bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibitory synaptic currents in stellate cells of rat cerebellar slices.
Authors: Llano I , Gerschenfeld H M
Published: December 2014

1. In thin cerebellar slices of rats aged 14‐21 days, voltage‐gated currents, synaptic currents and GABA responses were studied with the tight‐seal whole‐cell recording technique from stellate cells (8‐9 micrograms soma diameter) located in the outer two‐thirds of the molecular layer. 2. In symmetrical Cl‐ conditions, stellate cells voltage‐clamped at ‐60 mV showed spontaneous inhibitory postsynaptic currents (IPSCs). As were the GABAA responses of the same cells, the IPSCs were blocked by bicuculline. The frequency of occurrence of IPSCs ranged from 0.2 to 1.9 events per second (21 cells). The mean amplitude of the events ranged from 61 to 226 pA (mean +/‐ S.E.M.: 132 +/‐ 11; n = 21). 3. The temporal course of IPSCs was characterized by a rapid rise (mean +/‐ S.E.M. of the time to peak: 1.1 +/‐ 0.1 ms, n = 7) and a slow decay. The decay phase was described by a double exponential function with time constants of 8.7 +/‐ 0.6 ms, and 40.9 +/‐ 3.7 ms respectively (means +/‐ S.E.M.; n = 7). 4. A minor fraction (15 to 20%) of the spontaneous synaptic events recorded in control saline had a faster onset than that of the IPSCs and decayed with a rapid mono‐exponential decay (time constant of 1.0‐1.3 ms). These were excitatory postsynaptic currents (EPSCs) unaffected by bicuculline and blocked by the glutamate receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX). 5. Bath application of TTX (0.5‐1 microM), which blocked voltage‐gated Na+ currents in stellate cells, induced a variable decrease in the frequency of IPSCs (mean +/‐ S.E.M. of the frequency ratio in TTX over control: 0.47 +/‐ 0.09; n = 12). However, the toxin had no significant effect either on the mean amplitude or on the kinetics of the IPSCs. The mean amplitude of the miniature IPSCs was 141 +/‐ 13 pA (mean +/‐ S.E.M.; n = 22). 6. In TTX‐containing solutions, the frequency of the IPSCs was unaffected when Ca2+ currents were eliminated either by removal of extracellular Ca2+ and addition of EGTA, or by addition of Cd2+. Miniature IPSCs of 200‐300 pA were still observed. 7. In symmetrical Cl‐ conditions, local application of GABA to stellate cells induced an inward current and an increase in membrane noise. Responses to prolonged applications of GABA showed desensitization in both whole‐cell mode and somatic outside‐out patches. The chord conductance estimated from recording single GABA channel events in somatic outside‐out patches was 28 pS.(ABSTRACT TRUNCATED AT 400 WORDS)
Effects of aerosol‐applied capsaicin, histamine and prostaglandin E2 on airway sensory receptors of anaesthetized cats.
Authors: Mohammed S P , Higenbottam T W , Adcock J J
Published: December 2014

1. Capsaicin, prostaglandin E2 (PGE2) and histamine are potent stimuli for reflex coughing and bronchoconstriction in many species including man. We have studied the effects of solutions of capsaicin, PGE2 and histamine on airway sensory receptors when administered as inhaled aerosols to the lower respiratory tract in anaesthetized, paralysed and artificially ventilated cats. 2. Histamine, administered by aerosol (6 breaths of a 1 mg ml‐1 solution) and intravenously (10 micrograms kg‐1), caused an increase in the rate of discharge from rapidly adapting stretch receptors (RARs) and caused bronchoconstriction. 3. Six breaths of a capsaicin aerosol generated from solutions of 0.1 or 1 mg ml‐1 stimulated six out of nine RARs tested. Bronchoconstriction occurred with and without RAR stimulation. The diluent for the capsaicin aerosol had no significant effect on pulmonary mechanics or rate of RAR discharge. 4. Administration of increasing concentrations (0.001‐1 mg ml‐1) of PGE2 aerosol given in six breaths (at 6 min intervals) caused a dose‐dependent increase in the rate of discharge of eight RARs tested and caused bronchoconstriction. The diluent for the PGE2 aerosol had no effect on pulmonary mechanics or rate of RAR discharge. 5. Inhalation of aerosols of histamine (6 breaths of 1 mg ml‐1 solution) and capsaicin (3 breaths of 0.1 mg ml‐1 solution) stimulated all six lung C fibre endings studied (3 pulmonary and 3 bronchial). These aerosols of capsaicin and histamine also caused bronchoconstriction. 6. We conclude that solutions of capsaicin and PGE2, when delivered by aerosol to the airway epithelial surface, are not selective stimulants of C fibres. Both agents can stimulate RARs. Activation of some but not all RARs tested, by inhaled capsaicin, suggests that there are subpopulations of capsaicin‐sensitive and ‐insensitive receptors. Stimulation of airway RARs by a range of pharmacologically active agents released by airway inflammation may contribute to reflex coughing and bronchoconstriction in man.
Local inhibitory synaptic inputs to neurones of the paraventricular nucleus in slices of rat hypothalamus.
Authors: Tasker J G , Dudek F E
Published: December 2014

