bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo voltammetric measurement of evoked extracellular dopamine in the rat basolateral amygdaloid nucleus.
Authors: Garris P A , Wightman R M
Published: December 2014

1. The in vivo measurement of evoked extracellular dopamine was established in the basolateral amygdaloid nucleus (BAN) using fast‐scan cyclic voltammetry at carbon‐fibre microelectrodes. 2. The identification of evoked extracellular dopamine in the BAN was based on anatomical, electrochemical and pharmacological criteria. Electrochemical and pharmacological evidence indicated that the species was a catecholamine. Mesencephalic sites eliciting overflow and amygdaloid sites supporting overflow correlated well with the mesoamygdaloid dopamine innervation. 3. Marked differences in the dynamics and magnitude of evoked dopamine overflow were observed in the BAN, caudate‐putamen and amygdalo‐striatal transition area. The results underscore the importance of making spatially resolved measurements of extracellular dopamine in the amygdala. 4. Mesoamygdaloid dopamine neurons have similar release characteristics as mesostriatal dopamine neurons but share with mesoprefrontal cortical dopamine neurons the ability to use a greater percentage of intraneuronal dopamine stores for release.
Concomitant activation of Cl‐ and K+ currents by secretory stimulation in human epithelial cells.
Authors: Baró I , Roch B , Hongre A S , Escande D
Published: December 2014

1. Whole‐cell currents were investigated in the model salt‐secreting epithelium, human T84 cell line, by means of the perforated patch‐clamp technique. In the control extracellular medium containing Cl‐, depolarizing voltage ramps evoked current responses which peaked at 5.43 +/‐ 0.81 pA pF‐1 at +60 mV and had a reversal potential (Erev) of ‐38.4 +/‐ 2.5 mV (n = 23). 2. Activation of the cAMP pathway with forskolin increased the current at +60 mV from 3.81 +/‐ 0.61 to 20.79 +/‐ 5.08 pA pF‐1 (n = 18). In thirteen cells, Erev was initially shifted towards positive potentials (Erev of the cAMP‐activated initial current was ‐18.2 +/‐ 1.2 mV) and subsequently shifted towards more negative potentials, consistent with the activation of both Cl‐ and K+ currents during cAMP stimulation. 3. Increasing the intracellular Ca2+ concentration, [Ca2+]i, with ionomycin (1 microM) or with acetylcholine (1 microM), increased the current at +60 mV from 7.79 +/‐ 1.57 to 57.50 +/‐ 12.10 pA pF‐1 (n = 6) and from 6.36 to 34.13 pA pF‐1 (n = 4), respectively. With both agonists, Erev was shifted either towards the reversal potential for potassium, EK, or towards the reversal potential for chloride, ECl, depending on the cell. 4. In the absence of chloride ions (gluconate substituted), stimulation of the Ca2+ pathway activated a time‐independent outward current of large amplitude. This current exhibited inward rectification at positive voltages, reverted at ‐89.5 +/‐ 0.2 mV and was markedly reduced by charybdotoxin (10 nM), a specific blocker of Ca(2+)‐activated K+ channels. When a voltage step protocol was used, increased [Ca2+]i also activated an outward current at potentials more positive than ‐40 mV which slowly relaxed during depolarizing steps. 5. The activation of both (i) a time‐dependent inwardly rectifying charybdotoxin‐sensitive K+ current, and (ii) a time‐dependent slowly inactivating current was also produced by cAMP stimulation. 6. We concluded that (i) in the T84 epithelial cells, both Cl‐ and K+ currents are concomitantly increased by secretagogue stimulation, and (ii) two different types of K+ conductances are activated by either the cAMP or the intracellular Ca2+ secreting pathways.
Inhibition of Ca2+ entry by Ca2+ overloading of intracellular Ca2+ stores in human platelets.
Authors: Kimura M , Cho J H , Reeves J P , Aviv A
Published: December 2014

1. This study examined the effect of overloading human platelet intracellular Ca2+ stores on the rate of agonist‐evoked external Ca2+ entry. To overload the Ca2+ stores (presumably dense tubules), external Na(+)‐dependent Ca2+ efflux via Na(+)‐Ca2+ exchange was inhibited by pretreating the cells with ouabain or Na(+)‐free medium. Ca2+ regulation was then examined after exposure to thrombin, ADP and thapsigargin. Cytosolic free Ca2+ levels were monitored using the fluorescent probe fura‐2 and external Ca2+ influx was assessed by the rates of extracellular Mn2+ or 45Ca2+ uptake. 2. Both ouabain and Na(+)‐free pretreatments caused a slight increase in the resting cytosolic free Ca2+. 3. In 1 mM Ca(2+)‐containing medium, Ca(2+)‐overloaded platelets showed similar thrombin‐evoked cytosolic free Ca2+ responses to those of control platelets. However, in Ca(2+)‐free medium, they showed substantially greater thrombin‐evoked cytosolic free Ca2+ responses than control platelets. Moreover, increased thrombin‐evoked Ca2+ mobilization from Ca2+ storage sites was accompanied by a diminished rate of thrombin‐evoked external Ca2+ entry. 4. Similar reductions in the rate of external Ca2+ entry were observed after treatment with ADP and thapsigargin. 5. Protein kinase C inhibitors (calphostin C and staurosporine) failed to reverse the effect of ouabain pretreatment on thrombin‐induced changes in the cytosolic free Ca2+ response. 6. Inositol 1,4,5‐trisphosphate profiles in ouabain‐treated and non‐treated platelets were not significantly different. 7. These data indicate that increased Ca2+ in the dense tubules is associated with diminished agonist‐evoked external Ca2+ influx.
Membrane potential dependence of the kinetics of cationic amino acid transport systems in human placenta.
Authors: Eleno N , Devés R , Boyd C A
Published: December 2014

