine whether tonic adaptation of vergence might protect young children from this conflict by reducing the impact of the neural coupling. Participants were 3- to 10-year-old typically-developing children and adults, who watched a video at a 33cm viewing distance. They initially viewed it monocularly to determine their baseline heterophoria (with a near IR filter and MCS PowerRefractor, 25Hz). They then watched the video binocularly for 5 or 60 sec, after which their alignment with the filter was measured again to determine the time constant over which they returned to their baseline heterphoria (Schor & Horner, 1989). The mean phoria at the task distance in adults was 4.4 pd (±2.9) exophoria and in children was 3.5 pd (±3.9) exophoria (p = 0.60). For these tasks the mean time constants only differed by 1.5sec (p = 0.40) (after 5sec viewing) and 1.8sec (p = 0.54) (after 60sec viewing) between the adults and children. Thus the balance of tonic adaptation and phasic activity appear similar for children and adults for these tasks. Given that tonic vergence is active in adults (Schor, 1979), the fact that the data from children were comparable to adults' indicates that tonic vergence could play a role in achieving alignment during development.

Proof that gmT2 is the reciprocal of gmR2

When performing multiexponential analysis of relaxation times in the slow exchange regime, it is often convenient to simplify the resulting distribution to one or more relaxation time constants. In doing so, what averaging method should be used and should the rate constant or time constant be reported? This note outlines why the geometric mean is most appropriate and provides a proof that the geometric mean rate and geometric mean time constants are reciprocals, which is counter-intuitive unless one considers that averaging values in a distribution is different than physically averaging relaxation species. © 2011 Wiley Periodicals, Inc. Concepts Magn Reson Part A 38A: 128–131, 2011.

Presynaptic impairment in Niemann–Pick C1-deficient neurons: Not dependent on presence of glial cells

Niemann–Pick disease type C (NPC) is a progressive neurodegenerative disorder characterized by accumulation of free cholesterol in late endosomes/lysosomes. The pathological basis for the disease has been poorly understood yet. In our previous study, we have demonstrated that synaptic function is impaired in this disease. In the current study, electrophysiological and fluorescent dyes studies were used to determine whether the synaptic defects result from presynaptic or postsynaptic contributions. Furthermore, we would like to ascertain whether such defects are caused by direct effect of NPC1 deficiency in neurons or indirect effect of NPC1 deficiency in glial cells. Both mean inter-event interval of miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) were significantly larger in NPC1 −/− neurons than those in the wild-type neurons, while the amplitudes and the receptor kinetics were not different compared with those in wild-type controls. Synaptic vesicle exocytosis was also slower in the NPC1 −/− neurons. The mean time constant of destaining was larger in NPC1 −/− neurons than in wild-type controls both in the presence and absence of glial cells. All these results indicated a general presynaptic functional impairment in the NPC1 −/− neurons and such defects were not dependent of glial cells. Therefore, neuropathology characteristics of NPC diseases may be a more possible consequence of neuronal presynaptic dysfunction than indirect defects in glial cells.

Neonatal Hyperoxia Contributes Additively to Cigarette Smoke–Induced Chronic Obstructive Pulmonary Disease Changes in Adult Mice

The extent by which early postnatal lung injury contributes to the development of chronic obstructive pulmonary disease (COPD) in the adult is unclear. We hypothesized that exposure to hyperoxia during early postnatal life can augment lung changes caused by adult chronic cigarette smoke (CS) exposure. C57BL/6J mice (1 d old) were exposed to hyperoxia (O(2)) for 5 days. At 1 month of age, half of the O(2)-exposed mice and half of the control mice were placed in a CS chamber for 6 months. After exposure to CS, mice underwent quasi-static pressure-volume curve and mean chord length measurements; quantification of pro-Sp-c expression; and measurement of lung IL-8/ KC, CXCR2/IL8Rα, TNF-α, and IL-6 mRNA by real-time PCR. Adult mice exposed to O(2)+CS had significantly larger chord length measurements (P < 0.02) and lung volumes at 35 cm H(2)O (P < 0.05) compared with all other groups. They also had significantly less pro-Sp-c protein and surfactant protein C mRNA expression (P < 0.003). Mice exposed to O(2)+CS and CS-only mice had significantly higher lung resistance and longer mean time constants (P < 0.01), significantly more inflammatory cells in the bronchoalveolar lavage fluid (P < 0.03), and significantly higher levels of lung CXCR2/IL8Rα mRNA compared with mice not exposed to smoke (P < 0.02). We conclude that exposure to early postnatal hyperoxia contributed additively to CS-induced COPD changes in adult mice. These results may be relevant to a growing population of preterm children who sustained lung injury in the newborn period and may be exposed to CS in later life.

