bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential contribution of the NR1‐ and NR2A‐subunits to the selectivity filter of recombinant NMDA receptor channels.
Authors: Wollmuth L P , Kuner T , Seeburg P H , Sakmann B
Published: December 2014

1. The molecular determinants for the narrow constriction of recombinant N‐methyl‐D‐aspartate (NMDA) receptor channels composed of wild‐type and mutant NR1‐ and NR2A‐subunits were studied in Xenopus oocytes. 2. The relative permeability of differently sized organic cations was used as an indicator of the size of the narrow constriction. From measured reversal potentials under bi‐ionic conditions with K+ as the reference solution, permeability ratios were calculated with the Lewis equation. 3. For wild‐type NMDA receptor channels, five organic cations showed clear reversal potentials, with permeability ratios (PX/PK): ammonium, 1.28; methylammonium, 0.48; dimethylammonium (DMA), 0.20; diethylammonium, 0.07; and dimethylethanol‐ammonium, 0.02. 4. Mutation of the N‐site asparagine (N) to glutamine (Q) at homologous positions in either NR1 (position 598) or NR2A (position 595) increased the permeability of DMA relative to wild‐type channels about equally. However, for larger sized organic cations, the NR1(N598Q) mutation had stronger effects on increasing their permeability whereas the NR2A(N595Q) mutation was without effect. These changes in organic cation permeability suggest that the NR1(N598Q) mutation increases the pore size while the NR2A(N595Q) mutation does not. 5. Channels in which the NR1 N‐site asparagine was replaced by the smaller glycine (G), NR1(N598G)‐NR2A, showed the largest increase in pore size of all sites examined in either subunit. In contrast, in the NR2A‐subunit the same N‐site substitution to glycine produced only small effects on pore size. 6. For the NR2A‐subunit, an asparagine residue (position 596) on the C‐terminal side of the N‐site, when mutated to larger or smaller sized amino acids, produced large, volume‐specific effects on pore size. The mutant channel NR1‐NR2A(N596G) had the largest increase in pore size of all sites examined in the NR2A‐subunit. In contrast, mutation of the homologous position in the NR1‐subunit had no effect on pore size. 7. The cross‐sectional diameter of the narrow constriction in wild‐type NMDA receptor channels was estimated to be 0.55 nm. The pore sizes of the NR1(N598G)‐NR2A and NR1‐NR2A(N596G) mutant channels increased to approximately 0.75 and 0.67 nm, respectively. The double mutation, NR1(N598G)‐NR2A(596G), increased the pore size to approximately 0.87 nm, essentially the sum of the increase produced by the individual mutations. 8. It is concluded that both the NR1‐ and NR2A‐subunits contribute to the narrow constriction of NMDA receptor channels with asparagines located at non‐homologous positions. The major determinants of the narrow constriction in NMDA receptor channels are the NR1 N‐site asparagine and an asparagine adjacent to the NR2A N‐site.
Postnatal changes in the mammalian respiratory network as revealed by the transverse brainstem slice of mice.
Authors: Ramirez J M , Quellmalz U J , Richter D W
Published: December 2014

1. Spontaneous rhythmic activity in hypoglossal (XII) rootlets is generated at all postnatal stages from postnatal day (P) 0 to P22 in the transverse brainstem slice of mice containing the pre‐Bötzinger complex (PBC). The PBC is known to be a region essential for respiratory rhythm generation. It contains neurones generating periodic bursts that occur in synchrony with rhythmic XII activity. This synchrony indicates that the rhythmic PBC activity generated by the transverse slice is the central respiratory rhythm. 2. The strength of coupling between XII bursts and PBC bursts decreased during early postnatal development. In younger mice (P0‐4) each burst in XII rootlets corresponded to one burst in the PBC. In older mice (P5‐18) one burst in XII rootlets occurred only every third to fourth burst in PBC neurones. 3. Cycle length and burst duration of rhythmic XII activity did not change significantly during the first three postnatal weeks. However, the pattern of XII bursts changed from decrementing (P0‐7) to bell shaped (P8‐18) while the rate of rise of XII bursts decreased significantly. 4. The rate of rise of rhythmic depolarizations in neurones of the PBC discharging in phase with XII bursts (‘inspiratory neurones’) decreased with postnatal development. During interburst intervals, membrane potentials of neurones of older mice (P6‐18) were characterized by waves of synaptic input that were not observed in neonatal animals (P0‐5). 5. Blockade of glycine receptors by strychnine increased the frequency of rhythmic XII activity in neonatal and older mice (P0‐22). Although in expiratory PBC neurones glycinergic transmission was blocked at 10 microM strychnine, in inspiratory PBC neurones and XII rootlets even higher concentrations of up to 50 microM strychnine failed to abolish rhythmic activity.
The peptide ACTH(1‐39), adrenal growth and steroidogenesis in the sheep fetus after disconnection of the hypothalamus and pituitary.
Authors: Phillips I D , Ross J T , Owens J A , Young I R , McMillen I C
Published: December 2014