1. Mediated influx of L‐lysine into human placental brush‐border membrane vesicles occurs through two systems, one of lower affinity but high capacity, the other of very high affinity but low capacity. These transporters have features characteristic of systems y+ (the classical system) and y+L (recently described in the erythrocyte), respectively. 2. In solutions containing sodium the entry of lysine through the high‐affinity system (y+L) is inhibited by the neutral amino acids L‐leucine, L‐methionine and L‐glutamine with comparable high affinity. The removal of sodium reduces the affinity but not the maximal extent of this inhibition. Leucine and methionine, but apparently not glutamine, inhibit lysine entry through system y+ with a much lower affinity. 3. The influx of lysine through system y+ changes markedly in response to alterations of membrane potential. In the presence of an inwardly directed negative diffusion potential created by an inwardly directed thiocyanate (SCN‐) gradient, the influx of lysine through this route is accelerated; with an inwardly directed positive potassium diffusion potential, lysine influx through this route is reduced. The influx of lysine through system y+L is much less sensitive to such alterations of potential. 4. Analysis of the kinetic constants characterizing system y+ shows that with a change of potential from zero to negative (approximately ‐60 mV) the maximum velocity (Vmax) is roughly doubled and the half‐saturation constant (Km) halved leading to a 4‐fold increase in permeability. For system y+L smaller changes are seen and Km does not change; the resulting increase in y+L permeability is 1.5‐fold. 5. These findings are discussed with respect both to the mechanism of membrane transport and placental epithelial function.
Alteration of the physiological responses to indomethacin by endotoxin tolerance in the rat: a possible role for central vasopressin.
Authors: Wilkinson M F , Pittman Q J
Published: December 2014

1. Previous studies suggest that arginine vasopressin (AVP) is released into the ventral septal area (VSA) of the rat brain during the antipyresis induced by the cyclo‐oxygenase inhibitor indomethacin. In addition, there is evidence for increased AVP transmission in the VSA of animals having a reduced pyretic response following three intravenous injections of bacterial endotoxin (LPS) (endotoxin tolerant). Since ventral septal AVP receptors can also become ‘sensitized’ following exposure to AVP, we questioned whether the antipyretic action of indomethacin would increase, via an action involving central AVP, if this drug were administered into LPS‐tolerant rats. 2. Intraperitoneal indomethacin (7.5 mg kg‐1) was effectively antipyretic when administered 2 h after an intravenous challenge with LPS (50 micrograms kg‐1) into conscious unrestrained rats. This dose of indomethacin had no effect on the core temperature of non‐febrile rats given intravenous 0.9% pyrogen‐free saline. 3. Three intravenous injections of LPS over a period of 3 days resulted in rats that were tolerant to the pyrogenic effects of LPS. When indomethacin was administered 2 h following the third LPS injection, a dose‐dependent hypothermia was observed. This effect was age dependent, as profound hypothermia was seen in 8 week but not 20 week old rats. 4. A mortality rate of 41% (P = 0.02) was observed within 24 h of indomethacin treatment in 8 week old tolerant rats compared with 0% in 8 week old non‐tolerant and 20 week old tolerant rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Voltage clamp analysis of excitatory synaptic transmission in the avian nucleus magnocellularis.
Authors: Zhang S , Trussell L O
Published: December 2014

1. The properties of evoked excitatory postsynaptic currents (EPSCs) and spontaneous miniature excitatory postsynaptic currents (mEPSCs) have been studied in neurons of the nucleus magnocellularis (nMAG), one of the avian cochlear nuclei which receive somatic, calyceal innervation from auditory nerve fibres. Whole‐cell patch clamp techniques were used to voltage clamp visually identified neurons in brain slices. 2. EPSCs resulting from activation of single axonal inputs were on average ‐5.3 nA at a driving force of ‐25 mV. Current‐voltage relationships for the peak of the EPSC were linear with a peak conductance of 211 nS. The rate of EPSC decay showed a linear increase with temperature, with a temperature coefficient (Q10) of 2.2 between 25 and 35 degrees C; in vivo (41 degrees C) the EPSC would decay in 0.2 ms. 3. The EPSC was composed of two pharmacologically and kinetically distinct components: an early phase due to non‐NMDA (N‐methyl‐D‐aspartate) receptors and a late phase resulting from NMDA receptors. Both components reversed near 0 mV. While both subtypes of glutamate receptor were activated by transmitter, NMDA receptors had a peak conductance at positive potentials which was only 11% of the peak non‐NMDA receptor component. 4. EPSCs during trains of stimuli exhibited a progressive decrease in amplitude. The extent of depression increased with the frequency of stimulation and was reduced by drugs which prevent receptor desensitization, indicating that, in part, postsynaptic factors limit synaptic strength during repetitive synaptic activity. Additionally, the coefficient of variation of the EPSC amplitude increased during trains, consistent with presynaptic depression. 5. mEPSCs occurred randomly in the presence of tetrodotoxin and presumably correspond to transmitter quanta. These synaptic events rose (10‐90%) within 100 microseconds and decayed with an exponential of 180 microseconds at 29‐32 degrees C. Despite the somatic location of the synapse, mEPSCs varied widely in amplitude, suggesting differences in the quantal synaptic current at each synaptic site. The ratio of the average peak conductance of the EPSC and mEPSC gave an estimated quantal content of 103.
Regional alternans in relaxation and the onset of pulsus alternans in the heart of the anaesthetized pig.
Authors: Murphy C F , Lab M J , Horner S M , Dick D J , Harrison F G
Published: December 2014