Neuronal Responses during and after the Presentation of Static Visual Stimuli in Macaque Primary Visual Cortex

Viewing static visual scenes for several seconds or longer can induce a wide variety of striking percepts, including negative afterimages, fading, and motion aftereffects. To characterize the neuronal bases of such phenomena and elucidate functional circuitry in the visual system, we recorded responses of neurons in primary visual cortex (V1) of anesthetized macaques during and after the presentation of prolonged static visual stimuli. We found that 72% of cells generated significant after-responses (ARs) that outlasted classical off-transients after the cessation of stimuli, and AR amplitude grew with stimulus duration. After the longest stimuli tested (32 s), the amplitude and the time course of the AR were on average comparable to, and correlated with, those of the maintained response evoked while stimuli were present. These observations generally held regardless of cell class: simple, complex, direction selective (DS) or non-DS. The average decay time constant of the AR for orientation-tuned cells was 0.65 s. This is strikingly shorter than time constants observed in the lateral geniculate nucleus, which were on the order of tens of seconds. Cells in V1 that lacked orientation tuning displayed an intermediate time course, with a mean time constant of 4.3 s. These results are consistent with a multistage model in which cells at successive stages adapt to their inputs with progressively shorter time constants. Our findings suggest that the perceptual phenomena of fading and afterimages are shaped by both cortical and subcortical dynamics and provide a physiological framework for the interpretation of recent and long-standing psychophysical observations.

A21 Altered calcium kinetics in skeletal muscle fibres of the R6/2 mouse model of HD

Background and aimsSome of the most obvious peripheral tissue changes in Huntington's disease (HD) are found in skeletal muscle. Alterations in morphology, gene expression pattern, energy metabolism and differentiation have...

Unusually Long Lifetime of Excited Charge-Transfer State of All-Inorganic Binuclear TiOMnII Unit Anchored on Silica Nanopore Surface

The lifetime and back electron transfer kinetics of an all-inorganic, oxo-bridged heterobinuclear TiIVOMnII group covalently anchored on a silica nanopore surface was investigated by transient optical absorption spectroscopy. Mesoporous silica particles of type SBA-15 loaded with TiOMn sites (1 wt %) were suspended in an index matching liquid for performing spectroscopy in transmission mode. Upon excitation of the TiIVOMnII → TiIIIOMnIII metal-to-metal charge-transfer transition (MMCT) by a visible laser pulse of 8 ns duration, a transient bleach was observed whose intensity versus pump wavelength dependence agreed with the MMCT absorption profile. The decay kinetics is well described by a superposition of first-order rates with a mean time constant of 1.8 ± 0.3 μs (room temperature). The dispersion of γ = 2 ± 0.2 (Albery model) is attributed to variations in the local silica coordination environment reflecting the disordered, amorphous nature of the silica nanopore surface. The result constitutes the first observation of the electron transfer kinetics of an all-inorganic heterobinuclear group. It is proposed that the microsecond lifetime, unusually long for such as small charge transfer chromophore, originates from strong polarization of the local and remote silica environment upon light-triggered electron transfer from Mn to Ti. This results in a substantial reorganization barrier for back electron transfer. The long lifetime makes oxo-bridged heterobinuclear units anchored on silica surfaces efficient visible light photocatalysts and suitable as charge-transfer chromophores for driving multielectron catalysts in artificial photosynthetic systems.