1. We have investigated the role of the fetal hypothalamo‐pituitary axis in the control of adrenocortical growth and steroidogenesis in the sheep fetus during late gestation. Plasma concentrations of ACTH(1‐39) increased between 120‐125 and 136‐142 days (P < 0.05), but did not change after surgical disconnection of the fetal hypothalamus and pituitary (HPD) at 106‐120 days gestation. There was no effect of either gestational age or HPD on the circulating concentrations of the ACTH‐containing precursors pro‐opiomelanocortin (POMC) and pro‐ACTH (the 22 kDa N‐terminal portion of POMC). 2. In the fetal sheep adrenal, the relative abundance of the mRNAs of the steroidogenic enzymes CYPIIA1 and CYP21A1 increased between 130‐135 and 136‐140 days gestation (P < 0.05) and remained high after 141 days, whereas that of CYP17 mRNA increased after 141 days gestation (P < 0.05). The abundance of adrenal 3 beta‐HSD mRNA did not change between 130 and 145 days. 3. Hypothalamo‐pituitary disconnection significantly reduced the abundance of of CYPIIA1 mRNA, 3 beta‐HSD mRNA and CYP17 mRNA by 3.4, 3.1 and 3.7 times, respectively, at 140‐142 days gestation (P < 0.05). 4. In the intact group of fetal sheep, adrenal weight increased between 130‐135 and 141‐145 days (P < 0.05), but there was no change in the abundance of adrenal insulin‐like growth factor II (IGF‐II) mRNA across this gestational age range. Hypothalamo‐pituitary disconnection significantly reduced fetal adrenal weight to 66% that of intact sheep (P < 0.01), but did not alter the abundance of IGF‐II mRNA in the fetal adrenal at 140‐142 days. 5. Our results suggest that the prepartum changes in adrenal growth and steroidogenesis are under the control of an intact hypothalamo‐pituitary axis in late gestation and are dependent on an increase in circulating ACTH(1‐39), rather than on ACTH precursors. We have found no evidence, however, for a direct‐relationship between fetal adrenal growth or steroidogenesis and adrenal IGF‐II mRNA between 130 and 145 days gestation.
Openings in frog microvascular endothelium induced by high intravascular pressures.
Authors: Neal C R , Michel C C
Published: December 2014

1. We have investigated the effects of microvascular pressures, high enough to rupture the vessel wall, upon the ultrastructure and permeability of mesenteric capillaries in pithed frogs. 2. The vessels were perfused via micropipettes with Ringer solutions containing bovine serum albumin (1 mg ml‐1) and a few human red cells. After a perfused section of vessel had been closed downstream, pressure (applied via the micropipette) was raised in a series of steps of 10 mmHg, each lasting approximately 10 s, until the vessel ruptured. Fluid filtration through the vessel wall prior to rupture was estimated from the movements of the red cells. 3. Seven vessels were fixed in glutaraldehyde immediately after rupture and prepared for electron microscopy. The electron micrographs revealed openings in the vessel walls and thirty‐six of these gaps were completely defined in runs of serial sections made on four of the vessels. Twenty‐nine of these gaps passed through the endothelial cells (transcellular) and seven were intercellular. 4. The pressure at which a vessel ruptured, Pb, was measured in twenty‐nine vessels and had a mean +/‐ S.E.M. value of 79.6 +/‐ 5.0 cmH2O. In ten of these vessels, which had a mean +/‐ S.E.M. Pb of 84.2 +/‐ 6.5 cmH2O, microvascular pressure was lowered immediately after the initial rupture and the vessel perfused at a pressure of 20 cmH2O. Pb was then remeasured and found to be 69.9 +/‐ 8.4 cmH2O, which was not significantly different from its initial value. 5. Hydraulic permeability (Lp) was measured in six vessels over the range of 15‐30 cmH2O before and 10 min after the vessel wall ruptured at high pressure. Mean values were 5.5 x 10(‐7) and 4.0 x 10(‐7) cms‐1 cmH2O‐1 and were not significantly different. 6. At pressures equal to and 10 mmHg below Pb, small short‐lived increases in filtration rate were observed. It is suggested that these may correspond to the increased permeability to fluid and macromolecules observed at high microvascular pressures in intact capillary beds.
Enhancement of ionic current and charge movement by coexpression of calcium channel beta 1A subunit with alpha 1C subunit in a human embryonic kidney cell line.
Authors: Kamp T J , Pérez-García M T , Marban E
Published: December 2014

1. Coexpression of the beta subunit with the alpha 1C subunit of the cardiac L‐type Ca2+ channel has been shown to increase ionic current. To examine the mechanism of this increase, ionic and gating currents were measured in transiently transfected HEK293 cells. 2. Beta 1A subunit coexpression increased the maximal whole‐cell conductance (Gmax) measured in 10 mM Ba2+ from 91 +/‐ 11 to 833 +/‐ 107 pS pF‐1 without a change in the voltage dependence of activation (V1/2: ‐6.1 +/‐ 1.1 and ‐6.6 +/‐ 0.9 mV, respectively). 3. Gating currents were smaller in cells expressing only the alpha 1C subunit (only four out of eleven cells exhibited gating currents above the limits of detection, whereas eight out of eight beta 1A coexpressing cells had measurable gating currents). The gating currents were integrated to measure the intramembrane charge movement (Q). The ON charge movement (Qon) could be described by a Boltzmann distribution reaching a maximal value of Qon,max. 4. The mean ratio of Gmax: Qon,max increased from 99 +/‐ 6 to 243 +/‐ 30 pS fC‐1 with beta 1A coexpression, demonstrating that the beta 1A subunit changes the gating of alpha 1C channels to favour the opening of the channels. However, this 2.5‐fold change in the Gmax: Qon,max ratio explains less than half of the 9.2‐fold increase in Gmax with beta 1A subunit coexpression. The major effect is due to a 3.7‐fold increase in Qon,max, demonstrating that beta 1A subunit coexpression increases the number of functional surface membrane channels.
Foveal cone mosaic and visual pigment density in dichromats.
Authors: Berendschot T T , van de Kraats J , van Norren D
Published: December 2014