1. The factors leading to the alternation in myocardial contractility believed primarily responsible for pulsus alternans are not known. We examine regional and global contraction patterns in the in situ heart at stimulation rates just below the threshold for pulsus alternans to determine if events occurring in the transition to alternans can give clues to cellular mechanisms. 2. Twelve pigs were anaesthetized, the chest wall removed and regional contraction measured in three areas of the left ventricle using tripodal strain gauges. We analysed regional and global dynamics during right atrial pacing at cycle lengths 50‐150 ms greater than the threshold for pulsus alternans. 3. At pacing cycle lengths 50 ms greater than that required to produce pulsus alternans seven of twelve pigs showed alternans in the maximum rate of ventricular pressure decay but none showed alternans in the maximum rate of pressure rise. Pigs showing alternans in global relaxation were more likely to show alternans in regional contracility (P < 0.05). 4. Twenty‐six of the thirty‐six areas sampled showed alternans in end‐diastolic length at pacing rates below the threshold for pulsus alternans. In fifteen of these areas alternation in end‐diastolic length occurred in the absence of alternans in measures of contractility. 5. Alternans in global measures of relaxation may simply be a manifestation of regional alternans in contractility. It is therefore not appropriate, from global haemodynamic data, to suppose that alternans in relaxation is the primary abnormality in the generation of pulsus alternans.(ABSTRACT TRUNCATED AT 250 WORDS)
Proximal tubular cell electrolytes during volume expansion in the rat.
Authors: Reddy S , Györy A Z , Boström T , Dyne M , Salipan‐Moore N , Field M J , Pollock C A , Cockayne D J
Published: December 2014

1. Proximal tubular intracellular elements were measured by electron microprobe X‐ray analysis (a) in rats volume‐expanded with albumin‐saline in which peritubular oncotic pressure remained normal and (b) in rats in which the renal artery was snared before volume expansion (the early snare model). Glomerular filtration rate and urine Na+ excretion were measured in addition to intracellular Rb+ following a 30 s infusion of RbCl as a marker for K+ transport. 2. In albumin‐saline volume‐expanded rats, intracellular levels of Na+ ([Na+]i) at 21.5 +/‐ 0.6 mmol (kg wet wt)‐1, Cl‐ ([Cl‐]i) at 18.0 +/‐ 0.4 mmol (kg wet wt)‐1 and Rb+ ([Rb+]i) at 9.4 +/‐ 0.4 mmol (kg wet wt)‐1 were significantly higher (P < 0.0001) than the levels in non‐expanded rats ([Na+]i, [Cl‐]i and [Rb+]i at 17.7 +/‐ 0.4, 14.6 +/‐ 0.3 and 4.7 +/‐ 0.4 mmol (kg wet wt)‐1, respectively; means +/‐ S.E.M.). The data are consistent with Na+ pump inhibition in the proximal tubule, although this cannot be directly derived from intracellular element measurements. 3. In an early snare model of volume expansion, [Na+]i, intracellular K+ ([K+]i) and [Rb+]i remained unchanged (16.1 +/‐ 0.4, 131.0 +/‐ 2.0 and 5.2 +/‐ 0.3 mmol (kg wet wt)‐1, respectively) compared to non‐expanded snared kidneys (15.9 +/‐ 0.6, 131.3 +/‐ 1.8 and 4.8 +/‐ 0.3 mmol (kg wet wt)‐1, respectively). [Cl‐]i at 18.3 +/‐ 0.5 mmol (kg wet wt)‐1 increased (P < 0.0008) compared to controls at 15.8 +/‐ 0.5 mmol (kg wet wt)‐1. Thus, in these rats, evidence for an inhibition of the Na+ pump was no longer observed. This points to a major intrinsic mechanism within the kidney for mediating natriuresis, since circulating factors were identical to those in the unsnared kidney, where significant natriuresis occurred.
Urinary kallikrein in the rat: stimulation with angiotensin infusion but depression with increasing sodium concentration.
Authors: Mills I H , Lee G , Brownlee A A
Published: December 2014

1. The kallikrein response to angiotensin II infusion in the conscious rat was studied to compare it with the response in the dog. 2. Active kallikrein was measured by the aprotinin‐suppressible esterase technique in 20 min periods. Angiotensin (5 x 10(‐9) to 5 x 10(‐2) micrograms min‐1) was infused in 10 mM saline in period 10 (group A), or in 90 mM saline in periods 10‐12 (group B). 3. In group A, no dose of angiotensin was antinatriuretic. Natriuresis and urinary sodium concentration were dose dependent. 4. Kallikrein excretion was dose dependent with angiotensin (P < 0.0001) and inversely correlated with urinary sodium concentration (P = 0.011). In natriuretic and non‐natriuretic rats, kallikrein excretion after angiotensin was inversely correlated with urinary sodium concentration in the preceding period. 5. In group B, natriuresis and urinary sodium concentration were dose dependent. Kallikrein excretion in periods 10‐13 was inversely correlated with urinary sodium concentration in the preceding period (P = 0.0001) and inversely correlated with urinary osmolality in periods 9‐13. 6. Infusion of angiotensin II at 5 x 10(‐6) micrograms min‐1 led to antinatriuresis. 7. Formulae were derived which enabled the opposing effects of angiotensin and urinary sodium concentration on kallikrein excretion to be separated. In group A both these effects were statistically significant only in the natriuretic rats (natriuresis > 20 mumols per period). In group B the formulae showed a dose‐dependent rise in kallikrein excretion, which was counteracted by the decrease in kallikrein excretion associated with the increasing urinary sodium concentration. 8. With infusions of 0.9% saline, kallikrein excretion in periods 10‐13 was inversely correlated with urinary sodium concentration in the preceding period (P = 0.001). 9. The overall effect in the rat differs from that in the dog, where kallikrein increases with angiotensin natriuresis and dilution of the urine occurs.
Effects of deep breaths on subsequent ventilation in man during rest and exercise.
Authors: Fernando S S , Saunders K B
Published: December 2014