Transient sweat response of the human head during cycling

This research aims at quantifying transient spatial gradients in sweat production on a human head while cycling. Six test persons were studied. Each test lasted 30 min while a change in work rate was applied after 5 min (from 80 to 150 W for males and from 50 to 125 W for females). Two conditions were analyzed in this research: warm (28.3 ± 0.1 °C, 38 ± 0.6% RH and 0.1 ± 0.1 m/s air velocity) and standard (16.1° ± 0.2 C, 45% ± 0.6 RH and 2.4 ± 0.2 m/s air velocity). Sweat production of the head was measured as a function of time on the right temple, left temple and forehead. This allowed modelling the dynamics of the sweat production response. Constant steady state sweat production, time delay in sweat production, time constant of sweat production and steady state gain of sweat production were quantified and analyzed. Time constants of sweat production were shorter in the warm condition compared to the standard condition. Mean and SEM time constant of sweat production varied from 561 ± 144 s (frontal region) to 1117 ± 230 s (left temple) and 1080 ± 232 s (right temple) in the warm condition. While, at the standard condition, the time constant of sweat production varied from 873 ± 121 s at the frontal region to 1431 ± 195 s at the left temple and 1727 ± 196 s at the right temple. Additionally, also constant steady state sweat production was 0.4–0.7 mg min −1 cm −2 higher in the warm compared to the standard condition ( P < 0.05). However, no differences ( P > 0.05) were observed for steady state gain and time delay of sweat production between the standard and warm condition. The results of this research can be used to enhance physiological insight of the sweating process and it can also help to develop sweating thermal manikins that behave more realistically to thermal changes. Knowledge of sweat production might also be valuable when designing active controlled headgear since the reaction time of the actuator should take the dynamics of sweat rate into account as a function of work rate and thermal environmental conditions. Relevance to industry Understanding of the dynamic behaviour of sweat production in relation to work rate under different environmental conditions allows the design of model based controllers in headgear that actively minimize sweat production. This could help a user's desire to wear a helmet as well as his ability to concentrate.

Characterization of the physiological- and genetic phenotype of CRF-expressing neurons in the bed nucleus of the stria terminalis

Characterization of the physiological- and genetic phenotype of CRF-expressing neurons in the bed nucleus of the stria terminalis

Effects of a 3-month rehabilitation program on muscle oxygenation in congestive heart failure patients as assessed by NIRS

In the present study the effect of an exercise rehabilitation program on exercise capacity and peripheral muscle oxygenation in patients with congestive heart failure (CHF) was evaluated. The population study consisted of 14 stable CHF patients that completed a rehabilitation program of 36 sessions. Eight patients were assigned to interval training (IT) that included alternations of 30 s of exercise with 60 s of rest, for 40 min per session [Meyer, K., Samek, L., Schwaibold, M., Westbrook, S., Hajric, R., Lehmann, M., et al. 1996. Physical responses to different modes of interval exercise in patients with chronic heart failure-application to exercise training. European Heart Journal 17, 1040–1047; Working group on cardiac rehabilitation and exercise physiology and working group on heart failure of the ESC, 2001. Recommendations for exercise training in chronic heart failure patients. European Heart Journal 22, 125–135], and six patients were assigned to IT for 20 min followed by strength training. Rehabilitation program resulted in a significant increase in exercise capacity as assessed by VO 2peak (17 ± 5 vs 20 ± 6 ml/kg per min, p = 0.002). This result was accompanied by a significant decrease of the mean time constant (mtc) of the exponential curve that describes the rise of tissue oxygenation after a submaximal cardiopulmonary exercise test (CPET) (from 287 ± 90 to 217 ± 93 s, p = 0.02). Such improved oxygen kinetics may be the result of increased oxygen delivery to working skeletal muscle or/and improvement of muscle oxidative metabolism. In conclusion, exercise training improves exercise capacity and peripheral microcirculation in CHF patients. NIRS during recovery of submaximal CPET can provide easily measured oxygenation indices for assessment of the functional status of these patients’ population. Relevance to industry. NIRS-derived indices are helpful in assessment of the influence of working activities upon functional status of healthy or diseased people under the particular conditions of the occupational environment.

Kvβ1.1 associates with Kvα1.4 in chinese hamster ovary cells and pigeon type II vestibular hair cells and enhances the amplitude, inactivation and negatively shifts the steady-state inactivation range