1. Optical reflectance spectra of the fovea were measured in ten subjects with normal colour vision, ten protanopes and seven deuteranopes. Four conditions were used: perpendicular and oblique angle of incident and reflected light on the retina, both in a dark‐adapted and a fully bleached state. 2. The spectra were analysed to assess the effects of dichromacy on the cone mosaic. A replacement model, i.e. one where the total number of cones remains unchanged and all cones are filled with a single type of pigment, was found to fit our data best. 3. The analysis of the spectral fundus reflectance also provided estimates for densities of photo‐labile and photo‐stable retinal pigments and fraction of long wavelength‐sensitive (LWS) cones. Visual pigment density was 0.39 for protanopes and 0.42 for deuteranopes, significantly lower than the 0.57 found for colour normals. Macular pigment density was 0.54 for colour normals, 0.46 for protanopes and 0.42 for deuteranopes. 4. For colour normals the LWS cone fraction was 0.56, in agreement with psychophysical literature. The LWS cone fraction for protanopes was ‐0.04, and for deuteranopes 0.96, consistent with their Rayleigh matches.
Carbachol‐induced [Ca2+]i oscillations in single smooth muscle cells of guinea‐pig ileum.
Authors: Kohda M , Komori S , Unno T , Ohashi H
Published: December 2014

1. Changes in cytosolic Ca2+ concentration ([Ca2+]i) produced by carbachol (CCh) were measured in single smooth muscle cells of guinea‐pig ileum using a Ca(2+)‐sensitive fluorescent dye, fura‐2, to clarify the underlying mechanisms of muscarinic [Ca2+]i oscillations. 2. Half of the cells, when exposed to 0.2 microM CCh, exhibited repeated changes in [Ca2+]i giving a serrated appearance. The oscillatory changes in [Ca2+]i were very similar to those evoked by increasing extracellular K(+) concentration ([K+]o) to 30 mM, which were abolished by removal of extracellular Ca2+, nifedipine and La3+, but remained unchanged after depletion of internal Ca2+ stores with cyclopiazonic acid, thapsigargin and ryanodine. 3. Every individual [Ca2+]i oscillation was just like a [Ca2+]i increase generated spontaneously in about 8% of cells or triggered by an action potential evoked by a current pulse in current‐clamped cells. 4. In the remaining half of the cells exposed to 0.2 microM CCh, slower [Ca2+]i oscillations were elicited and every individual [Ca2+]i oscillation was always preceded by the fast brief increase in [Ca2+]i. 5. [Ca2+]i oscillations elicited by 2 microM CCh were temporally and functionally distinct from those induced by high [K+]o. They were more or less regular in the periodicity and pattern, comprised pacemaker potential‐like [Ca2+]i increases or sinusoidal types of [Ca2+]i increases, and could be elicited even in 100 mM K+(o). 6. Removal of extracellular Ca2+ or application of nifedipine, methoxyverapamil (D600), diltiazem or La3+ during CCh (2 micro M)‐induced [Ca2+]i oscillations caused them to disappear. In cells i which internal Ca2+ stores were depleted, 2 microM CCh did not evoke [Ca2+]i oscillations but occasionally induced single or repeated generation of the increase in [Ca2+]i with a serrated appearance. 7. The results indicate that CCh can induce two types of [Ca2+]i oscillation in guinea‐pig ileal smooth muscle cells; one arises from Ca2+ influx associated with action potential discharges and the other from periodic release of Ca2+ from internal stores. The latter [Ca2+]i oscillation requires extracellular Ca2+ to sustain it.
Ca2+ clearance mechanisms in isolated rat adrenal chromaffin cells.
Authors: Park Y B , Herrington J , Babcock D F , Hille B
Published: December 2014

1. Intracellular Ca2+ clearance mechanisms were studied in rat adrenal chromaffin cells, by measuring slow tail currents through small‐conductance Ca(2+)‐activated K+ channels and using indo‐1 photometry following depolarization‐induced Ca2+ loading. 2. Following several‐hundred millisecond depolarizations, [Ca2+]i decayed in three phases. An initial fast decay was followed by a long‐lasting, low plateau, then [Ca2+]i returned to the resting level slowly. 3. Replacement of external Na+ moderately slowed [Ca2+]i decay, indicating a contribution of plasma membrane Na(+)‐Ca2+ exchange. 4. Raising external pH or application of extracellular Eosin of La3+ prolonged slow tail currents, indicating a contribution of plasma membrane Ca(2+)‐ATPase to Ca2+ clearance. 5. Ca(2+)‐induced Ca2+ release from caffeine‐sensitive stores occurred during depolarization. 6. Inhibition of endoplasmic reticulum Ca(2+)‐ATPase had little effect on Ca2+ clearance. 7. Slow tail currents and [Ca2+]i decay following 0.2 ‐ 2 s depolarizations were much prolonged by mitochondrial inhibition with carbonyl cyanide m‐chlorophenylhydrazone (CCCP) or Ruthenium Red, which abolished the initial rapid decay and plateau of [Ca2+]i. 8. In conclusion, mitochondrial Ca2+ uptake plays a major role in Ca2+ clearance by rapidly and reversibly sequestering Ca2+ during depolarization‐evoked Ca2+ loads.
Muscarinic receptors mediating depression and long‐term potentiation in rat hippocampus.
Authors: Auerbach J M , Segal M
Published: December 2014