1. We examined the effects of twenty‐four to thirty inspiratory capacity (IC), expiratory capacity (EC) and vital capacity (VC) breaths on subsequent breathing pattern in five normal subjects at rest. 2. During IC breaths and following EC and VC breaths at rest, end‐tidal CO2 pressure (PET,CO2) fell by 7.5, 8.5 and 9.5 mmHg, respectively. In the group analysis significant inhibition of ventilation of 1.5 l min‐1 was seen after the IC breath but not after EC or VC breaths. 3. We repeated the study with five normal subjects under conditions of higher ventilatory drive, namely 50 W exercise (one subject was common to both groups). 4. During exercise, the drop in PET,CO2 was smaller (4.0, 3.5 and 4.0 mmHg, respectively, with IC, EC and VC breaths) but ventilation was inhibited to a greater extent. Ventilatory undershoot was seen after all three types of deep breaths. 5. We propose that the expiration to residual volume in EC and VC breaths abolished the hypocapnic inhibition of ventilation at rest, possibly by a deflation reflex which was not sufficiently powerful to overcome the ventilatory undershoot during exercise. Our results also support the view that the slope of the CO2 response curve is steeper near the control point during exercise.
Sensitization of the contractile system of canine colonic smooth muscle by agonists and phorbol ester.
Authors: Sato K , Leposavic R , Publicover N G , Sanders K M , Gerthoffer W T
Published: December 2014

1. Sensitization of the contractile system in response to combinations of excitatory agonists acetylcholine (ACh), methacholine, histamine and neurokinin A (NKA) was investigated in colonic circular smooth muscle of dog, NKA (1 nM) potentiated the contractile response to 1 microM ACh, but did not increase the fura‐2 fluorescence ratio (R340/380). Contraction in response to low concentrations of either methacholine or histamine was potentiated significantly by 0.1 microM 4‐phorbol 12,13‐dibutyrate (PDBu), suggesting that activation of protein kinase C can potentiate contraction at threshold concentrations of agonists. 2. Variability in the sensitivity of the contractile system to Ca2+ was demonstrated over a range of agonist concentrations. KCl, ACh, histamine and NKA each produced a concentration‐dependent increase in the amplitude of phasic contractions and R340/380. However, ACh, histamine and NKA each induced maximal increases in R340/380 at concentrations less than that needed to induce maximum force. 3. In depolarized muscles, NKA (50 nM) and PDBu (1 microM) each increased the magnitude of tonic contraction with no change or a decrease in both R340/380 and myosin light chain phosphorylation. In alpha‐toxin‐permeabilized fibres, 0.1 microM PDBu and 1 microM NKA shifted the Ca(2+)‐force response to the left. Ca(2+)‐induced contractions were also potentiated by 100 microM GTP‐gamma‐S or 1 microM NKA plus 10 microM GTP. Potentiation of contraction by NKA and GTP was antagonized by 10 microM GDP‐beta‐S. 4. The results suggest that endogenous agonists acting via G‐proteins sensitize the contractile element of colonic smooth muscle in part by activation of protein kinase C. In some cases, sensitization may be secondary to increased myosin phosphorylation (ACh), but in other cases it appears to be independent of increased myosin light chain phosphorylation (NKA and PDBu). Therefore regulatory mechanisms in addition to myosin phosphorylation contribute to the apparent sensitization of the contractile system to Ca2+.
Nerve growth factor depletion reduces collateral sprouting of cutaneous mechanoreceptive and tooth‐pulp axons in ferrets.
Authors: Doubleday B , Robinson P P
Published: December 2014

1. Electrophysiological experiments were carried out to determine whether or not collateral sprouting of cutaneous low‐threshold mechanoreceptive fibres could be detected and to investigate the effect of nerve growth factor (NGF) deprivation on the sprouting of these fibres and the fibres innervating tooth pulps. 2. In twenty‐one ferrets (eleven of which had been autoimmunized against NGF) the right inferior alveolar nerve (IAN) was sectioned and prevented from regenerating. After 12 weeks, transmedian innervation from the left IAN was determined by stimulating the nerve whilst recording from electrodes implanted in the contralateral anterior teeth and also by single unit recordings from the nerve whilst mechanically and electrically stimulating the skin. The results were compared with those from ten control animals. 3. Transmedian innervation of contralateral teeth was found in none of the control animals; in all ten of the animals which had undergone denervation without immunization (4/10 canines, 17/20 incisors); but in only six of the eleven immunized and denervated animals (0/11 canines, 7/22 incisors). 4. Of 270 cutaneous mechanoreceptive units sampled in the controls, only four units had transmedian receptive fields, extending a maximum of 1 mm across the mid‐line. After denervation, significantly more units (42 of 274) crossed the mid‐line and extended up to 4 mm. After immunization and denervation only eleven of 305 units crossed the midline by a maximum of 1 mm. 5. These data show that cutaneous low‐threshold mechanoreceptive A beta and A delta fibres, as well as A delta tooth pulp fibres, are able to undergo collateral sprouting. This sprouting is partially blocked by NGF depletion, suggesting that NGF plays an essential role in the process.
Potentiation of Ca2+ transients in the presynaptic terminals of goldfish retinal bipolar cells.
Authors: Kobayashi K , Sakaba T , Tachibana M
Published: December 2014

1. To study a possible contribution of intracellular Ca2+ stores to the presynaptic Ca2+ regulation, the Ca2+ current (ICa) and the intracellular free Ca2+ concentration ([Ca2+]i) were simultaneously monitored in isolated goldfish retinal bipolar cells using the whole‐cell voltage clamp procedure and fura‐2 fluorimetry. 2. The Ca2+ transient triggered by the activation of ICa was potentiated when [Ca2+]i was increased by applying either a prepulse or a small steady depolarization. The potentiation seemed to be partly due to the release of Ca2+ from intracellular Ca2+ stores. 3. The intracellular Ca2+ release was reversibly inhibited by caffeine but was not affected by ryanodine, suggesting that Ca2+ is released through intracellular Ca2+ channels which differ from ryanodine receptor channels. 4. These results suggest that the intracellular Ca2+ release may contribute to the facilitation of transmitter release.
ATP utilization for calcium uptake and force production in skinned muscle fibres of Xenopus laevis.
Authors: Stienen G J , Zaremba R , Elzinga G
Published: December 2014