Although A-type potassium currents are found in type II hair cells in the inner ear of most species, the molecular mechanisms for activation and inactivation of the A-type potassium current (I A) remain unknown. In frog semicircular canal hair cells, for example, there appear to be two classes of currents having either fast or slow inactivation [Norris CH, Ricci AJ, Housley GD, Guth PS (1992) The inactivating potassium currents of hair cells isolated from the crista ampullaris of the frog. J Neurophysiol 68:1642–1653; Russo G, Calzi D, Martini M, Rossi ML, Fesce R, Prigioni I (2007) Potassium currents in the hair cells of vestibular epithelium: position-dependent expression of two types of A channels. Eur J Neurosci 25:695–704]. It has been suggested that somehow the “ball and chain” mechanism (NH 3 (N) terminus motif) is modified by alternative splicing to account for the two classes of inactivation. To examine other possibilities, we cloned alpha and beta subunits that comprise the A-type potassium channel complex in adult and embryonic pigeon brain, cochlea and labyrinth. By sequence homology, we concluded that the subunits present were Kvα1.4 and Kvβ1.1. The sequence of the open reading frame for Kvα1.4 contained the N-terminus, pore and COOH (C) terminus motifs for N-and C-type inactivation. The sequence for Kvβ1.1 displayed amino acids consistent with assembly and association with Kvα1.4 alpha subunits. Kvα1.4 and Kvβ1.1 were transfected either singly or in combination into Chinese hamster ovary (CHO) cells. These cells and native hair cells from the pigeon utricle were patch clamped and the inactivation properties of the A-type current were studied. In the native hair cells, the A-type current was identified by its pharmacological (4-aminopyridine (4-AP); IC 50=11 μM) and voltage dependent inactivation properties. A comparison of the mean time constants from best-fitted single exponential and sum of two exponential equations to the ionic current inactivation revealed the following. In CHO cells when Kvα1.4 was expressed alone, the mean time constant (τ 1=107 ms±19, N=32) was significantly ( P<0.001) longer and the mean peak amplitude (2.28 nA±0.39, N=32) was smaller than when Kvα1.4 and Kvβ1.1 were expressed in CHO cells. Moreover, the co-transfection of Kvα1.4 and Kvβ1.1 into CHO cells caused a shift in the steady state inactivation curve parameter Vo 30 mV in the hyperpolarized direction relative to CHO cells expressing only Kvα1.4. Similarly, Kvα1.4-transfected CHO cells produced longer time constants and smaller amplitudes than those found for native utricular hair cells. These data lead us to conclude that while the amino acid motifs are present in Kvα1.4 and Kvβ1.1 to suggest N-and C-type inactivation, co-assembly and association of Kvα1.4 and Kvβ1.1 may also produce changes in the time dependent inactivation properties of vestibular hair cells.

Axonal Speeding: Shaping Synaptic Potentials in Small Neurons by the Axonal Membrane Compartment

The role of the axonal membrane compartment in synaptic integration is usually neglected. We show here that in interneurons of the cerebellar molecular layer, where dendrites are so short that the somatodendritic domain can be considered isopotential, the axonal membrane contributes a significant part of the cell input capacitance. We examine the impact of axonal membrane on synaptic integration by cutting the axon with two-photon illumination. We find that the axonal compartment acts as a sink for signals generated at fast conductance synapses, thus increasing the initial decay rate of corresponding synaptic potentials over the value predicted from the resistance-capacitance (RC) product of the cell membrane; signals generated at slower synapses are much less affected. This mechanism sharpens the spike firing precision of fast glutamatergic inputs without resorting to multisynaptic pathways.

Effect of low-xenon and krypton supplementation on signal/noise of regional CT-based ventilation measurements

Xenon computed tomography (Xe-CT) is used to estimate regional ventilation by measuring regional attenuation changes over multiple breaths while rebreathing a constant Xe concentration ([Xe]). Xe-CT has potential human applications, although anesthetic properties limit [Xe] to <or=35%. We investigate effects of lower [Xe], including a low [Xe]-krypton (Kr) combination, on time constant (TC) determination. Six anesthetized sheep were scanned prone and supine using multidetector row CT. Lungs were imaged by respiratory gating during washin of a 30%, 40%, 55% Xe, and a 30% Xe/30% Kr mixture. Using Kr avoids unwanted effects of Xe. Mean TCs, coefficients of variation (CV), and half confidence intervals (CI)/mean served as indexes of sensitivity to noise. Mean supine and prone TCs of three [Xe] values were not significantly different. Average CVs of TCs increased from 57% (55% Xe), 58% (40% Xe), and 73% (30% Xe) (P < 0.05: paired t-tests; 30% Xe vs. higher [Xe]). Monte Carlo simulation indicated a CV based on inherent image noise was 8% for 55% Xe and 17% for 30% Xe (P < 0.05). Adding 30% Kr to 30% Xe gave a washin signal equivalent to 40% Xe. Half CI/mean using the 30% Xe/30% Kr mixture was not significantly different from 55 and 40% Xe. Although average TCs were not affected by changes in [Xe], the higher CV and half CI/mean suggested reduced signal-to-noise ratio at the 30% [Xe]. The 30% Xe/30% Kr mixture was comparable to that of 40% Xe, providing an important agent for CT-based assessment of regional ventilation in humans.