1. Two concentration‐dependent effects of the muscarinic agonist carbachol (CCh) were characterized in submerged slices of rat hippocampus using extracellular recordings of excitatory postsynaptic potentials (EPSPs): muscarinic long‐term potentiation (LTP(m)) and depression. 2. LTP(m) of the EPSP slope was seen following long exposure (20 min) of the slice to low concentrations of CCh (0.2‐0.5 microM). This LTP(m) was not accompanied by a change in the size of the afferent fibre volley or by a change in paired‐pulse potentiation, consistent with a postsynaptic locus of CCh action. 3. Intracellular recordings from voltage‐clamped neurons of inward current evoked by iontophoretically applied alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) and N‐methyl‐D‐aspartate (NMDA) revealed that, while cellular responses to NMDA rose transiently upon superfusion with 0.5 microM CCh, responses to AMPA increased gradually and remained potentiated after washout of CCh. 4. LTP(m) is mediated by an M2 muscarinic receptor. Two M2 muscarinic receptor antagonists, methoctramine and AFDX‐116, blocked LTP(m). The M2 agonist oxotremorine induced LTP(m) at low agonist concentrations. None of the M1 and M3 receptor agonists and antagonists tested affected LTP(m). 5. Muscarinic fast onset depression of the EPSP was seen in response to higher concentrations of CCh (2‐5 mu M). This depression was accompanied by an increase in paired‐pulse potentiation, indicating a possible presynaptic locus of action. The M3 muscarinic receptor antagonist 4‐diphenylacetoxy‐N‐methylpiperidine methiodide (4‐DAMP) blocked the muscarinic depression of the EPSP slope. M1, M2 and M4 muscarinic antagonists did not block this response. 6. Blockade of the muscarinic depression by 4‐DAMP did not uncover a suppressed LTP(m). However, addition of picrotoxin facilitated the expression of LTP(m) induced by high concentrations of CCh, indicating an involvement of interneurons in regulation of LTP(m). 7. Cholinergic denervation produced by fimbria‐fornix transection resulted in supersensitivity of both M2‐ and M3‐mediated effects, indicating that the receptors mediating these effects are not located on presynaptic cholinergic fibres. In the presence of 4‐DAMP and picrotoxin the dose‐response curve for CCh‐induced effects in slices from lesioned animals was shifted to the left relative to that of normal animals, indicating a supersensitivity of both receptor types.
Respiratory activity in the facial nucleus in an in vitro brainstem of tadpole, Rana catesbeiana.
Authors: Liao G-S  , Kubin L , Galante R J , Fishman A P , Pack A I
Published: December 2014

1. In studies of the central neural control of breathing, little advantage has been taken of comparative approaches. We have developed an in vitro brainstem preparation using larval Rana catesbeiana which generates two rhythmic neural activities characteristic of lung and gill ventilation. Based on the pattern of the facial (VII) nerve activity both lung and gill rhythm‐related respiratory cycles were divided into three distinct phases. The purpose of this study was to characterize and classify membrane potential trajectories of respiratory motoneurons in the VII nucleus at intermediate stages (XII‐XVII) of development. 2. Seventy‐five respiratory‐modulated neurons were recorded intracellularly within the facial motor nucleus region. Their resting membrane potential was between ‐40 and ‐80 mV. Sixty of them were identified as VII motoneurons and fifteen were non‐antidromically activated. Membrane potentials of fifty‐six of the seventy‐five neurons were modulated with both lung (5‐27 mV) and gill rhythms (3‐15 mV) and the remaining nineteen neurons had only a modulation with lung rhythmicity (6‐23 mV). No cells with gill modulation alone were observed. 3. All of the cells modulated with lung rhythmicity had only phase‐bound depolarizing or hyperpolarizing membrane potential swings which could be categorized into four distinct patterns. In contrast, of the fifty‐six cells modulated with gill rhythmicity, thirty‐two were phasically depolarized during distinct phases of the gill cycle (four patterns were distinguished), whereas the remaining twenty‐four were phase spanning with two distinct patterns. The magnitudes of lung and gill modulations were proportionally related to each other in the cells modulated with both rhythms. 4. In all sixteen neurons studied, a reduction or a reversal of phasic inhibitory inputs during a portion of the lung or gill respiratory cycle was observed following a negative current or chloride ion (Cl‐) injection. The phasic membrane resistance modulation in relation to the gill rhythm was analysed in six neurons and a relative decrease in the somatic membrane resistance (0.7‐8.1 M omega) was detected during the periods of hyperpolarization. 5. We propose that, at these intermediate stages of development: (a) both gill and lung respiratory oscillations in motoneurons are generated by respiratory premotor neurons having only a few distinct activity patterns; (b) these patterns delineate distinct portions of the centrally generated respiratory cycles; and (c) phasic synaptic inhibition, mediated by Cl‐, contributes to shaping the membrane potential trajectories of respiratory motoneurons.
Role of chloride‐mediated inhibition in respiratory rhythmogenesis in an in vitro brainstem of tadpole, Rana catesbeiana.
Authors: Galante R J , Kubin L , Fishman A P , Pack A I
Published: December 2014

1. The isolated brainstem of larval Rana catesbeiana maintained in vitro generates neural bursts that correspond to the lung and gill ventilatory activity generated in the intact specimen. To investigate the role of chloride channel‐dependent inhibitory mechanisms mediated by GABA(A) and/or glycine receptors on fictive lung and gill ventilation, we superfused the isolated brainstems with agonists, antagonists (bicuculline and/or strychnine) or a chloride‐free solution while recording multi‐unit activity from the facial motor nucleus. 2. Superfusion with the agonists (GABA or glycine) produced differential effects on frequency, amplitude and duration of the neural bursts related to lung and gill ventilation. At a GABA or glycine concentration of 1.0 mM, fictive gill bursts were abolished while fictive lung bursts persisted, albeit with reduced amplitude and frequency. 3. At the lowest concentrations used (1.0‐2.5 microM), the GABA(A) receptor antagonist bicuculline produced an increase in the frequency of lung bursts. At higher concentrations (5.0‐2.0 microM) bicuculline produced non‐specific excitatory effects. The glycine antagonist strychnine, at concentrations lower than 5.0 microM, caused a progressive decrease in the frequency and amplitude of the gill bursts and eventually abolished the rhythmic activity. At higher concentrations (7.5 microM), non‐specific excitatory effects occurred. Superfusion with bicuculline (10 microM) and strychnine (5 microM) combined abolished the neural output for gill ventilation but increased the frequency, amplitude and duration of lung bursts. 4. Superfusion with Cl(‐)‐free solution also abolished the rhythmic neural bursts associated with gill ventilation, while it significantly increased the amplitude (228 +/‐ 51%; P < 0.05) (mean +/‐ S.E.M.) and duration of the lung bursts (3.5 +/‐ 0.1 to 35.3 +/‐ 3.7 s; P < 0.05) and improved the regularity of their occurrence. 5. We conclude that different neural systems generate rhythmic activity for lung and gill ventilation. Chloride‐mediated inhibition may be essential for generation of neural bursts associated with gill ventilation. In contrast, the burst associated with lung ventilation can be generated in the absence of Cl(‐)‐mediated inhibition although the latter plays a role in shaping the normal lung burst.
P2U receptor is linked to cytosolic Ca2+ transient and release of vasorelaxing factor in bovine endothelial cells in situ.
Authors: Miyagi Y , Kobayashi S , Nishimura J , Fukui M , Kanaide H
Published: December 2014