1. Intracellular recordings were obtained from neurones in the region of the paraventricular nucleus in slices of rat hypothalamus. Glutamate microdrops were applied to the surface of the slices at sites dorsal, lateral and ventral to the paraventricular nucleus to selectively activate local presynaptic neurones. The gamma‐aminobutyric acidA (GABAA)‐receptor antagonists picrotoxin or bicuculline were bath‐applied to block synaptic inhibition. 2. Glutamate microapplication caused a tonic depolarization and often repetitive action potentials in twenty of forty‐seven recorded cells. This was probably caused by the direct exposure of the dendrites of the recorded cells to the glutamate microdrops. 3. Glutamate microstimulation elicited inhibitory synaptic responses in nine of forty‐seven neurones tested. Glutamate microdrops caused discrete, hyperpolarizing postsynaptic potentials (PSPs) in four cells recorded with microelectrodes containing potassium acetate and evoked depolarizing PSPs in four cells recorded with KCl‐filled microelectrodes. Glutamate microapplication inhibited spontaneous spike firing in another cell recorded with a potassium acetate microelectrode. 4. Bath application of GABAA‐receptor antagonists completely blocked the hyperpolarizing PSPs elicited by glutamate microstimulation in three of three cells recorded with potassium acetate electrodes and the depolarizing PSPs in two of two cells recorded with KCl electrodes, indicating they were inhibitory PSPs caused by the release of GABA. Suppression of GABAA‐mediated synaptic inhibition did not reveal any glutamate‐evoked excitatory PSPs. 5. Recorded cells were identified as magnocellular, parvocellular or non‐paraventricular bursting neurones on the basis of their electrophysiological properties. Direct depolarization and local inhibitory synaptic responses were observed in all three cell types. 6. Several conclusions can be drawn from these data: (1) functional glutamate receptors are distributed throughout neuronal populations in the paraventricular region of the hypothalamus, confirming and extending previous observations; (2) local synaptic inputs to neurones in the paraventricular nucleus are primarily inhibitory, supplied by perinuclear GABAergic neurones; (3) both magnocellular and parvocellular subpopulations receive local inhibitory synaptic inputs. The possibility that these local GABAergic circuits mediate inhibitory inputs to paraventricular neurones from limbic structures is discussed.
Block of current through T‐type calcium channels by trivalent metal cations and nickel in neural rat and human cells.
Authors: Mlinar B , Enyeart J J
Published: December 2014

1. The effects of the trivalent cations yttrium (Y3+), lanthanum (La3+), cerium (Ce3+), neodymium (Nd3+), gadolinium (Gd3+), holmium (Ho3+), erbium (Er3+), ytterbium (Yb3+) and the divalent cation nickel (Ni2+) on the T‐type voltage gated calcium channel (VGCC) were characterized by the whole‐cell patch clamp technique using rat and human thyroid C cell lines. 2. All the metal cations (M3+) studied, blocked current through T‐type VGCC (IT) in a concentration‐dependent manner. Smaller trivalents were the best T‐channel antagonists and potency varied inversely with ionic radii for the larger M3+ ions. Estimation of half‐maximal blocking concentrations (IC50s) for IT carried by 10 mM Ca2+ resulted in the following potency sequence: Ho3+ (IC50 = 0.107 microM) approximately Y3+ (0.117) approximately Yb3+ (0.124) > or = Er3+ (0.153) > Gd3+ (0.267) > Nd3+ (0.429) > Ce3+ (0.728) > La3+ (1.015) >> Ni2+ (5.65). 3. Tail current measurements and conditioning protocols were used to study the influence of membrane voltage on the potency of these antagonists. Block of IT by Ni2+, Y3+, La3+ and the lanthanides was voltage independent in the range from ‐200 to +80 mV. In addition, the antagonists did not affect macroscopic inactivation and deactivation of T‐type VGCC. 4. Increasing the extracellular Ca2+ concentration reduced the potency of IT block by Ho3+, indicative of competitive antagonism between this blocker and the permeant ion for a binding site. 5. The results suggest that the mechanism of metal cation block of T‐type VGCC is occlusion of the channel pore by the antagonist binding to a Ca2+/M3+ binding site, located out of the membrane electric field. 6. Block of T‐type VGCC by Y3+, lanthanides and La3+ differ from the inhibition of high voltage‐activated VGCC block in several respects: smaller cations are more potent IT antagonists; block is voltage independent and the antagonists do not permeate T‐type channels. These differences suggest corresponding structural dissimilarities in the permeation pathways of low and high voltage‐activated Ca2+ channels.
Mechanisms of endothelin‐1‐induced pulmonary vasodilatation in neonatal pigs.
Authors: Pinheiro J M , Malik A B
Published: December 2014

1. We determined the contributions of three independent vasodilator mechanisms (cyclo‐oxygenase metabolites, nitric oxide and ATP‐sensitive potassium channels) in the mediation of pulmonary vasomotor effects of endothelin‐1 (ET‐1) in neonatal pigs. 2. Lungs of piglets (2.7 +/‐ 0.3 days old) were perfused at constant flow (60 ml min‐1) with recirculating Ringer‐albumin solution. We measured pulmonary artery pressure (Ppa) and the distribution of pulmonary vascular resistance using the double‐occlusion method. 3. ET‐1 (10(‐12)‐10(‐9) M) produced concentration‐dependent pulmonary vasodilatation. ET‐1 (10(‐9) M) decreased Ppa from 24.5 +/‐ 3.1 to 17.0 +/‐ 3.0 cmH2O with a nadir occurring at 1 min, followed by a slow return to baseline over 60 min (time for half‐recovery (t1/2R) of 17.2 min). The decrease in Ppa was the result of pulmonary precapillary vasodilatation. Endothelin‐3 (ET‐3) (10(‐12) and 10(‐11) M) also induced vasodilatation comparable to equimolar concentrations of ET‐1, whereas the selective ETB receptor agonist IRL 1620 at equimolar concentrations caused a more protracted vasodilatation response. 4. Neither the cyclo‐oxygenase inhibitor indomethacin (10(‐5) M) nor the KATP+ (ATP‐sensitive) potassium channel blocker glibenclamide (10(‐5) M) significantly altered the baseline Ppa; moreover, neither inhibitor affected the ET‐1‐induced vasodilatation, indicating the lack of involvement of cyclo‐oxygenase metabolites and KATP+ channel activity in the mediation of the pulmonary vasodilator response to ET‐1. 5. Addition of 10(‐5) M reduced haemoglobin, which antagonizes the action of nitric oxide (NO), increased Ppa over prehaemoglobin levels. Haemoglobin significantly decreased the duration (t1/2R, 3.8 +/‐ 0.7 min) of pulmonary vasodilatation to ET‐1, but did not abolish the initial phase of the response. L‐N‐Monomethylarginine, an inhibitor of NO synthesis, either alone or in combination with haemoglobin, similarly reduced the duration of ET‐1‐induced pulmonary vasodilatation. 6. The ETA receptor antagonist [Dpr1‐Asp15]‐ET‐1 (Dpr, diaminoproprionic acid) had no effect on pulmonary vasodilatation induced by ET‐1, ET‐3 or IRL 1620 (suc‐(Glu9,Ala11,15)‐ET‐1(8‐21)). This finding combined with the observed relative potencies of the peptides (IRL 1620 > ET‐1 = ET‐3) suggests that pulmonary vasodilatation was mediated by activation of the non‐selective ETB receptor. 7. The results indicate that the sustained ET‐1‐induced pulmonary vasodilatation in neonates is probably mediated via ETB receptor activation and that it is critically dependent on NO.
Mitogenic factors regulate ion channels in Schwann cells cultured from newborn rat sciatic nerve.
Authors: Wilson G F , Chiu S Y
Published: December 2014