1. A method has been developed to discriminate between the rate of ATP hydrolysis associated with calcium uptake into the sarcoplasmic reticulum (SR) and force development of the contractile apparatus in mechanically or saponin‐skinned skeletal muscle fibres. The rate of ATP hydrolysis was determined in fibres of different types from the iliofibularis muscle of Xenopus laevis by enzymatic coupling of ATP re‐synthesis to the oxidation of NADH. 2. The ATPase activity was determined before and after exposure of the preparations for 30 min to a solution containing 0.5% Triton X‐100, which effectively abolishes the SR ATPase activity. The fibres were activated in a solution containing 5 mM caffeine to ensure that calcium uptake into the SR was maximal. 3. At saturating Ca2+ concentrations the actomyosin (AM) and SR ATPase activities in fast‐twitch fibres, at 4.3 degrees C, amounted to 1.52 +/‐ 0.07 and 0.58 +/‐ 0.10 mumol s‐1 (g dry wt)‐1, respectively (means +/‐ S.E.M.; n = 25). The SR ATPase activity was 25% of the total ATPase activity. At submaximal calcium concentrations the AM ATPase activity varied in proportion to the isometric force. 4. The calcium sensitivity of the SR ATPase was larger than that of the AM ATPase and its dependence on [Ca2+] was less steep. The AM ATPase activity was half‐maximal at a pCa of 6.11 (pCa = ‐log [Ca2+]) whereas the SR ATPase activity was half‐maximal at a pCa of 6.62. 5. In Triton X‐100‐treated fibres, at different 2,3‐butanedione monoxime (BDM) concentrations, the AM ATPase activity and isometric force varied proportionally. The SR ATPase activity determined by extrapolation of the total ATPase activity in mechanically skinned or saponin‐treated fibres to zero force, was independent of the BDM concentration in the range studied (0‐20 mM). The values obtained for the SR ATPase activity in this way were similar to those obtained with Triton X‐100 treatment. 6. The AM ATPase activity in slow‐twitch fibres amounted to 0.74 +/‐ 0.13 mumol s‐1 (g dry wt)‐1, i.e. about a factor of two smaller than in fast‐twitch fibres. The SR ATPase activity amounted to 0.47 +/‐ 0.07 mumol s‐1 (g dry wt)‐1, i.e. rather similar to the value in fast‐twitch fibres. The proportion of the total ATPase activity that was due to SR ATPase (40%) was larger than in fast‐twitch fibres. 7. The temperature dependence of the AM and SR ATPase activities in fast‐twitch fibres differed. In the temperature range 5‐10 degrees C, the relative changes in AM and SR ATPase activities for a 10 degrees C temperature change (Q10) were 3.9 +/‐ 0.3 and 7.2 +/‐ 1.5, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
Cytochrome P450 mono‐oxygenase‐regulated signalling of Ca2+ entry in human and bovine endothelial cells.
Authors: Graier W F , Simecek S , Sturek M
Published: December 2014

1. We tested the hypothesis that agonist‐stimulated Ca2+ entry, and thus formation of endothelium‐derived nitric oxide (EDNO) in vascular endothelial cells, is related to activation of microsomal P450 mono‐oxygenase (P450 MO) and the biosynthesis of 5,6‐epoxyeicosatrienoic acid (5,6‐EET). 2. Several P450 inhibitors diminished the sustained [Ca2+]i plateau response to agonist or intracellular Ca2+ store depletion with ATPase inhibitors by 31‐69% (fura‐2 technique). Mn2+ influx stimulated by agonists or ATPase inhibitors was prevented by P450 inhibitors. 3. Histamine‐ or ATPase inhibitor‐stimulated formation of EDNO was strongly attenuated (50‐83%) by P450 inhibitors, without any effect on EDNO formation by the Ca2+ ionophore A23187, indicating that decreased EDNO synthesis is due specifically to the inhibition of Ca2+ entry by these compounds. 4. Induction of P450 MO by beta‐naphthoflavone potentiated agonist‐induced Ca2+ and Mn2+ influx by 60 and 53%, respectively. Intracellular Ca2+ release remained unchanged. 5. The P450 MO product, 5,6‐EET (< 156 nmol l‐1), activated Ca2+/Mn2+ entry without any depletion of intracellular Ca2+ stores. The 5,6‐EET‐stimulated Ca2+/Mn2+ entry was not affected by P450 inhibitors. 6. As with the bradykinin‐stimulated Ca2+ entry pathway, the 5,6‐EET‐activated Ca2+ entry pathway was permeable to Mn2+ and Ba2+, sensitive to Ni2+, La3+ and membrane depolarization, and insensitive to the removal of extracellular Na+ or the organic Ca2+ antagonist, nitrendipine. 7. In the presence of 5,6‐EET, stimulation with bradykinin only transiently increased [Ca2+]i. Vice versa, 5,6‐EET failed to increase [Ca2+]i further in bradykinin‐stimulated cells. The sustained [Ca2+]i plateau phase induced by a co‐stimulation with bradykinin and 5,6‐EET was identical to that observed with bradykinin or 5,6‐EET alone. 8. These results demonstrate that Ca2+ entry induced by the P450 MO product, 5,6‐EET, is indistinguishable to that observed by stimulation with bradykinin. 9. All data support our hypothesis that depletion of endothelial Ca2+ stores activates microsomal P450 MO which in turn synthesizes 5,6‐EET. We propose that the arachidonic acid metabolite 5,6‐EET or one of its metabolites is a second messenger for activation of endothelial Ca2+ entry.
Activation of Ca(2+)‐dependent Cl‐ currents in cultured rat sensory neurones by flash photolysis of DM‐nitrophen.
Authors: Currie K P , Wootton J F , Scott R H
Published: December 2014