1. With the use of front‐surface fluorimetry and fura‐2‐loaded strips of bovine aortic valve, we characterized the [Ca2+]i transients induced in endothelial cells in situ using a non‐selective purinergic agonist (adenosine 5'‐triphosphate (ATP)), and selective agonists for P2X (alpha, beta‐methylene ATP), P2Y (2‐methylthio‐ATP (2MeSATP)) and P2U (uridine 5'‐triphosphate (UTP)) purinoceptors and an unrelated agonist bradykinin (BK). 2. Double staining with fura‐2 and acetylated low‐density lipoprotein labelled with 1,1'‐dioctadecyl‐3,3,3',3'‐tetramethyl‐indo‐carbocyanine perchlorate showed that the fura‐2 fluorescence arose exclusively from a single monolayer of endothelial cells covering the surface of the valvular strips. 3. All nucleotides (ATP, UTP and 2MeSATP) induced an elevation of the intracellular Ca2+ concentration ([Ca2+]i), with an initial transient peak and a subsequent lower sustained elevation. Blockade of the Ca2+ influx with 1 mM Ni2+ did not affect the peak levels of the [Ca2+]i transients, whereas it abolished the sustained increases in [Ca2+]i induced by these nucleotides. 4. The potency order of these nucleotides was 2MeSATP > ATP > UTP, while the order of the maximum responses was UTP = ATP > 2MeSATP. alpha, beta‐Methylene ATP (up to 1 mM) had only a minimal effect. 5. Prolonged exposure to ATP or UTP, at concentrations giving a maximum response, desensitized the responses to ATP, UTP and 2MeSATP, but not to BK. Prolonged exposure to 2MeSATP at concentrations giving a maximum response did not desensitize the responses to UTP or BK, but did desensitize those to ATP and 2MeSATP. Prolonged exposure to BK did not induce heterologous desensitization to any of the three nucleotides. 6. [Ca2+]i elevation in valvular endothelial cells induced by UTP was associated with the relaxation of adjacent vascular medial strips precontracted with U‐46619, the stable analogue of thromboxane A2. 7. We conclude that: (1) the peak elevation of the [Ca2+]i transient induced by these nucleotides is independent of extracellular Ca2+, which therefore suggests the release of intracellular Ca2+ and, (2) mature endothelial cells in situ, in a valvular preparation, have a common receptor for ATP and UTP (nucleotide or P2U receptor), which coexists with the P2Y receptor. Thus we propose that the activation of the nucleotide receptor, P2U, induces [Ca2+]i elevation in endothelial cells in situ, and thus leads to the release of vasorelaxing factors.
Selective activation of exocytosis by low concentrations of ACh in rat pancreatic acinar cells.
Authors: Maruyama Y
Published: December 2014

1. We have monitored changes in membrane capacitance (delta C) and conductance (delta G) induced by muscarinic acetylcholine stimulation in single rat pancreatic acinar cells. 2. Acetylcholine (ACh, 500 nM) induced simultaneous increases of delta C and delta G. In contrast, a low concentration (50 nM) of ACh exclusively induced delta C increases without delta G. These responses were abolished by the internal perfusion of heparin. This indicates that inositol 1,4,5‐trisphosphate‐mediated internal Ca2+ mobilization either simultaneously activates exocytosis and ion channels or exclusively initiates exocytosis. In comparison, a low concentration of A23187 selectively activated ion channels but a high concentration activated exocytosis and ion channels simultaneously. 3. These selective response patterns of delta C and delta G depend on the choice of agonist and the internal EGTA concentration. From this, we postulated two explanations for the selective action of muscarinic ACh stimulation on exocytosis. First, an area of high [Ca2+]i, spatially close to secretory granules, activates exocytosis. Second, an as yet unknown signalling factor sensitizes the Ca2+ affinity of the exocytotic apparatus.
Specific thermal responsiveness of ventromedial hypothalamic neurons to localized scrotal heating and cooling in rats.
Authors: Li Q , Thornhill J
Published: December 2014

1. The specificity of thermoresponsive ventromedial hypothalamic (VMH) neurons to localized, incremental scrotal thermal cooling and heating of urethane‐anaesthetized male Sprague‐Dawley rats (maintained at 37 degrees C colonically) was investigated. 2. Ventromedial hypothalamic extracellular neuronal activity and surface (scalp) electroencephalogram (EEG) activity from the parietal region were recorded. Intrascapular brown adipose tissue (TIBAT), surface tail (Tt) and scrotal (Tsc) temperatures, where thermal stimulation was evoked, were also monitored. 3. One hundred and twenty‐five VMH neurons were recorded, with forty (32%) VMH neurons classified as warm‐responsive neurons (WRNs), twenty‐three (18%) as coldresponsive neurons (CRNs) and sixty‐two (50%) as thermal non‐responsive neurons (TNRNs) based on their thermal coefficients. Of VMH WRNs, 60% (i.e. 24) were classified as having biphasic neuronal activity responses, as were 60% (i.e. 14 of 23) of the CRNs. Forty per cent of WRNs and CRNs were classified as having monophasic changes in neuronal activity. 4. Scrotal heating or cooling from 5 to 40 degrees C resulted in specific firing rate changes of VMH WRNs and CRNs without any associated change in EEG activity (i.e. no significant change in EEG frequency or amplitude from initial baseline EEG activity when Tsc was 20 degrees C). EEG desynchronization (increased EEG frequency, decreased amplitude) was only observed when scrotal temperatures were at 45 degrees C or after each tail pinch (noxious stimulation) but not with scrotal brushing (mechanical stimulation). 5. With core temperature maintained at 37 degrees C, localized, scrotal heating and cooling of rats did not induce IBAT temperature changes indicative of brown adipose tissue activation, but delayed changes in tail temperature, indicative of vasoactive effector responses, did occur.
Water compartmentalization and extracellular tortuosity after osmotic changes in cerebellum of Trachemys scripta.
Authors: Krizaj D , Rice M E , Wardle R A , Nicholson C
Published: December 2014