1. Patch clamp studies were carried out in Schwann cells cultured from newborn rat sciatic nerve to determine the effects of mitogens on voltage‐gated currents without the confounding influences of axonal contact and myelin present in vivo. The relevance of the various Schwann cell currents to proliferation was assessed using assays of [3H]thymidine incorporation. 2. Treatment of cultured Schwann cells with known mitogens, namely axon fragments (AF), myelin fragments (MF), or glial growth factor in combination with forskolin (GGF+F), increased the magnitudes of delayed rectifying potassium (K+) and sodium (Na+) currents. 3. In both control and mitogen‐treated cells, the magnitude of net outward current paralleled clearly the magnitude of the cells' proliferative response. 4. The K+ channel‐blocking quaternary ammonium ions, tetrabutylammonium (TBuA), tetrapentylammonium (TPeA) and tetrahexylammonium (THeA), but not the Na+ channel blocker tetrodotoxin (TTX), reduced proliferation in a dose‐dependent fashion offering further evidence for a role for K+ channels in Schwann cell proliferation. 5. Voltage‐gated chloride (Cl‐) currents were observed in both control and mitogen‐treated cells. Addition of the Cl‐ channel blockers, 4‐acetamido‐4'‐isocyanatostilbene‐2,2'‐disulphonate (SITS) or 4,4'‐diisothiocyanatostilbene‐2,2'‐disulphonate (DIDS), to the culture media enhanced proliferation. 6. The possible intermediary role of the Schwann cell resting potential was explored in ion substitution experiments by increasing the K+ concentration of the media and by adding ouabain. Both manipulations inhibited Schwann cell mitosis. 7. Comparison of the expression of functional ion channels in vitro with that previously described for Schwann cells in vivo suggests a difference in the Schwann cell response to the membrane fragment mitogens and their intact counterparts in regard to the regulation of ion channels. MF up‐regulates the number of functional channels, whereas the elaboration of myelin (or a factor related to its presence) in vivo appears to down‐regulate channel expression, at the cell soma of myelinating Schwann cells. In addition, axonal contact may be required for normal expression of functional inwardly rectifying K+ channels.
Ultra‐deep blockade of Na+ channels by a quaternary ammonium ion: catalysis by a transition‐intermediate state?
Authors: Gingrich K J , Beardsley D , Yue D T
Published: December 2014

1. Individual Na+ channels from isolated guinea‐pig ventricular heart cells were studied using the patch‐clamp technique. To localize the selectivity region of the channels we investigated their blockade by a permanently charged quaternary ammonium ion (QX‐314, 2‐(triethylamino)‐N‐(2,6‐dimethylphenyl)acetamide, 0‐5 mM) that was applied to the cytoplasmic side of the channel. 2. Resolution of individual blocking events was enhanced by covalent removal of fast inactivation following brief internal exposure to the enzyme papain. The improved resolution reveals the existence of two distinct modalities of blockade: reduction of unitary current, and millisecond interruptions of current. 3. Both modes of internal block could be potentiated by lowering external Na+ concentration. This finding argues that the two corresponding sites of interaction are both located within the channel pore. 4. Analysis of the voltage dependence of block placed both binding sites deep within the pore, at 70% of the electric field from the cytoplasmic entrance. Combined with recent studies localizing block by external Cd2+, the present results argue that the selectivity region of Na+ channels is quite narrow (spanning about 10% of the electric field), and located near the external side of the channel. 5. The manner in which the two blocking processes interact, along with the physical proximity of their binding sites, leads us to propose that the block configuration responsible for the reduction in unitary current serves as a transition intermediate that catalyses formation of the discrete‐block complex.
Regulation of fluid secretion and intracellular messengers in isolated rat pancreatic ducts by acetylcholine.
Authors: Ashton N , Evans R L , Elliott A C , Green R , Argent B E
Published: December 2014

