bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium released by photolysis of DM‐nitrophen stimulates transmitter release at squid giant synapse.
Authors: Delaney K R , Zucker R S
Published: December 2014

1. Transmitter release at the squid giant synapse was stimulated by photolytic release of Ca2+ from the ‘caged’ Ca2+ compound DM‐nitrophen (Kaplan & Ellis‐Davies, 1988) inserted into presynaptic terminals. 2. Competing binding reactions cause the amount of Ca2+ released by DM‐nitrophen photolysis to depend on the concentrations of DM‐nitrophen, total Ca2+, Mg+, ATP and native cytoplasmic Ca2+ buffer. Measurements of presynaptic [Ca2+] changes by co‐injection of the fluorescent indicator dye Fura‐2 show that DM‐nitrophen photolysis causes a transient rise in Ca2+ followed by decay within about 150 ms to an increased steady‐state level. 3. Rapid photolysis of Ca2(+)‐loaded nitrophen within the presynaptic terminal was followed in less than a millisecond by depolarization of the postsynaptic membrane. As with action potential‐evoked excitatory postsynaptic potentials (EPSPs), the light‐evoked response was partially and reversibly blocked by 1‐3 mM‐kainic acid which desensitizes postsynaptic glutamate receptors. 4. Release was similar in magnitude and rate to normal action potential‐mediated EPSPs. 5. The release of transmitter by photolysis of Ca2(+)‐loaded DM‐nitrophen was not affected by removal of Ca2+ from the saline or addition of tetrodotoxin. Photolysis of DM‐nitrophen injected into presynaptic terminals without added Ca2+ did not stimulate release of transmitter nor did it interfere with normal action potential‐mediated release. 6. Stimulation of presynaptic action potentials in Ca2(+)‐free saline during the light‐evoked response did not elicit increased release of transmitter if the ganglion was bathed in Ca2(+)‐free saline, i.e. in the absence of Ca2+ influx. Increasing the intensity of the light or stimulating presynaptic action potentials in Ca2(+)‐containing saline increased the release of transmitter. Therefore the failure of presynaptic voltage change to increase transmitter release resulting from release of caged Ca2+ was not due to saturation or inhibition of the release mechanism by light‐released Ca2+. 7. Decreasing the temperature of the preparation increased the delay to onset of the light‐evoked response and reduced its amplitude and rate of rise to an extent similar to that observed for action potential‐evoked EPSPs.
The role of adenosine in exercise hyperaemia of the gracilis muscle in anaesthetized cats.
Authors: Poucher S M , Nowell C G , Collis M G
Published: December 2014

1. A number of metabolites have been proposed to control the vascular tone of skeletal muscle during exercise. The present study was designed to investigate the role of adenosine in this response by determining the effect of the adenosine receptor antagonist 8‐phenyltheophylline. 2. The gracilis muscle of anaesthetized cats was exposed and made to contract by stimulating the obturator nerve (at 1 Hz, 5 V, 0.1 ms) for 20 min. Gracilis muscle blood flow and tension were measured during exercise and for 20 min following exercise. Initially this was performed in each animal during the infusion of a vehicle solution (50% polyethylene glycol 400, 50% 0.1 M‐NaOH, 0.1 ml min‐1 I.V.). Exercise was then repeated during infusion of either further vehicle (group I), 8‐phenyltheophylline (group II) or 3‐propylxanthine (group III), both at 2.7 x 10(7) mol min‐1 kg‐1. 3. In group 1 (n = 4) gracilis muscle blood flow during the first exercise period increased by 47.5 +/‐ 11.3 ml min‐1 (110 g)‐1 and gracilis muscle tension by 8.6 +/‐ 1.3 kg (100 g muscle mass)‐1 at 20 min of exercise. These responses were not significantly different when repeated. 4. In group II (n = 5), blood flow increased by 46.9 +/‐ 9.9 ml min‐1 (100 g)‐1 and tension by 6.5 +/‐ 0.7 kg (100 g muscle mass)‐1 during vehicle infusion. Infusion of 8‐phenyltheophylline at a rate which abolished the vasodilatation response to 2‐chloroadenosine, significantly reduced the muscle blood flow increase to 19.8 +/‐ 2.7 ml min‐1 (100 g muscle mass)‐1 (P less than 0.05) but the tension response was unaffected (increased by 7.0 +/‐ 0.8 kg (100 g muscle mass)‐1). 8‐Phenyltheophylline did not affect gracilis muscle blood flow or tension at rest. 5. Administration of 3‐propylxanthine, which did not modify the vasodilatation response to 2‐chloroadenosine, failed to alter the vascular responses to muscle contraction. 6. These results suggest that activation of adenosine receptors can contribute to up to 40% of the vasodilatation observed during isometric twitch contraction of the gracilis muscle of cats.
Rapid co‐transport of sodium and chloride ions in giant salivary gland cells of the leech Haementeria ghilianii.
Authors: Wuttke W A , Berry M S
Published: December 2014