Spatial heterogeneity of the restitution portrait in rabbit epicardium.

Spatial heterogeneity of repolarization can provide a substrate for reentry to occur in myocardium. This heterogeneity may result from spatial differences in action potential duration (APD) restitution. The restitution portrait (RP) measures many aspects of rate-dependent restitution: the dynamic restitution curve (RC), S1-S2 RC, and short-term memory response. We used the RP to characterize epicardial patterns of spatial heterogeneity of restitution that were repeatable across animals. New Zealand White rabbit ventricles were paced from the epicardial apex, midventricle, or base, and optical action potentials were recorded from the same three regions. A perturbed downsweep pacing protocol was applied that measured the RP over a range of cycle lengths from 1,000 to 140 ms. The time constant of short-term memory measured close to the stimulus was dependent on location. In the midventricle the mean time constant was 19.1 +/- 1.1 s, but it was 39% longer at the apex (P < 0.01) and 23% longer at the base (P = 0.03). The S1-S2 RC slope was dependent on pacing site (P = 0.015), with steeper slope when pacing from the apex than from the base. There were no significant repeatable spatial patterns in steady-state APD at all cycle lengths or in dynamic RC slope. These results indicate that transient patterns of epicardial heterogeneity of APD may occur after a change in pacing rate. Thus it may affect cardiac electrical stability at the onset of a tachycardia or during a series of ectopic beats. Differences in restitution with respect to pacing site suggest that vulnerability may be affected by the location of reentry or ectopic foci.

Carrier recombination and diffusion in GaN revealed by transient luminescence under one-photon and two-photon excitations

Carrier recombination and diffusion dynamics in a 100-μm-thick GaN grown by hydride vapor phase epitaxy on semi-insulating GaN:Mg substrate have been studied by means of transient photoluminescence under one-photon (1P) and two-photon (2P) excitations. For 2P bulk excitation the luminescence transients featured an exponential decay with the time constant of 1100ps, which was mainly due to carrier capture to nonradiative deep traps. Meanwhile for 1P surface excitation, the luminescence transients showed a nonexponential decay with the mean time constant of 440ps, which was shown to be due to both carrier in-depth diffusion and recombination.

Shaping the pupil’s response to light in the hooded rat

The contribution of iris muscle steady state and dynamic response characteristics to the shaping of the pupil response to light in the hooded rat were studied using electrical stimulation of the parasympathetic fibers in the III nerve. The waveforms of pupillary contractions to single or brief trains of electrical impulses applied to the III nerve were virtually identical to those elicited with short duration light flashes. Individual contractions could be resolved at stimulation rates of 2 Hz and below, and the size of the contractions increased with the decrease in frequency. The pupil responded to long trains of stimuli above 2 Hz with smooth tonic contractions. Steady state contraction amplitude was linearly related to log stimulation frequency. The mean time constant of pupil constriction to stimulus trains was 1.41 s (SD +/- 0.71 s) and the shortest mean latency was 292 ms (SD +/- 30 ms). The fastest mean latency of pupil constriction to the brightest light flash used was 295 ms. In contrast, the time constant of pupillary dilation was 7 s (SD +/- 1.4 s) and the shortest latency was 485 ms (SD +/- 74 ms). Therefore, the sluggish dynamic properties of the iris musculature are responsible for the asymmetries in pupil contraction, dilation, and latencies as well as low flicker fusion frequency and constriction amplitude characteristics of pupil responses to light.

A time‐domain induced‐polarization method for estimating permeability in a shaly sand reservoir