1. We have studied the effects of acetylcholine (ACh) on fluid secretion and intracellular messengers in interlobular ducts isolated from the rat pancreas and maintained in short‐term tissue culture. 2. Ductal fluid secretion was measured using micropuncture techniques. Intracellular free calcium ([Ca2+]i) and cyclic AMP concentrations were measured in single ducts using fura‐2 microspectrofluorimetry and radioimmunoassay techniques respectively. Changes in the levels of these intracellular messengers were correlated with fluid secretion. 3. ACh stimulated ductal fluid secretion. The dose required for a half‐maximal response was about 0.4 microM and maximal secretion was achieved with 10 microM ACh. These effects of ACh were blocked by atropine and by removal of extracellular Ca2+. 4. ACh was about four orders of magnitude less potent as an activator of ductal fluid transport than the hormone secretin; however, the maximal rates of fluid secretion evoked by these two agonists were similar. 5. ACh caused a dose‐dependent rise in duct cell [Ca2+]i, but had no effect on cyclic AMP. In contrast, secretin increased duct cell cyclic AMP, but had no effect on [Ca2+]i. 6. The [Ca2+]i response evoked by ACh resulted from both mobilization of intracellular Ca2+ stores and influx of Ca2+ from the extracellular space. 7. The Ca2+ ionophore, ionomycin, mimicked the effect of ACh on ductal [Ca2+]i and fluid secretion. 8. We conclude that ACh stimulates fluid secretion from rat pancreatic duct cells by activating a ‘Ca2+ pathway’ which is distinct from the well documented ‘cyclic AMP pathway’ utilized by secretin.
The modulatory effects of endothelin‐1, carbachol and isoprenaline upon Na(+)‐H+ exchange in dog cardiac Purkinje fibres.
Authors: Wu M L , Tseng Y Z
Published: December 2014

1. The modulatory effects of carbachol, endothelin‐1 and isoprenaline upon Na(+)‐H+ exchange were examined in dog cardiac Purkinje fibres. Intracellular pH (pHi) and intracellular sodium activity (aiNa) were recorded using pH and Na(+)‐selective microelectrodes. Acid extrusion via Na(+)‐H+ exchange was estimated from the pHi recovery rate (multiplied by intrinsic buffering power (beta i) and adding mean background acid load) in response to an internal acid load induced by the removal of 20 mM NH4Cl. All experiments in this work were performed in Hepes‐buffered solutions at 37 degrees C. 2. beta i was estimated at various values of pHi in the range of 7.4‐6.4 and was calculated from the fall of pHi induced by the addition and removal of NH4Cl. Experiments were performed when Na(+)‐H+ exchange was blocked. The values of beta i in this tissue were only slightly dependent on pHi in the range of 7.4‐6.4 with an empirical relationship: beta i = ‐4.69 pHi + 64.59. 3. Endothelin‐1 (10(‐8) M) alkalinized the resting pHi by approximately 0.1 pH unit and accelerated acid extrusion, by approximately 96%, at pHi approximately 6.9. A reduction of background acid loading within the cell cannot account for the augmentation of pHi recovery, since the rate of acid extrusion was not changed either at resting pHi or at internal acidification in Na(+)‐free solution (a measure of background loading) by the addition of endothelin‐1. The protein kinase C inhibitors staurosporin (10(‐6) M) and 1‐(5‐isoquinolinylsulphonyl)‐2‐methyl‐piperazine (H‐7, 50 microM) could not block the effect of endothelin‐1 on the antiporter. 4. At pHi approximately 6.8, carbachol (7.5 x 10(‐4) M) accelerated pHi recovery by approximately 68% and alkalinized the resting pHi by approximately 0.1 pH unit. This stimulatory effect of carbachol was completely blocked by pretreatment with atropine (10(‐4) M) and staurosporine 10(‐6) M. The background acid load was not reduced by adding carbachol, since the acid extrusion during pHi recovery or at the resting state was not affected by the addition of carbachol to a sodium‐free solution. 5. Isoprenaline (10(‐6) M) slowed pHi recovery by approximately 45% measured at pHi 6.9 with no change in resting pHi. A rise in background acid loading could not account for the reduction of acid extrusion. Pretreated with atenolol (10(‐6) M), a beta 1‐selective antagonist, completely blocked the effect of isoprenaline.(ABSTRACT TRUNCATED AT 400 WORDS)
Analysis of the propagation of disinhibition‐induced after‐discharges along the guinea‐pig hippocampal slice in vitro.
Authors: Traub R D , Jefferys J G , Miles R
Published: December 2014

1. A model has been proposed of picrotoxin‐induced hippocampal in vitro after‐discharges; it depends critically upon alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) and N‐methyl‐D‐aspartate (NMDA) receptors in the recurrent excitatory connections between pyramidal neurones, and upon the ability of pyramidal neurones to generate bursts at about 10 Hz when their dendrites are sufficiently depolarized. 2. We study here the question of whether this model can account for spatial‐‐as well as temporal‐‐aspects of after‐discharges in guinea‐pig hippocampal slices. For example, can the model explain the propagation along a transverse slice of the initial burst and the secondary bursts at about the same velocity, approximately 0.10‐0.20 m s‐1? Under what conditions might the secondary bursts exhibit a different spatial pattern to the initial burst, as we now show can occur in longitudinal slices? To examine these questions, we increased the number of cells in our model from 100 to 8000 (in a 20 x 400 array), arranging the excitatory synaptic connections in a spatially restricted fashion, with an average extent of 1.0 mm (as suggested experimentally). 3. Our model suggests that both AMPA and NMDA receptors contribute to the propagation pattern and velocity of the initial and the secondary bursts in an after‐discharge. 4. When unitary AMPA and NMDA conductances are in the range where the primary burst lasts for 100‐200 ms, and there are three or four secondary bursts, then both primary and secondary bursts propagate near to the experimentally observed velocity for transverse slices. In the model, however, secondary bursts propagate at somewhat slower velocities than the initial burst. 5. The mechanisms of propagation are different for the initial and for the secondary bursts: propagation of the primary burst depends upon the initiation of electrogenesis in ‘resting’ dendrites by AMPA and NMDA inputs that are rapidly increasing in time. Propagation of secondary bursts depends upon the timing of calcium spikes in depolarized dendrites with slowly varying NMDA inputs; the timing of calcium spikes can be influenced by a ‘wave’ of AMPA input, but calcium spikes‐‐we predict‐‐should occur even without the AMPA input, once the after‐discharge has been initiated. The blockade of firing in an intermediate region of the disinhibited slice is predicted to have different effects on the primary burst and on secondary bursts distal to the region of blockade.(ABSTRACT TRUNCATED AT 400 WORDS)
Potassium currents operated by thyrotrophin‐releasing hormone in dissociated CA1 pyramidal neurones of rat hippocampus.
Authors: Ebihara S , Akaike N
Published: December 2014