1. Water compartmentalization in the turtle cerebellum subject to media of different osmolalities was quantified by combining extracellular diffusion analysis with wet weight and dry weight measurements. The diffusion analysis also determined the tortuosity of the extracellular space. 2. Isolated cerebella were immersed in normal, oxygenated physiological saline (302 mosmol kg‐1), hypotonic saline (238 mosmol kg‐1) and a series of hypertonic salines (up to 668 mosmol kg‐1). The osmolality was varied by altering the NaCl content. 3. Extracellular volume fraction and tortuosity of the granular layer of the cerebellum were determined from measurements of ionophoretically induced diffusion profiles of tetramethylammonium, using ion‐selective microelectrodes. The volume fraction was 0.22 in normal saline, 0.12 in hypotonic medium and 0.60 in the most hypertonic medium. Tortuosity was 1.70 in the normal saline, 1.79 in the hypotonic and 1.50 in the most hypertonic saline. 4. The water content, defined as (wet weight‐dry weight)/wet weight, of a typical isolated cerebellum (including granular, Purkinje cell and molecular layers) was 82.9%. It increased to 85.2% in hypotonic saline and decreased to 80.1% in the most hypertonic saline. 5. Measurements of extracellular volume fraction and water content were combined to show that hypotonic solutions caused water to move from the extracellular to the intracellular compartment while hypertonic solutions caused water to move from the intracellular to extracellular compartment, with only a relatively small changes in total water in both cases. 6. These results suggest the use of the isolated turtle cerebellum as a model system for studying light scattering or diffusion‐weighted magnetic resonance imaging.
In vivo synchronous membrane potential oscillations in mouse pancreatic beta‐cells: lack of co‐ordination between islets.
Authors: Valdeolmillos M , Gomis A , Sánchez-Andrés J V
Published: December 2014

1. The properties of the oscillations in electrical activity of different beta‐cells within the same islet of Langerhans, and of different islets within the same pancreas, recorded in vivo, are described. 2. Simultaneous recordings of two cells within the same islet showed that the oscillations were synchronous. A rapid increase in blood glucose led to the simultaneous appearance of a transitory phase of continuous electrical activity in both cells. These results indicate that under physiological conditions, the islets operate as a functional syncytium. 3. Simultaneous recordings of cells from two different islets within the same pancreas showed that the oscillations in the electrical activity were not synchronous, which suggests that each islet is a functionally independent unit. Rapid changes in blood glucose led to the appearance of a transitory phase of increased electrical activity in both islets, although of different duration. These results suggest that the endocrine pancreas lacks a pacemaker driving the electrical activity of all the islets. 4. The comparison of the degree of activation of different islets, simultaneously recorded at different glucose concentrations, indicated that all the islets had a similar sensitivity to glucose. Furthermore, when the glucose concentration was increased, the electrical activity in both islets increased in parallel, suggesting that the amount of insulin released due to the increase in glycaemia was produced by the simultaneous response of all the islets and not by the recruitment of islets with different sensitivities to glucose. 5. Our results predict that the synchronous electrical activity of all the cells within an islet will result in widespread intracellular calcium oscillations and pulsatile insulin secretion. The periodicity of the pulses of insulin secretion in different islets is suggested to be of slightly different length and asynchronous.
Effect on the fura‐2 transient of rapidly blocking the Ca2+ channel in electrically stimulated rabbit heart cells.
Authors: Levi A J , Issberner J
Published: December 2014

1. We used a rapid solution switcher technique to investigate mechanisms that might trigger intracellular Ca2+ release in rabbit ventricular myocytes. The study was carried out at 36 degrees C, intracellular Ca2+ (Ca2+i) was monitored with fura‐2, and myocytes were electrically stimulated. 2. In patch‐clamped cells, using the switcher to apply 20 microM nifedipine (an L‐type Ca2+ current (ICa,L) blocker) 4 s before a depolarization to +10 mV reduced the amplitude of ICa,L to 10.25 +/‐ 2.25% of control (mean +/‐ S.E.M., n = 7 cells). 3. In externally stimulated cells, a rapid switch to 20 microM nifedipine 4 s before a stimulus reduced the amplitude of the fura‐2 transient to 64.01 +/‐ 2.09% of control (mean +/‐ S.E.M., n = 19 cells). Using an in vivo calibration curve for fura‐2, this was equivalent to a reduction in the Ca2+ transient to 50% during nifedipine application. Since an identical nifedipine switch reduced ICa,L to 10.25%, it would seem that blocking a large fraction of ICa,L inhibited only half the Ca2+ transient. 4. The Na(+)‐Ca2+ exchanger is inhibited by 5 mM nickel. Switching to 20 microM nifedipine +5 mM nickel 4 s before a stimulus abolished the fura‐2 transient completely, consistent with the hypothesis that Ca2+ entry via reverse Na(+)‐Ca2+ exchange might trigger a fraction of the fura‐2 transient that remained during nifedipine. 5. After the Na(+)‐K+ pump was inhibited by strophanthidin to increase intracellular Na+ (Na+i), a switch to 20 microM nifedipine became progressively less effective in reducing the fura‐2 transient. This suggests that as Na+i rose, other mechanisms (perhaps reverse Na(+)‐Ca2+ exchange) appeared able to substitute for ICa,L in triggering the Ca2+ transient. 6. In cells depleted of Nai+ to inhibit the triggering of sarcoplasmic reticulum (SR) Ca2+ release by reverse Na(+)‐Ca2+ exchange, a nifedipine switch reduced the fura‐2 transient to 10.9 +/‐ 4.19% (mean +/‐ S.E.M., n = 7; equivalent to 6.5% of the Ca2+ transient). 7. A switch to Na(+)‐free (Li+) solution 100 ms before an electrical stimulus caused an increase in the fura‐2 transient of 12.2 +/‐ 1.5% (mean +/‐ S.E.M., n = 7; equivalent to a 22% increase in the Ca2+ transient). 8. The results confirm that ICa,L is an important trigger for SR Ca2+ release and the resulting Ca2+ transient. However, since 50% of the Ca2+ transient remained when ICa,L was largely inhibited, it would seem likely that other SR trigger mechanisms might exist in addition. These data are consistent with the idea that Ca2+ entry via reverse Na(+)‐Ca2+ exchange during the upstroke of the normal cardiac action potential might trigger a fraction of SR Ca2+ release and the resulting Ca2+ transient.
Characterization of acid extrusion mechanisms in cultured fetal rat hippocampal neurones.
Authors: Baxter K A , Church J
Published: December 2014