1. Double‐barrelled Cl(‐)‐selective microelectrodes were used to measure the apparent intracellular Cl‐ activity (aiCl) and membrane potential (Em) of leech salivary gland cells. In standard physiological solution buffered with HEPES (10 mM), intracellular Cl‐ activity (corrected for interference) was 38 +/‐ 8 mM (n = 11) compared to a value of 12.8 mM expected for passive Cl‐ distribution. The mean Em was ‐49.4 +/‐ 8.2 mV (n = 21) which was about 27 mV negative to the Cl‐ equilibrium potential. 2. Removal of external Cl‐ led to a slow fall in aiCl until a steady‐state level of 4‐11 mM was reached in 30‐60 min. Recovery of aiCl on readdition of external Cl‐ took only 2‐3 min. The uptake followed an exponential time course having a single rate constant of 1.73 +/‐ 0.1 min‐1 (n = 5) whereas the loss appeared to occur in two phases. Changes in external Cl‐ produced immediate changes in Em which were the opposite of those expected for a high Cl‐ permeability, i.e. Cl‐ removal produced an immediate hyperpolarization (3‐18 mV) and readdition of Cl‐ produced a transient depolarization (5‐22 mV). 3. The intracellular accumulation of Cl‐ was dependent on the external Cl‐ activity. Even when the external Cl‐ concentration was reduced to 3%, the cells accumulated Cl‐ against an electrochemical gradient. 4. Cl‐ accumulation was temperature sensitive (Q10 approximately 2). 5. On removal of external Na+, aiCl fell to a level which was close to that expected for passive distribution. The active reaccumulation of Cl‐, after intracellular Cl‐ depletion, was abolished in the absence of external Na+; aiCl slowly increased to its passive level. Steady‐state aiCl or its recovery by Cl(‐)‐depleted cells was not affected by the absence of K+ in the bathing solution. 6. The reaccumulation of Cl‐ was not affected by furosemide (1‐5 mM), bumetanide (10(‐4) M), amiloride (10(‐3) M) or 4‐acetamido‐4'‐isothiocyanostilbene‐2,2'‐disulphonic acid (SITS, 10(‐4) M). 7. Removal of external Cl‐ caused a fall in intracellular Na+ activity (aiNa, measured with Na(+)‐selective microelectrodes) from 15.9 +/‐ 6.8 mM (n = 9) to 2.5 +/‐ 1.3 mM (n = 3). When external Cl‐ was readded, aiNa rose to 46.5 +/‐ 6.6 mM (n = 3) before slowly recovering towards its original value. The maximal change in aiNa was 41.7 +/‐ 4.5 mM (n = 3) and the rate constant for Na+ uptake was 1.8 +/‐ 0.4 min‐1 (n = 3).(ABSTRACT TRUNCATED AT 400 WORDS)
Muscarinic modulation of calcium current in neurones from the interatrial septum of bull‐frog heart.
Authors: Tse A , Clark R B , Giles W R
Published: December 2014