1. Membrane currents activated by thyrotrophin‐releasing hormone (TRH) were investigated in the dissociated rat hippocampal CA1 pyramidal neurone using the nystatin perforated patch recording configuration. 2. Under current‐clamp condition, TRH caused a transient hyperpolarization accompanied by a decrease of firing activity and a successive long‐lasting depolarization. The latter induced a blockade of firing. 3. When neurones were held at a holding potential (VH) of ‐40 mV under voltage clamp, TRH elicited a transient outward current with an increase in the membrane conductance, which was followed by a sustained inward current with a decrease in membrane conductance. The inactive TRH metabolite, TRH free acid, did not induce any currents. 4. The reversal potential of TRH‐induced outward current (ETRH) was close to the K+ equilibrium potential (EK). The change in ETRH for a 10‐fold change in extracellular K+ concentration was 56.4 mV, indicating that the membrane behaves like a K+ electrode in the presence of TRH. On the other hand, the TRH‐induced inward current was due to suppression of a slow inward current relaxation during hyperpolarizing voltage commands to ‐50 mV from a VH of ‐40 mV, indicating the suppression of the voltage‐ and time‐dependent component of the K+ current (M‐current). 5. The TRH‐induced outward current (ITRH) increased in a concentration‐dependent manner over the concentration range 10(‐8)‐10(‐6) M. The half‐maximum concentration was 7.4 x 10(‐8) M and the Hill coefficient was 1.5. 6. The TRH‐induced outward current (ITRH) was antagonized by K+ channel blockers such as tetraethylammonium (TEA), 4‐aminopyridine (4‐AP) and Ba2+ in a concentration‐dependent manner. ITRH was insensitive to both apamin and iberiotoxin. 7. The first application of TRH to neurones perfused with Ca(2+)‐free external solution containing 2 mM EGTA could induce ITRH but the TRH response diminished dramatically with successive applications. Intracellular perfusion with a Ca2+ chelator, 1,2‐bis(O‐aminophenoxy)ethane‐N,N,N',N'‐tetraacetic acid (BAPTA), also diminished the TRH response. 8. The depletion of Ca2+ from the intracellular Ca2+ store by thapsigargin blocked the TRH response without affecting the caffeine response. Pretreatment with Li+ significantly enhanced ITRH, suggesting that ITRH is involved in the elevation of intracellular free Ca2+ released from the inositol 1,4,5‐trisphosphate (IP3)‐sensitive Ca2+ store site but not from the caffeine‐sensitive one. 9. Staurosporine, a protein kinase C (PKC) inhibitor, suppressed ITRH in a concentration‐dependent manner (the half‐maximum inhibitory concentration (IC50), was 2.45 x 10(‐8) M).(ABSTRACT TRUNCATED AT 400 WORDS)
Charge conservation in intact frog skeletal muscle fibres in gluconate‐containing solutions.
Authors: Huang C L
Published: December 2014

1. The conservation of intramembrane charge was investigated in intact voltage‐clamped frog skeletal muscle fibres under conditions that minimized time‐dependent ionic currents and so facilitated precise determination of capacitative charge. 2. Prolonged (q gamma) transients were demonstrated in 3,4‐diaminopyridine and tetraethyl‐ammonium gluconate‐containing low [Ca2+] solutions in response to 125 ms pulses that explored the voltage range ‐90 to ‐20 mV. The tetracaine‐sensitive, q gamma, component then accounted for a significant proportion (over 50%) of available charge. 3. Both delayed ‘on’ q gamma currents and ‘off’ current tails decayed to steady direct current (DC) baselines without significant residual ionic current slopes in the chosen extracellular solutions. This suggested that the current transients represented capacitative decays. It was also compatible with the precise determination of effective charge by integration. 4. The advent of ‘on’ q gamma current was accompanied by increased ‘off’ charge. Thus, charge was conserved through all ‘on’ and ‘off’ steps and through test voltages that extended from the threshold appearance of q gamma as a slow transient to its full merger with the earlier q beta decay at stronger depolarizations. 5. Charge conservation persisted through a wide range of ‘on’ pulse durations between 60 and 370 ms and was therefore independent of the interval following the q gamma decay. 6. The quantity of q gamma charge remained a monotonic single‐valued function of test voltage, whether this potential was reached directly from the ‐90 mV holding potential or following a prepulse to ‐10 mV. 7. These findings suggest that the q gamma charge movement represents the electrical signature of an intramembrane entity whose transitions are primarily driven by, and therefore conserved with, the steady‐state potential.
Stretch reflex responses in the human elbow joint during a voluntary movement.
Authors: Bennett D J
Published: December 2014