1. We investigated the mechanisms regulating acid extrusion in cultured fetal rat hippocampal neurones loaded with 2',7'‐bis‐(2‐carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein. 2. In the absence of HCO3‐, removal of external Na+ by substitution with N‐methyl‐D‐glucamine caused a sustained intracellular acidification that was not observed when Na+ was replaced by Li+, but neither steady‐state intracellular pH (pHi) nor the rate of pHi recovery from an imposed acid load were influenced by amiloride analogues or HOE 694, inhibitors of Na(+)‐H+ exchange in other cell types. In the presence of HCO3‐, removal of external Na+ or Cl‐ evoked an intracellular acidification and a 4,4'‐diisothiocyanatostilbene‐2,2'‐disulphonic acid‐sensitive (DIDS‐sensitive) intracellular alkalinization, respectively. Applied alone, however, DIDS elicited a fall in steady‐state pHi at room temperature but not at 37 degrees C. The DIDS‐evoked fall in steady‐state pHi and the 0 Cl(‐)‐evoked intracellular alkalinization observed in the presence of HCO3‐ at room temperature were dependent on external Na+. 3. At room temperature (18‐22 degrees C), but not at 37 degrees C, the transition from HCO3(‐)‐free to HCO3(‐)‐containing medium at a constant pHo produced a net alkalinization that was dependent on external Na+ and was inhibited by DIDS or the depletion of internal Cl‐. 4. Recovery of pHi from an acid load imposed in the absence of HCO3‐ was dependent on external Na+. Addition of HCO3‐ to the perfusion medium increased the rate of pHi recovery from an acid load at room temperature but not at 37 degrees C. In the presence of HCO3‐, DIDS slowed the rate of recovery of pHi from an acid load at both room temperature and at 37 degrees C. 5. Recovery of pHi following an imposed intracellular acidification to pH < 6.5 could occur in the absence of external Na+, providing that HCO3‐ was present in the perfusate. This slow, Na(+)‐independent recovery of pHi from very low levels of intracellular pH was sensitive to DIDS. 6. The results indicate that acid extrusion in cultured fetal rat hippocampal neurones involves primarily two Na(+)‐dependent mechanisms, one HCO3‐ dependent (a HCO3(‐)‐Cl‐ exchanger) and the other HCO3‐ independent (possibly a Na(+)‐H+ exchanger). Although both mechanisms participate in the maintenance of steady‐state pHi at room temperature, only the HCO3(‐)‐independent mechanism does so at 37 degrees C.
Contractile properties and protein isoforms of single fibres from the chicken pectoralis red strip muscle.
Authors: Reiser P J , Greaser M L , Moss R L
Published: December 2014

1. The contractile properties of single muscle fibres of the red strip region of adult chicken pectoralis major (PM) muscle, some of which are known to express an embryonic isoform of myosin heavy chain (MHC), were determined and compared with the properties of the fast white fibres of the PM and the slow tonic fibres of the anterior latissimus dorsi (ALD) muscle. 2. The red strip fibres could be classified into two groups, fast and slow. The mean velocity of unloaded shortening (Vmax) in fast red strip fibres was approximately half the Vmax of fast white fibres. Vmax of slow red strip fibres was less than 20% of the value for fast red strip fibres and was not different from Vmax of ALD fibres. 3. The tension‐generating ability, i.e. the maximal isometric tension/fibre cross‐sectional area (P0/CSA), was the same in fast red strip fibres and fast white fibres. P0/CSA was approximately 30% lower in slow red strip fibres compared with fast red strip fibres but was 70% greater in slow red strip fibres compared with ALD fibres. 4. The tension‐pCa relation of fast red strip fibres was shifted to lower pCa values, indicating a lower calcium sensitivity compared with fast white fibres, and this difference was associated with a difference in troponin T isoform composition. The tension‐pCa relation of slow red strip fibres was not different from that in ALD fibres. 5. The difference in Vmax between fast red strip fibres and fast white fibres was associated with different MHC compositions of these fibres. 6. The myofibrillar protein isoform composition of slow red strip fibres was identical to that of the slow tonic fibres of ALD muscle and these two groups of fibres had very similar contractile properties.
Disappearance and unexpected reappearance of progesterone in the circulation of the monkey: novel hormone kinetics.
Authors: Kowalski W B , Chatterton R T , Kazer R R , Severini T A
Published: December 2014