1. The effects of activation of muscarinic receptors on the voltage‐dependent calcium current, ICa, in parasympathetic neurones were examined. 2. Neurones were enzymatically isolated from the interatrial septum of bull‐frog (Rana catesbeiana) heart, and were maintained in short‐term (1‐6 day) tissue culture. ICa was recorded from the cells using whole‐cell patch‐clamp methods (Clark, Tse & Giles, 1990). 3. External application of 2 nM to 10 microM acetylcholine (ACh) reduced the amplitude and slowed the time course of activation of ICa. These effects were dependent on membrane potential; they were most pronounced at potentials near the peak of the current‐voltage relation for ICa (i.e. +10 to +15 mV), whereas at more‐negative potentials (i.e. ‐15 to ‐25 mV) the effects on both amplitude and time course were relatively small. 4. Atropine (1 microM) completely blocked the action of 1 microM‐ACh, indicating that the effects of ACh on ICa were mediated by activation of muscarinic receptors. 5. Other muscarinic agonists, such as carbamylcholine (0.1‐10 microM), DL‐muscarine (0.1‐2.5 microM) and oxotremorine (5 microM), had similar effects on ICa to ACh. 6. A guanine nucleotide‐binding protein (G‐protein) is involved in this muscarinic inhibition of ICa. Inclusion of the non‐hydrolysable guanosine triphosphate analogue guanosine 5'‐O‐(3‐thiotriphosphate) (GTP‐gamma‐S; 200 microM) in the intracellular solutions mimicked the effects of ACh, and application of external ACh in the presence of internal GTP‐gamma‐S produced smaller changes in ICa than in control conditions. Inclusion of another non‐hydrolysable analogue, guanosine 5'‐O‐(2‐thiodiphosphate) (GDP‐beta‐S; 0.5‐5 mM), blocked the inhibitory effect of ACh on ICa. 7. The G‐protein involved in the inhibition of ICa was sensitive to pertussis toxin (islet‐activating protein; IAP). The inhibition of ICa by carbamylcholine (5 microM) was reduced by about 90% after incubating cells for 12‐15 h in culture medium containing 200 ng/ml IAP. 8. The possible roles of cyclic AMP or cyclic GMP‐dependent protein kinases, or protein kinase C, in the muscarinic inhibition of ICa were tested, but these enzymes appear not to be directly involved.
The influence of temperature on mechanics of red muscle in carp.
Authors: Rome L C , Sosnicki A A
Published: December 2014

1. We measured the influence of temperature on maximum velocity of shortening (Vmax) of red muscle in carp in order to better understand the influence of temperature on locomotory performance. 2. A stable red muscle bundle preparation containing about 100 muscle fibres was developed. The bundles could not be activated directly by electrical stimulation, but rather contained sufficient nervous tissue so that acetylcholine released from the nerve terminals caused activation of the muscle. A high level of activation was achieved (116 kN/m2) by adding a combination of a 1 mM‐caffeine and 10(‐5) g/ml eserine to physiological Ringer solution and electrically stimulating the preparation. 3. Force‐velocity characteristics were determined at 10 and 20 degrees C by the force clamp method. The data were well fitted by a hyperbola not constrained to pass through P0 = 1 (where P0 is the isometric force). The mean Vmax at 10 degrees C was 3.55 +/‐ 0.26 muscle lengths/s (ML/s) (n = 6) and at 20 degrees C, 5.71 +/‐ 0.29 ML/s (n = 6). The mean Q10 for Vmax was 1.63 +/‐ 0.07 (n = 6). The a/P0* (Hill constant) and Po* (where P0* is the extrapolated load at zero velocity) were 0.49 +/‐ 0.06 (n = 6) and 1.19 +/‐ 0.04 (n = 6) respectively at 10 degrees C and 0.29 +/‐ 0.06 (n = 6) and 1.51 +/‐ 0.20 (n = 6) respectively at 20 degrees C. 4. The mean Q10 for maximum isometric tension was 1.13 +/‐ 0.02 (n = 6). The maximal power generation was 59.7 +/‐ 2.3 W/kg (n = 6) at 10 degrees C and 94.3 +/‐ 3.2 W/kg (n = 6) at 20 degrees C representing a Q10 of 1.58. The Q10 is less than the product of Q10s for P0 and Vmax because of the greater curvature of the force‐velocity curve at 20 degrees C. 5. The 1.63‐fold higher Vmax at 20 degrees C than at 10 degrees C enables fish to swim with a 1.6‐fold faster muscle shortening velocity, V, at the higher temperature. Thus at both 10 and 20 degrees C, red muscle is used only over the same narrow range of V/Vmax (0.18‐0.36), where isolated muscle experiments suggest that power and efficiency are maximal. Thus V/Vmax appears to be an effective design constraint which limits the range of velocities over which muscle is used in vivo at different temperatures.
Neurones in a discrete region of the nucleus tractus solitarius are required for the Breuer‐Hering reflex in rat.
Authors: Bonham A C , McCrimmon D R
Published: December 2014