1. The responsiveness of the stretch reflex is modulated during human voluntary limb movements. The influence of this modulation on the limb mechanical properties (stiffness) was investigated. 2. Subjects were taught to replicate accurately a rapid (4.0 rad s‐1) targeted elbow flexion movement of 1 rad. From the onset of 12% of the trials a sinusoidal position disturbance (0.05 rad) was superimposed on the normal (trained) movement trajectory. The net joint torque (muscle torque) resisting these stretches was computed from measurements of applied torque, acceleration and limb inertia. Electromyographic (EMG) responses in the triceps brachii (TB), brachialis (Br) and biceps brachii (BB) were monitored. 3. The EMG responses to sinusoidal stretches applied early in the movement were less than those responses to perturbations applied when the arm neared the target (especially in the antagonist muscle TB). These EMG responses caused fluctuations in the resistance to the perturbation (stiffness), as described below. 4. When the perturbation frequency was low (< 4 Hz) the resistance of the elbow muscles to the stretch increased as the arm approached the target (48% increase). In contrast, when the stretch frequency was 7 Hz the resistance decreased by 63%. This decrease can be explained by the increased reflex response, since at 7 Hz the reflex response is probably timed so that it assists, rather than resists, the stretching as a result of loop delays. This reflex timing was confirmed by observing that, after abruptly stopping the sinusoidal stretch, the reflex response persisted for 100 ms and was indeed in a direction that would have reduced the resistance, had the perturbation continued. 5. The time course of the net muscle stiffness was estimated for frequencies ranging from 4 to 8 Hz and for each 40 ms interval a Nyquist plot was constructed, forming a C‐shaped curve as frequency was varied. The size of this curve gave a measure of the stiffness resulting from reflex activity. When the arm neared the target this reflexive stiffness reached a maximum, and was probably comparable in size to the intrinsic (non‐reflexive) muscle stiffness. Also, in four of the five subjects the viscous component of stiffness at 7 Hz dropped significantly below zero when the arm neared the target, again indicating that at this frequency the reflex was large and acted inappropriately.(ABSTRACT TRUNCATED AT 400 WORDS)
Activation properties of chemically skinned fibres from human isolated bronchial smooth muscle.
Authors: Savineau J P , Marthan R
Published: December 2014

1. The contractile activation properties of human isolated bronchial smooth muscle were investigated using chemically (beta‐escin) skinned strips. 2. Concentration‐dependent contractions were induced by free ion concentrations of Ca2+ (0.5‐3 microM), Sr2+ (2‐200 microM) and Ba2+ (50‐1000 microM). The resulting ‐log[cation]‐tension relationships were fitted by sigmoidal curves with EC50 values (cation concentration required to produce half‐maximal tension) and co‐operativity factors (Hill coefficient, nH) of, respectively, 0.25 microM and 3.4 for Ca2+, 12 microM and 2.64 for Sr2+ and 100 microM and 1.73 for Ba2+. Maximal responses to Sr2+ and Ba2+ were 125.5 +/‐ 15.4 and 96 +/‐ 8.1% (n = 5) respectively of the maximum tension induced by Ca2+. 3. Trifluoperazine (5‐100 microM), cyclic AMP (50‐300 microM) and cyclic GMP (50‐100 microM) each antagonized Ca2+ in a concentration‐dependent manner. On the other hand, okadaic acid (OA, 0.2‐1 microM) potentiated Ca2+ and increased the maximum response to Ca2+ (+25 +/‐ 5.4%, n = 5, for 1 microM OA). 4. This study has demonstrated the high Ca2+ sensitivity of the activation mechanism of human isolated bronchial smooth muscle. It also suggests that control of the contractile machinery in the human bronchus involves processes of phosphorylation and dephosphorylation. The beta‐escin‐treated human bronchus may be a useful model for investigating the cellular basis of some pathophysiological processes such as bronchial hyper‐responsiveness.
P‐type calcium channels in rat neocortical neurones.
Authors: Brown A M , Sayer R J , Schwindt P C , Crill W E
Published: December 2014

1. The high threshold, voltage‐activated (HVA) calcium current was recorded from acutely isolated rat neocortical pyramidal neurones using the whole‐cell patch technique to examine the effect of agents that block P‐type calcium channels and to compare their effects to those of omega‐conotoxin GVIA (omega‐CgTX) and nifedipine. 2. When applied at a saturating concentration (100 nM) the peptide toxins omega‐Aga‐IVA and synthetic omega‐Aga‐IVA blocked 31.5 and 33.0% of the HVA current respectively. 3. A saturating concentration of nifedipine (10 microM) inhibited 48.2% of the omega‐Aga‐IVA‐sensitive current, whereas saturating concentrations of both omega‐Aga‐IVA (100 nM) and omega‐CgTX (10 microM) blocked separate specific components of the HVA current. 4. Partially purified funnel web spider toxin (FTX) at a dilution of 1:1000 blocked 81.4% of the HVA current and occluded the inhibitory effect of omega‐Aga‐IVA. Synthetic FTX 3.3 arginine polyamine (sFTX) at a concentration of 1 mM blocked 61.2% of the HVA current rapidly and reversibly. The effects of sFTX were partially occluded by pre‐application of omega‐Aga‐IVA. We conclude that neither FTX nor sFTX blocked a specific component of the HVA current in these cells. 5. In view of the specificity of omega‐Aga‐IVA for P‐type calcium channels in other preparations and for a specific component of the HVA current in dissociated neocortical neurones we conclude that about 30% of the HVA current in these neurones flow through P‐channels.
Contribution of nitric oxide to the endothelium‐dependent hyperpolarization in rat aorta.
Authors: Vanheel B , Van de Voorde J , Leusen I
Published: December 2014