1. Intravenous injection of [3H]progesterone in non‐pregnant monkeys resulted in total disappearance of the labelled hormone from the circulation within 3 h. However, 0.5‐1.75 h after disappearance the hormone reappeared, reaching 20% (median, 5%) of the initial maximal concentration. 2. Reappearance of labelled hormone was accompanied by similar fluctuations in the levels of labelled metabolites, [3H]20 alpha‐dihydroprogesterone and [3H]17 alpha‐hydroxyprogesterone, which reached 61% (median, 14%) and 120% (median, 13%), respectively, of the initial maximal concentrations. 3. Chromatography was used to separate labelled progesterone and its metabolites. Efficiency of the procedure was determined separately in each sample and for each steroid. All data were corrected for percentage recovery. 4. Analytical equations were devised, based on the theory of compartmental systems with continuously distributed time lags, to describe the unexpected kinetics of progesterone levels. The coefficients of determination ranged from 86 to 99% (median, 96%) which indicates that the equations enabled reliable prediction of hormone levels in blood within the time range studied. 5. The unexpected reappearance of labelled progesterone cannot be explained by hormone secretion but only by a delayed release from tissue stores, since progesterone does not undergo enterohepatic recirculation. Thus, a previously undescribed mechanism affecting circulating progesterone levels, and perhaps those of other hormones, exists.
P2 purinoceptor‐activated inward currents in follicular oocytes of Xenopus laevis.
Authors: King B F , Wang S , Burnstock G
Published: December 2014

1. ATP, UTP and 2MeSATP (10 microM) evoked fast, transient (2‐20 s duration) and slow, sustained (60‐120 s duration) inward currents in 118 of 285 follicular Xenopus oocytes but failed to activate inward currents in defolliculated Xenopus oocytes (n = 136). 2. The potency order for nucleotides was (at 10 microM): UTP = 2MeSATP > 2ClATP = ATP > ADP > alpha, beta‐methylene‐ATP > adenosine > ITP, with beta, gamma‐methylene‐ATP, CTP and GTP inactive. This potency order is atypical for any single P2 purinoceptor subtype. 3. EC50 values (for fast and slow inward currents) were (microM): ATP, 5.6 +/‐ 2.1 and 6.7 +/‐ 3.0; UTP, 1.0 +/‐ 0.3 and 0.9 +/‐ 0.3; 2MeSATP, 0.6 +/‐ 0.2 and 9.2 +/‐ 7.4 (n = 3). Suramin inhibited fast (IC50: 65 +/‐ 20 microM; n = 7) and slow (IC50: 57 +/‐ 10 microM; n = 7) inward currents evoked by ATP. Suramin also inhibited slow inward currents produced by UTP but potentiated those caused by 2MeSATP. 4. Fast and slow inward currents evoked in response to ATP and UTP reversed at ‐12 +/‐ 3 mV (n = 3), a value close to Erev for Na(+)‐permeable ion channels present in oocytes. These same currents were reduced significantly when extracellular Na+ was substituted with choline and Erev changed to ‐30 +/‐ 5 mV (n = 3). Inward currents evoked by ATP and UTP were inhibited in a dose‐dependent manner by amiloride (10‐100 microM). 5. Fast and slow inward currents caused by 2MeSATP reversed at ‐29 +/‐ 3 mV (n = 3), a value close to Erev for Cl‐ channels in oocytes. Fast and slow responses to 2MeSATP were inhibited in a dose‐dependent manner by the Cl‐ channel blocker NPPB (10‐100 microM) and were only reduced slightly by amiloride (100 microM). 6. We conclude that two P2 purinoceptor subtypes are present on follicular Xenopus oocytes. 2MeSATP activates fast and slow Cl‐ currents (Erev = ‐30 mV) via a suramin‐insensitive P2Y purinoceptor, while UTP and ATP activate fast and slow Na(+)‐dependent currents (Erev = ‐10 mV) via a suramin‐sensitive P2U purinoceptor.
Activation of L‐type calcium channel in twitch skeletal muscle fibres of the frog.
Authors: Francini F , Bencini C , Squecco R
Published: December 2014

1. The activation of the L‐type calcium current (ICa) was studied in normally polarized (‐100 mV) cut skeletal muscle fibres of the frog with the double Vaseline‐gap voltage‐clamp technique. Both external and internal solutions were Ca2+ buffered. Solutions were made in order to minimize all but the Ca2+ current. 2. The voltage‐dependent components of the time course of activation were determined by two procedures: fast and slow components were evaluated by multiexponential fitting to current traces elicited by long voltage pulses (5 s) after removing inactivation; fast components were also determined by short voltage pulses having different duration (0.5‐70 ms). 3. The components of deactivation were evaluated after removing the charge‐movement current from the total tail current by the difference between two short (50 and 70 ms) voltage pulses to 10 mV, moving the same intramembrane charge. Two exponential components, fast and slow (time constants, 6 +/‐ 0.3 and 90 +/‐ 7 ms at ‐100 mV; n = 26), were found. 4. The time onset of ICa was evaluated either by multiexponential fitting to the ICa activation or by pulses of different duration to test the beginning of the ‘on’ and ‘off’ inequality. This was at about 2 ms, denoting that it was very early. 5. The time constant vs. voltage plots indicated the presence of four voltage‐dependent components in the activation pathway. Various kinetic models are discussed. Models with independent transitions, like a Hodgkin‐Huxley scheme, were excluded. Suitable models were a five‐state sequential and a four‐state cyclic with a branch scheme. The latter gave the best simulation of the data. 6. The steady‐state activation curve saturated at high potentials. It had a half‐voltage value of 1 +/‐ 0.2 mV and the opening probability was only 0.82 +/‐ 0.2 at 20 mV (n = 32). This result implies a larger number of functional calcium channels than was previously supposed and is in agreement with the number of dihydropyridine (DHP) receptors calculated for the tubular system.