1. Voltage‐gated Ca2+ currents (ICa) and Ca(2+)‐activated Cl‐ currents (ICl(Ca)) were recorded from cultured rat dorsal root ganglion (DRG) neurones using the whole‐cell configuration of the patch clamp technique. Intracellular photorelease of Ca2+ by flash photolysis of DM‐nitrophen elicited transient inward currents only in those cells which possessed Ca(2+)‐activated Cl‐ tail currents following ICa. The reversal potential of the flash responses was hyperpolarized when extracellular Cl‐ was replaced by SCN‐. The flash responses and the Ca(2+)‐activated Cl‐ tail currents were inhibited by the Cl‐ channel blockers niflumic acid (10‐100 microM) and 5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid (NPPB) (10 microM). 2. After activation by ICa, the Ca(2+)‐activated Cl‐ current could be reactivated during its decay by photorelease of caged Ca2+. Experiments carried out on neurones held at 0 mV demonstrated that ICl(Ca) could be chronically activated due to residual Ca2+ influx. These data directly demonstrated that the decay of ICl(Ca) is not due to inactivation but rather to deactivation as a result of removal of the Ca2+ load from the cell cytoplasm. 3. Photorelease of caged inositol 1,4,5‐trisphosphate (IP3) failed to activate any Ca(2+)‐dependent current responses in cultured DRG neurones, although application of caffeine elicited transient inward currents, and responses to photoreleased IP3 could be obtained from freshly dissociated smooth muscle cells. 4. Photorelease of Ca2+ provides a useful method for investigating the properties of ICl(Ca) independently from other physiological parameters. In addition, we have directly demonstrated that ICl(Ca) in DRG neurones does not inactivate, and so may continue to modulate membrane excitability as long as the intracellular Ca2+ concentration ([Ca2+]i) close to the cell membrane is elevated.(ABSTRACT TRUNCATED AT 250 WORDS)
Ca2+ regulation in the presynaptic terminals of goldfish retinal bipolar cells.
Authors: Kobayashi K , Tachibana M
Published: December 2014

1. To investigate regulation of the intracellular free Ca2+ concentration ([Ca2+]i) in presynaptic terminals, the Ca2+ current (ICa) and [Ca2+]i in axon terminals were simultaneously monitored in acutely dissociated retinal bipolar cells under whole‐cell voltage clamp. 2. The recovery phase of the Ca2+ transient, which was evoked by activation of ICa, became slower when the Na(+)‐Ca2+ exchanger was suppressed by removing extracellular Na+. 3. Inhibition of the plasma membrane Ca2+ pump produced by raising extracellular pH to 8.4 increased the basal [Ca2+]i and caused incomplete recovery from the Ca2+ transient. These effects were not observed in orthovanadate‐loaded bipolar cells. 4. The Ca2+ transient was not significantly affected by ryanodine, caffeine, thapsigargin, Ruthenium Red or FCCP. Internal Ca2+ stores may not participate in shaping the Ca2+ transient. 5. The ratio of the peak amplitude of the Ca2+ transient to the total amount of Ca2+ influx became smaller as the size of the Ca2+ influx increased. This action was not affected by blockage of Ca2+ transporters in the plasma membrane, or by reduction of the rate of Ca2+ influx. The peak amplitude of the Ca2+ transient seemed to be determined by Ca2+ buffering substances with a positive co‐operativity.
Two components of [Ca2+]i‐activated Cl‐ current during large [Ca2+]i transients in single rabbit heart Purkinje cells.
Authors: Papp Z , Sipido K R , Callewaert G , Carmeliet E
Published: December 2014

1. Single Purkinje cells, enzymatically isolated from rabbit ventricle, were studied under whole‐cell voltage clamp conditions and internally perfused with the fluorescent Ca2+ indicator fura‐2(100 microM). 2. Ca2+ release from the sarcoplasmic reticulum was either induced by external application of caffeine or occurred spontaneously in Ca2+i‐overloaded cells. Membrane currents accompanying these Ca(2+)‐release signals were studied at steady membrane potentials. 3. [Ca2+]i transients were accompanied by transient membrane currents. In the absence of Na(+)‐Ca2+ exchange, two current components could be observed. The first component peaked well before the [Ca2+]i transient (Ifast) and relaxed before peak [Ca2+]i. The second component, on the other hand, peaked at the time when [Ca2+]i was maximal (Islow). 4. In symmetrical Cl‐ solutions both current components had a reversal potential close to O mV. A reduction of external or internal [Cl‐] shifted this reversal potential in accordance with the change of the Cl‐ equilibrium potential. 5. Each [Ca2+]i transient was accompanied by Ifast. Properties of Ifast suggest that this current component is the [Ca2+]i‐dependent Cl‐ current, ICl(Ca), previously observed during depolarizing pulses. 6. Islow was only detected in cells that displayed a large [Ca2+]i transient with or without elevated resting [Ca2+]i. 7. It is concluded that during large [Ca2+]i transients a slow component of ICl(Ca) can be activated. This second component may arise from the same channel population as the previously described fast component and be related to the presence of spatial and temporal inhomogeneities of [Ca2+]i. Alternatively, this current component may arise from a different Cl‐ channel population with a different Ca2+ sensitivity.
Stimulation of repetitive calcium transients in mouse eggs.
Authors: Ozil J P , Swann K
Published: December 2014