ABSTRACTIt is known that the time‐domain induced‐polarization decay curve for a shaly sand reservoir depends on the pore structure of the reservoir, and this curve can be used to estimate permeability, which is a determining factor in making production decisions in the petroleum industry. Compared with NMR logging tools, induced polarization has several advantages, such as a deep depth of investigation and a high signal‐to‐noise ratio. The purpose of this paper is to establish an appropriate model using induced polarization to estimate the permeability. The curve can be modelled as a weighted superposition of exponential relaxations. The plot of weight versus the relaxation time constant is defined as the relaxation time spectrum.Induced‐polarization decay‐curve measurements were performed on 123 samples from the Daqing oilfield using a four‐electrode technique. A singular‐value decomposition method was used to transform the induced‐polarization decay data into a spectrum. Different models to estimate the permeability were discussed. The results of the research indicate that the induced‐polarization measurements greatly improve the statistical significance of permeability correlations. Compared with the traditional forms, AφC and AFC, the forms, ATBφC and ATBFC, have lower error factors, where T, Φ and F are the geometric mean time constant of the induced‐polarization relaxation time spectrum, the porosity and the resistivity formation factor, respectively, and A, B and C are constants. The mean time constant is the decisive parameter in the permeability estimation and it is not completely independent of the resistivity formation factor. The additional use of the porosity and the resistivity formation factor leads to an appreciable improvement. It is concluded that this new model will make it possible to estimate the permeability of a shaly sand reservoir downhole.

Glutamate‐induced protease‐mediated loss of plasma membrane Ca2+ pump activity in rat hippocampal neurons

Ca2+ dysregulation is a hallmark of excitotoxicity, a process that underlies multiple neurodegenerative disorders. The plasma membrane Ca2+ ATPase (PMCA) plays a major role in clearing Ca2+ from the neuronal cytoplasm. Here, we show that the rate of PMCA-mediated Ca2+ efflux from rat hippocampal neurons decreased following treatment with an excitotoxic concentration of glutamate. PMCA-mediated Ca2+ extrusion following a brief train of action potentials exhibited an exponential decay with a mean time constant (tau) of 8.8 +/- 0.2 s. Four hours following the start of a 30 min treatment with 200 microm glutamate, a second population of cells emerged with slowed recovery kinetics (tau = 16.5 +/- 0.3 s). Confocal imaging of cells expressing an enhanced green fluorescent protein (EGFP)-PMCA4b fusion protein revealed that glutamate treatment internalized EGFP and that cells with reduced plasma membrane fluorescence had impaired Ca2+ clearance. Treatment with inhibitors of the Ca2+-activated protease calpain protected PMCA function and prevented EGFP-PMCA internalization. PMCA internalization was triggered by activation of NMDA receptors and was less pronounced for a non-toxic concentration of glutamate relative to one that produces excitotoxicity. PMCA isoform 2 also internalized following exposure to glutamate, although the Na+/K+ ATPase did not. These data suggest that glutamate exposure initiated protease-mediated internalization of PMCAs with a corresponding loss of function that may contribute to the Ca2+ dysregulation that accompanies excitotoxicity.

Calcium currents in interstitial cells from the guinea‐pig bladder

To determine whether there are inward currents in interstitial cells (IC) isolated from the guinea-pig detrusor and if so, to characterise them using the patch-clamp technique and pharmacological agents. Using the whole-cell patch-clamp technique, inward currents were studied in IC enzymatically isolated from the detrusor of the guinea-pig bladder. Currents were evoked by stepping positively from a holding potential of - 80 mV. Outward K+ currents were blocked by Cs+ internal solution to reveal inward currents, which activated at voltages more positive than - 50 mV, peaked at 0 mV, reversed near + 50 mV and were half-maximally activated at - 27 mV. The inward currents showed voltage-dependent inactivation and were half-maximally inactivated at - 36 mV. Fitting the activation and inactivation data with a Boltzmann function revealed a window current between - 40 mV and + 20 mV. The decay of the current evoked at 0 mV could be fitted with a single exponential with a mean time-constant of 88 ms. Replacing external Ca2+ with Ba2+ significantly increased this to 344 ms. The current amplitude was augmented by Ba2+, and by Bay K 8644. Inward currents were significantly reduced by 1 microm nifedipine, across the voltage range, but the blockade was more effective on the current evoked at 0 mV than that evoked by a step to - 20 mV, perhaps indicating voltage-dependence of the action of nifedipine or another component of inward current. Increasing the concentration of the drug to 10 microm caused no further significant reduction either at 0 mV or at -20 mV. However, in the presence of 1 microm nifedipine the latter current was significantly reduced by 100 microm Ni2+. Both currents were significantly reduced in Ca2+-free solution. IC from the guinea-pig detrusor possess inward currents with typical characteristics of L-type Ca2+ current. They also have a component of inward Ca2+ current, which was resistant to nifedipine, but sensitive to Ni2+. Further work is needed to characterise the latter conductance.