1. The Breuer‐Hering reflex consists of a shortening of inspiration and lengthening of expiration in response to afferent input from slowly adapting pulmonary stretch receptors (SAR). We hypothesized that neurones in a discrete region of the nucleus tractus solitarius (NTS) are required for producing the reflex. Accordingly, the present studies were undertaken to: (1) identify sites in the NTS in which chemical excitation of neurones inhibited phrenic nerve discharge in a manner consistent with SAR activation, (2) determine whether localized interruption of synaptic transmission prevented the Breuer‐Hering reflex, and (3) determine whether these regions contained pump cells and SAR terminal afferents. Studies were carried out in urethane‐anaesthetized rats. 2. Injection of picomoles of an excitatory amino acid, DL‐homocysteic acid (DLH), in the NTS, at the rostrocaudal level of the area postrema and immediately medial to the tractus solitarius, silenced phrenic nerve activity similarly to that expected from SAR activation. These apnoeas lasted from 3 to 43 s and were produced with little or no change in arterial pressure or heart rate. 3. The Breuer‐Hering reflex, physiologically activated by maintaining lung inflation, was transiently impaired by interruption of synaptic transmission following injections of cobalt chloride in the DLH‐responsive region. 4. Pump cell (SAR interneurone) and SAR afferent activity were recorded at the site in which DLH produced apnoea. 5. Taken together, the results of chemical excitation, interruption of synaptic transmission and extracellular recording, suggest that cells within a discrete region of the NTS, probably pump cells, are necessary for the production of the Breuer‐Hering reflex.
Ca2+ release from the sarcoplasmic reticulum of barnacle myofibrillar bundles initiated by photolysis of caged Ca2+.
Authors: Lea T J , Ashley C C
Published: December 2014

1. Ca2(+)‐induced Ca2+ release (CICR) from the sarcoplasmic reticulum was measured by isometric tension recording from barnacle myofibrillar bundles. Laser‐induced photolysis of the caged calcium molecule, nitr‐5, was used to generate a rapid jump in free Ca2+ (within 1 ms) at the site of the sarcoplasmic reticulum, thus overcoming delays due to Ca2+ diffusion from the bathing solution. 2. The method consisted of equilibrating a myofibrillar bundle (100 micrograms diameter) in a solution containing 0.1 mM‐nitr‐5 (initial pCa 6.8‐6.6) and then exposing it to a UV laser pulse. The resulting phasic contraction had an amplitude of up to 100% maximum tension (P0) in some preparations and a mean half‐time for the rise of tension of 2.3 s at 12 degrees C. Longer half‐times were obtained at low pulse energies. 3. Pre‐treatment of the myofibrillar bundles with ryanodine (10(‐4) M) or the detergent Triton X‐100 abolished a large part of the phasic contraction, confirming its dependence on SR Ca2+ release. The small tonic response which remained had a shorter rise half‐time than the Ca2(+)‐induced Ca2+ release response and was attributed to direct activation of the myofibrils by Ca2+ released from the nitr‐5. 4. The size of the photolytic Ca2+ jump was estimated from the amplitude of the fast tension component. By increasing the laser pulse energy or the initial Ca2+ loading of the nitr‐5, the post‐photolysis pCa was varied from 6.7 to 6.0; the CICR response increased in size over this pCa range. 5. Direct activation of Triton‐treated myofibrils by photolysis of 2.0 mM‐nitr‐5 (initial pCa 6.4) gave contractions of up to 100% P0 and a mean rise half‐time of 164 ms at 12 degrees C (n = 9 for contractions greater than 40% P0). Both the amplitude and the rate of these contractions were dependent on the laser pulse energy. 6. The Ca2(+)‐induced Ca2+ release responses obtained with nitr‐5 photolysis were significantly slower than the fastest rate of tetanus development which has been recorded from intact fibres of barnacle muscle (mean half‐time = 177 ms at 12 degrees C). This could mean that either Ca2(+)‐induced Ca2+ release is less efficient in isolated myofibrillar bundles than in intact fibres or that Ca2(+)‐induced Ca2+ release is not the primary Ca2+ releasing mechanism in excitation‐contraction coupling in barnacle muscle.
The vasopressin response to centrally administered hypertonic solutions in the conscious rat.
Authors: Wells T , Forsling M L , Windle R J
Published: December 2014