1. We have combined cell membrane electroporation by electrical field (EF) stimulation with a rapid perfusion system in order to stimulate repetitive increases in cytoplasmic free [Ca2+] ([Ca2+]i) in mouse eggs. [Ca2+]i was monitored by ratio fluorescent measurements of intracellular indo‐1 on individual eggs. The conditions required to cause different types of [Ca2+]i increases were established and the effects of these [Ca2+]i changes upon egg activation examined. 2. The rapid perfusion of non‐ionic medium caused a single [Ca2+]i increase. However, to generate repetitive [Ca2+]i increases, eggs were exposed to EF pulses in the presence of Ca2+ and then washed rapidly with culture medium. Sequential EF pulse application led to prolonged elevation of [Ca2+]i levels and eventual cell lysis unless rapid reperfusion with culture medium was achieved. Transient increases in [Ca2+]i in eggs could also be generated by EF pulses in the presence of inositol 1,4,5‐trisphosphate (InsP3). 3. In response to EF stimulation fertilized eggs showed [Ca2+]i increases that were enhanced relative to unfertilized eggs. The responses in these fertilized eggs were often followed by repetitive [Ca2+]i oscillations, despite the fact that the [Ca2+]i oscillations associated with sperm penetration had ceased by this stage. 4. In unfertilized mouse eggs the [Ca2+]i increases appeared to be due to direct cation influx since repeated EF pulses caused repeated influx of Mn2+ as monitored by quenching of fluorescence of fura‐2 loaded eggs. 5. Under conditions that stimulated reproducible patterns of [Ca2+]i transients we found that a single large [Ca2+]i transient did not cause significant egg activation, but that inducing repetitive [Ca2+]i transients was effective in activating eggs. The speed of activation as judged by the rate of pronuclear formation was also dependent upon the frequency of pulse application. 6. These data show that combining EF pulses with a rapid and precise sequential perfusion system can be used to manipulate [Ca2+]i levels in mammalian eggs. This provides a means of artificial mimicry of the [Ca2+]i transients seen after fertilization. It appears that Ca2+ influx during EF pulses does not cause significant Ca2+ release from internal stores in unfertilized eggs, but after fertilization Ca2+ influx does induce Ca2+ release. It is also apparent that mouse eggs are more successfully activated by repetitive [Ca2+]i increases than by single large [Ca2+]i rises. We suggest that our data provide direct evidence for the hypothesis that a cellular response to oscillations of intracellular [Ca2+]i can be distinct from that to monotonic rises in [Ca2+]i.
Cation regulation of anion current activated by cell swelling in two types of human epithelial cancer cells.
Authors: Anderson J W , Jirsch J D , Fedida D
Published: December 2014

1. In epithelial cells, hyposmotic stress induces visible cell swelling and large Cl‐ currents, which deactivate on return to isotonic solutions and are abolished by 0.1‐0.5 mM DIDS (4,4'‐diisothiocyanatostilbene‐2,2'‐disulphonic acid). During depolarizing voltage clamp pulses, the currents activate rapidly and show time‐dependent relaxation with associated tail currents on return to negative potentials. 2. We used whole‐cell and outside‐out patch recording to study volume activation of Cl‐ currents in the epithelial cancer cell lines H69AR and HeLa S5. In a 210 or 160 mosmol l‐1 hyposmotic bathing solution containing 90 mM NaCl, 1 mM Ca2+ and 1 mM Mg2+, current relaxation was rapid, occurred positive to the Cl‐ reversal potential and reduced current to < 30% of its peak level at +100 mV. 3. Replacement of most bath inorganic cations by N‐methyl‐D‐glucamine (NMDG) at constant Cl‐ concentration and osmolarity eliminated most of the current relaxation and caused an increase in steady‐state current levels. Steady‐state current was 85 +/‐ 6% of peak current at +100 mV in NMDG‐Cl bath solution. This ratio fell to 55 +/‐ 2% (n = 5) when 1 mM Mg2+ was re‐added to the bath. 4. Re‐addition of Mg2+ or other Group II metals (Ca2+, Sr2+, Ba2+) induced immediate changes in current relaxation in a dose‐ and species‐dependent manner. Concentrations of Mg2+ as low as 0.1 mM were effective in causing Cl‐ current relaxation. The IC50 for steady‐state current block by external Mg2+ was 1.75 mM.(ABSTRACT TRUNCATED AT 250 WORDS)
Hyperpolarization induced by vasoactive substances in intact guinea‐pig endocardial endothelial cells.
Authors: Manabe K , Ito H , Matsuda H , Noma A
Published: December 2014

1. The responses of guinea‐pig endocardial endothelial (EE) cells to various vasoactive substances were investigated in either the small tissue preparation or freshly isolated cells using the patch clamp technique. 2. The mean resting potential of the EE cell was ‐44 mV in the small tissue preparation, and applications of ATP, ADP, AMP, adenosine, histamine and substance P induced transient hyperpolarizations of ‐22, ‐21, ‐9, ‐10, ‐23 and ‐15 mV, respectively. The membrane potential of EE cells failed to respond to acetylcholine, bradykinin, thrombin, atrial natriuretic peptide, vasopressin and serotonin. 3. The whole‐cell voltage clamp of dissociated cells revealed a transient increase of K+ conductance underlying the ATP and histamine responses. The agonist‐induced current showed no time‐dependent change during voltage steps. The response was showed no time‐dependent change during voltage steps. The response was prevented by adding 10 mM EGTA to the pipette solution. 4. In the cell‐attached single channel recordings, ATP induced transient K+ channel activities having a slope conductance of 34 pS. In inside‐out patches, similar K+ channels were activated by applying Ca2+ of more than 0.1 microM. 5. These findings are consistent with the idea that the Ca(2+)‐dependent K+ channel is involved in the hyperpolarizing response of EE cells, as described in vascular endothelial cells.
The control of mono‐articular muscles in multijoint leg extensions in man.
Authors: van Ingen Schenau G J , Dorssers W M , Welter T G , Beelen A , de Groot G , Jacobs R
Published: December 2014