1. The effect of endogenous and exogenous nitric oxide on the membrane potential (Em) of smooth muscle cells of the thoracic aorta of rats was investigated. 2. In tissues with intact endothelium, application of ACh or carbachol generated a change of the membrane potential consisting of an initial hyperpolarization by 10‐12 mV, followed by a partial recovery toward a level which was at 10 min still 6‐8 mV more negative than in control conditions. 3. Application of NG‐nitro‐L‐arginine methylester (L‐NAME), an inhibitor of endogenous NO production, had no significant effect on the resting membrane potential. The initial peak endothelium‐dependent hyperpolarization elicited by ACh or carbachol was not significantly diminished. However, the recovery was more accentuated. Similarly, NG‐monomethyl‐L‐arginine (L‐NMMA) significantly diminished the second component of the endothelium‐dependent hyperpolarization without affecting the magnitude of the first transient peak Em change. 4. Nitroglycerin produced a small sustained hyperpolarization of 1‐2 mV, and the NO donor SIN‐1, the active metabolite of molsidomine, similarly increased Em by about 1 mV. Infusion of high doses of acidified NaNO2 solution caused a hyperpolarization smaller than that evoked by ACh or carbachol. 5. 8‐Bromo‐cyclic GMP caused little change of membrane potential. In the presence of 8‐Br‐cGMP, ACh evoked a membrane electrical response similar to that observed in the absence of the nucleotide. 6. It is concluded that, in the rat aorta, the initial peak endothelium‐dependent hyperpolarization observed under the influence of ACh or carbachol is not directly related to the synthesis of NO.(ABSTRACT TRUNCATED AT 250 WORDS)
Capsaicin‐sensitive stretch responses in ferret trachealis muscle.
Authors: Coburn R F , Mitchell H , Dey R D , Alkon J
Published: December 2014

1. Stretch‐induced electrical and mechanical responses in segments of ferret trachealis muscle were studied. Stretches and post‐stretch length changes were quantified by measuring distances between two marker spheres placed on the muscle surface. Electrical responses were determined by measuring membrane potential in the muscle cell syncytium. 2. Smooth muscle mechanical and electrical responses to the stretch manoeuvre were characterized by an initial shortening and depolarization phase and a reversal‐repolarization phase. Both phases were resistant to atropine and tetrodotoxin. During the initial phase, the membrane depolarized to potentials as low as ‐20 mV. For stretches to 1.0 Lmax, from a holding length of 0.75 Lmax, 50% repolarization occurred at 6.8 +/‐ 0.4 min post‐stretch; 50% reversal of shortening of the stretched segment occurred at 6.9 +/‐ 0.8 min post‐stretch. 3. Depolarizing currents generated within muscle cells in the stretched segment spread into cells in non‐stretched muscle. Space constants in the transverse and longitudinal directions averaged 480 +/‐ 46 and 146 +/‐ 50 microns, respectively. 4. During infusion of capsaicin (10 microM), muscle cells depolarized by 5.5 +/‐ 2.3 mV. Maximal depolarization was achieved after 15‐20 min. After inhibition of neutral enkephalinase, capsaicin‐evoked depolarization occurred more rapidly. Muscles depolarized by 11.2 +/‐ 2.1 mV after about 10 min of capsaicin and then slowly repolarized during continued treatment. When muscle segments were stretched during administration of capsaicin, the initial phase was similar to that observed before capsaicin, but the reversal‐repolarization phase was prolonged. Following wash exposure to capsaicin, maximal stretch‐induced depolarization was unchanged, but the time for 50% repolarization (t50‐repolarization) decreased from the pre‐capsaicin value of 8.4 +/‐ 1.3 to 4.1 +/‐ 0.5 min. The t50‐reversal of stretch‐evoked muscle shortening decreased to 54% of control values. 5. Short exposures (< 2 min) to substance P (SP, 1‐7.5 microM) depolarized smooth muscle cells. Maximal depolarization was delayed, and occurred after [SP] had decreased to < 10 nM. Repolarization was delayed as long as 6 min following wash‐out of SP. Stretches performed when SP‐induced depolarization had nearly reversed showed no changes in the initial mechanical or electrical responses, but t50‐repolarization increased to 162% of control values. 6. Immunochemical studies showed networks of neurones which react with SP antibodies. 7. These findings suggest that stretch induces SP release from capsaicin‐sensitive C fibres, and that released SP affects smooth muscle ionic mechanisms which control and delay the reversal of stretch‐induced membrane depolarization and shortening.
pH dependence of inositol 1,4,5‐trisphosphate‐induced Ca2+ release in permeabilized smooth muscle cells of the guinea‐pig.
Authors: Tsukioka M , Iino M , Endo M
Published: December 2014

1. The dependence on pH of inositol 1,4,5‐trisphosphate (IP3)‐induced Ca2+ release was studied in saponin‐skinned smooth muscle cells from guinea‐pig portal vein, using the indicator fura‐2 to monitor Ca2+ release. 2. Increasing pH between 6.7 and 7.3 enhanced the rate of IP3‐induced Ca2+ release at all the Ca2+ concentrations above 30 nM without changing the bell‐shaped dependence of the Ca2+ release rate on Ca2+ concentration with a peak near 300 nM. 3. The ascending limb of the biphasic Ca2+ dependence was shifted slightly toward the lower Ca2+ concentration at pH 7.3, suggesting an increase in the Ca2+ sensitivity of IP3‐induced Ca2+ release at the higher pH. 4. With the elevation in pH from 6.7 to 7.3 at 100 nM Ca2+, about 7‐fold higher IP3 concentration was required to release half of the Ca2+ in the store within 15 s. This pH‐dependent change in the IP3 sensitivity was smaller at 1 microM Ca2+ and was indiscernible in the absence of Ca2+. 5. These results suggest that H+ may inhibit binding of IP3 and Ca2+ to the modulator sites of the Ca2+ release mechanism. However, these effects on the binding sites may not fully explain the complex effect of pH, and there may be pH‐dependent step(s) involved in the gating mechanism of IP3 channels. The present study demonstrates the importance of pH as a modulator of IP3‐induced Ca2+ release.
Calcium channels responsible for potassium‐induced transmitter release at rat cerebellar synapses.
Authors: Momiyama A , Takahashi T
Published: December 2014