1. Movements often require control of direction and a magnitude of force exerted externally on the environment. Bi‐articular upper leg muscles appear to play a unique role in the regulation of the net torques about the hip and knee joints, necessary for the control of this external force. 2. The aim of this study was to test the hypothesis that the mono‐articular muscles act as work generators in powerful dynamic leg extensions, which means that they should be activated primarily in the phases during which they can contribute to work, irrespective of the net joint torques required to control the external force. 3. Cycling movements of six trained subjects were analysed by means of inverse dynamics, yielding net joint torques as well as activity patterns and shortening velocities of four mono‐ and four bi‐articular leg muscles. 4. The results show that the mono‐articular muscles exert force only in the phase in which these muscles shorten, whereas this appears not to be the case for the bi‐articular muscles. 5. Reciprocal patterns of activation of the rectus femoris and hamstring muscles appear to tune the distribution of net joint torques about the hip and knee joints, necessary to control the (changing) direction of the force on the pedal. 6. An analysis of running in man and additional related literature based on animal studies appears to provide further support for the hypothesis that mono‐ and bi‐articular muscles have essentially different roles in these powerful multijoint leg extension tasks.
Release of Ca2+ by noradrenaline and ATP from the same Ca2+ store sensitive to both InsP3 and Ca2+ in rat portal vein myocytes.
Authors: Pacaud P , Loirand G
Published: December 2014

1. Changes in cytosolic free Ca2+ concentration ([Ca2+]i) induced by noradrenaline (NA) and ATP were investigated using indo‐1 microspectrofluorimetry in single smooth muscle cells of rat portal vein. 2. Activation of P2x‐purinoceptors by ATP (10 microM) increased [Ca2+]i from 92 +/‐ 7 nM (n = 18) to 557 +/‐ 30 nM (n = 11). In the presence of NA (10 microM), the ATP‐induced rise in [Ca2+]i was reduced to 23.6 +/‐ 1.5% (n = 7) of the control response (in the absence of NA). 3. Tetracaine (10 microM to 2 mM) inhibited in a concentration‐dependent manner the Ca(2+)‐induced Ca2+ release (CICR) evoked by 5 mM caffeine. In the presence of 1 mM tetracaine, the rise in [Ca2+]i induced by ATP (10(‐8)‐10(‐4) M) was strongly inhibited. A tetracaine‐resistant rise in [Ca2+]i, corresponding to 26.4 +/‐ 2.3% (n = 14) of control values, was recorded in response to 10 microM ATP. 4. The amplitude of the NA‐induced [Ca2+]i rise depended on NA concentrations (10(‐8)‐10(‐5) M) and was not modified by tetracaine (1 mM). 5. This study suggests that Ca2+ ions released through the InsP3 receptor‐channel upon NA application do not activate CICR and the InsP3‐ and Ca(2+)‐sensitive Ca2+ store appears to represent, at least functionally, a single releasable Ca2+ pool.
Two distinct functional effects of protein phosphatase inhibitors on guinea‐pig cardiac L‐type Ca2+ channels.
Authors: Wiechen K , Yue D T , Herzig S
Published: December 2014

1. The effects of the phosphatase inhibitors okadaic acid and calyculin A on single guinea‐pig ventricular L‐type Ca2+ channels were studied. The inactive derivative norokadaone was used as a negative control. 2. The two known effects of cAMP‐dependent stimulation are mimicked by the phosphatase inhibitors to a varying extent. Only okadaic acid promotes the high‐activity gating mode (‘mode 2’), while calyculin A increases channel availability to a larger extent. As revealed by kinetic analysis of slow gating, the two phosphatase inhibitors retard a slow rate constant, which is assumed to represent exit from the available state by dephosphorylation. Norokadaone was inactive in both regards. 3. Mode 2 gating elicited by very positive prepulses is augmented by okadaic acid, and mode 2 lifetime is prolonged. Calyculin A fails to affect these parameters. Thus, voltage‐facilitated mode 2 gating reveals the same pharmacological properties as the mode 2 sweeps observed using conventional pulse protocols. 4. The results are interpreted in terms of the different sensitivity of protein phosphatase subtypes towards the inhibitors: channel availability appears to be controlled by a phosphorylation site dephosphorylated by a type 1‐like phosphatase, while mode 2 gating is coupled to a distinct site, dephosphorylated by a type 2A‐like phosphatase.
Volume‐sensitive taurine transport in bovine articular chondrocytes.
Authors: Hall A C
Published: December 2014

1. The swelling of bovine articular chondrocytes isolated from, or in situ within, cartilage by hypotonic shock rapidly activated the efflux or influx of radiolabelled taurine, an amino acid involved in volume regulation in a range of other cell types. 2. When chondrocytes were isolated by the use of collagenase into media of 280 or 380 mosmol l‐1, the activation of taurine efflux was at about the osmolarity of the isolating medium, but it was more marked for a given hypotonic shock in the cells isolated at the lower osmolarity. The volume of chondrocytes following isolation in these two osmolarities was the same, suggesting that the cells possess volume regulatory capacity. 3. In isolated chondrocytes, the induced pathway had some of the characteristics of a volume‐activated channel, i.e. no transport saturation with increasing substrate concentration, and lack of trans acceleration. The pattern of inhibition of the volume‐activated pathway by pharmacological blockers (e.g. pimozide, [(dihydro‐indenyl)oxy]alkanoic acid (DIOA) and tamoxifen) differed from that described for a similar pathway in other cell types. 4. The transport of other potential osmolytes (uridine, sorbitol and inositol) was stimulated by cell swelling, independent of sodium and inhibited by pimozide with a selectivity ratio of taurine, 1.00; uridine, 0.84; sorbitol, 0.66; and inositol, 0.38. The selectivity of taurine: inositol was not altered at different cell volumes. 5. The intracellular taurine concentration of chondrocytes within cartilage was low (about 2 mmol (l cell water)‐1) showing a negligible role for taurine as an osmolyte during recovery from cell swelling. The swelling‐induced loss of taurine from chondrocytes in situ was largely inhibited by pimozide and other drugs, showing that despite the rigid nature of cartilage, the chondrocytes were osmotically sensitive within the extracellular matrix.