The effects of calcium channel blockers on potassium‐induced transmitter release were studied in thin slices of cerebellum from neonatal rats using whole‐cell patch clamp methods. Miniature inhibitory postsynaptic currents (mIPSCs) mediated by gamma‐aminobutyric acid (GABA) were recorded from deep cerebellar nuclear neurones in the presence of tetrodotoxin. The frequency of mIPSCs was reproducibly increased by a brief application of high‐potassium solution. In the presence of the L‐type Ca2+ channel blocker nicardipine (10 microM), the potassium‐induced increase in mIPSC frequency was suppressed by 49%. Neither the mean amplitude nor the time course of mIPSCs was affected by the blocker. The N‐type Ca2+ channel blocker omega‐conotoxin GVIA (omega‐CgTX, 3 microM) had no effect on the frequency of potassium‐induced mIPSCs. The P‐type Ca2+ channel blocker omega‐Aga‐IVA (200 nM) suppressed the potassium‐induced increase in mIPSC frequency by 83% without affecting the mean amplitude or time course of mIPSCs. Comparing these data with previous studies of neurally evoked transmission, it is concluded that the Ca2+ channel subtypes responsible for potassium‐induced transmitter release may be different from those mediating fast synaptic transmission.
Ca(2+)‐dependent negative control mechanism for Ca(2+)‐induced Ca2+ release in crayfish muscle.
Authors: Györke S , Palade P
Published: December 2014

The mechanism of termination of Ca(2+)‐induced Ca2+ release (CICR) from the sarcoplasmic reticulum has been investigated in voltage clamped cut crayfish muscle fibres loaded with rhod‐2. During depolarizing steps evoking calcium current (ICa), Ca2+ release was first activated. Then the release rapidly (tau approximately 6 ms) declined, as evidenced by the rate of change of the intracellular fluorescence signal representing a Ca2+ transient. The rapid termination of release was not accounted for by inactivation of the trigger ICa or depletion of Ca2+ from the SR, since the rate at which release declined was constant under conditions where the rate of ICa inactivation and the amount of Ca2+ released varied widely. Pre‐elevations of [Ca2+]i with prepulses or photolysis of caged Ca2+ caused depression of Ca2+ release during a subsequent test pulse. When the rate of ICa onset was varied by applying voltage ramps with different slopes, currents with fast onset elicited larger Ca2+ release than calcium currents with slower onset, even though the amplitude of the currents was the same. These results suggest that a Ca(2+)‐dependent negative control mechanism exists which mediates the termination of CICR independently of the duration of the trigger ICa and before significant depletion of Ca2+ in the SR occurs.
Stretch reflexes in human masseter.
Authors: Poliakov A V , Miles T S
Published: December 2014

The reflex response to stretch in most contracting human muscles includes both a short‐latency, probably monosynaptic, excitatory component, and a longer‐latency, polysynaptic excitation. However, it has been claimed that stretch of the jaw‐closing muscles evokes only the short‐latency response in masseter. This question was re‐examined, using controlled stretches of varied rates and durations. Very brief, rapid stretches analogous to the stimuli used to investigate the ‘jaw‐jerk’ reflex in earlier studies evoked a prominent excitatory peak in the electromyogram at monosynaptic latency excitation, but little or no longer‐latency excitation. This response could be produced even by stimuli that were barely detectable by the subject. However, this prominent electrical response did not produce a measurable increase in biting force. In contrast, slower stretches evoked both a short‐ and a longer‐latency excitatory response in the surface electromyogram, as in most limb muscles. It is shown that the absence of a long‐latency excitatory response in earlier studies can be explained by the powerful reflex disfacilitation of the motoneurones that occurred at the end of the brief stretches used. Depending on the duration of the stretch, this disfacilitation is often sufficient to mask or abolish the long‐latency reflex. The reflex response to stretches was not markedly affected by blocking the activation of mechanoreceptors around the teeth with local anaesthetic, indicating that receptors around the teeth cannot be playing more than a minor role in the response. The stretch‐induced increase in force became greater as the velocity of the stretch decreased.
Effect of carotid denervation on plasma vasopressin levels during acute hypoxia in the late‐gestation sheep fetus.
Authors: Giussani D A , McGarrigle H H , Spencer J A , Moore P J , Bennet L , Hanson M A
Published: December 2014

1. We measured plasma concentrations of arginine vasopressin (AVP), arterial, venous and amniotic pressures, and carotid and femoral blood flows in fifteen chronically instrumented fetal sheep at 119‐125 days of gestation. In eight of the fetuses the carotid sinus nerves were cut (denervated fetuses); the other seven remained intact and served as controls (intact fetuses). 2. In the intact fetuses during hypoxia there was an increase in plasma [AVP] and in perfusion (arterial‐venous) pressure, a transient bradycardia, and an increase in carotid and a decrease in femoral blood flow. Whilst femoral vascular resistance (perfusion pressure/femoral blood flow) increased, there were no changes in carotid vascular resistance during hypoxia. 3. In the denervated fetuses no significant bradycardia, fall in femoral blood flow or increase in femoral vascular resistance was present soon after the onset of hypoxia but plasma AVP increased to similar concentrations to those observed in intact fetuses during hypoxia. 4. We conclude that carotid denervation does not affect plasma [AVP] during hypoxia in fetal sheep. This suggests that (1) AVP release during hypoxia is not mediated by a carotid chemoreflex and (2) AVP does not play an important role in these initial fetal cardiovascular responses. Furthermore, we previously reported that intact fetuses survive acute hypoxia better than denervated fetuses following phentolamine treatment, and we believe this to be due to the action of a non‐alpha‐adrenergic vasoconstrictor released in part via a carotid chemoreflex. The present results suggest that this vasoconstrictor is not AVP.