1. Intracerebroventricular (I.C.V.) injections of isotonic and hypertonic solutions into the dorsal (D3V) and ventral (V3V) third ventricle were employed to examine the release of vasopressin (AVP) and the mean arterial pressure (MAP) response to elevated cerebrospinal fluid (CSF) osmolality in the conscious rat. 2. The D3V injection of hypertonic sodium chloride solution was associated with a concentration‐dependent, transient increase in plasma AVP concentration and MAP. 3. The D3V injection of 5 microliters 0.85 M‐sodium chloride elicited a 7‐fold increase in plasma AVP and oxytocin concentrations, but had no effect on plasma ACTH concentration. The D3V injection of 1.11 M‐mannitol in 0.15 M‐sodium chloride had no effect on plasma AVP concentration or MAP. However, the D3V injection of 0.746 M‐mannitol in 0.4 M‐sodium chloride elicited a significant transient increase in plasma AVP, but had no effect on MAP. 4. The V3V injection of 5 microliters 0.85 M‐sodium chloride elicited a prolonged increase in plasma AVP concentration and a transient increase in MAP. The V3V injection of 5 microliters 1.11 M‐mannitol in 0.15 M‐sodium chloride elicited an equal, but transient, increase in plasma AVP concentration, but had no effect on MAP. 5. The pressor effect of a D3V injection of 0.85 M‐sodium chloride was unaffected by prior administration of the V1 (pressor) receptor antagonist (beta‐mercapto‐beta,beta‐cyclopentamethylene propionyl1, O‐Me‐Tyr2, Arg8)‐vasopressin. 6. These results indicate that osmotically induced AVP secretion may be mediated by both sodium receptors and osmoreceptors, although expression of the response may depend upon the maintenance of a ‘permissive’ concentration of sodium in the CSF. 7. It appears also that the pressor effect is not due to increased plasma AVP concentration, but only results from elevation of the CSF sodium concentration.
Opioid actions on single nucleus raphe magnus neurons from rat and guinea‐pig in vitro.
Authors: Pan Z Z , Williams J T , Osborne P B
Published: December 2014

1. Intracellular recordings were made from neurons of the nucleus raphe magnus (NRM) from rat (n = 128) and guinea‐pig (n = 115). Two types of cells were found in each, primary (103 in rat, 27 in guinea‐pig) and secondary cells (25 in rat, 88 in guinea‐pig). 2. Primary cells had input resistances of 186 +/‐ 9 M omega (n = 9) in rat and 255 +/‐ 50 M omega (n = 11) in guinea‐pig. The action potential in each was about 1.5 ms in duration. Synaptic potentials were evoked by focal electrical stimulation and consisted of both gamma‐aminobutyric acid (GABA) and excitatory amino acid components. 3. Morphine, [Met5]enkephalin (ME) and [D‐Ala2,N‐Me‐Phe4, Gly5‐ol]enkephalin (DAMGO) depressed the amplitude of the GABA‐mediated synaptic potential by a maximum of 50‐65% and had little effect on the excitatory amino acid‐mediated synaptic potential. There was no effect of these opioids on the resting membrane potential or input resistance of primary cells in rat or guinea‐pig. 4. Secondary cells had short duration action potentials (less than 1 ms) and an input resistance of 354 +/‐ 47 M omega in rat (n = 6) and 290 +/‐ 40 M omega in guinea‐pig (n = 15). The synaptic potential observed in the cells of this group was mediated by activation of only excitatory amino acid receptors. 5. ME hyperpolarized and/or abolished the spontaneous firing in sixteen out of twenty‐four neurons in the secondary group from rat and eight out of eighty‐four neurons from guinea‐pig. ME induced an outward current at ‐60 mV that reversed polarity at potentials more negative than ‐92 +/‐ 3 mV in rat (n = 6) and ‐98 +/‐ 2 mV in guinea‐pig (n = 18). The reversal potential of the opioid current was shifted to less negative potentials when the external potassium concentration was increased, as predicted by the Nernst equation. 6. The morphology of the two types of cells were distinguishable in that primary cells were oval (29 x 18 microns in rat; 36 x 19 microns in guinea‐pig) with two to four thick tapering dendrites that branched within 50 microns of the cell body. Secondary cells were generally round or oval (about 24 x 13 microns in rat; 27 x 17 microns in guinea‐pig) with two to five thin non‐tapering dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)
Rectification of currents activated by nicotinic acetylcholine receptors in rat sympathetic ganglion neurones.
Authors: Mathie A , Colquhoun D , Cull‐Candy S G
Published: December 2014

1. The inward rectification of the whole‐cell current evoked by acetylcholine (ACh) and other nicotinic agonists in rat sympathetic ganglion neurones has been studied using patch‐clamp recording techniques. The selective nicotinic agonist 1,1‐dimethyl‐4‐phenylpiperazinium iodide (DMPP) (20 microM) induced an average peak current of ‐367 pA at ‐50 mV but no detectable outward current at +50 mV. Similar observations were made with ACh and carbachol. 2. The current‐voltage relation of the whole‐cell response induced by DMPP was linear in the negative voltage range; however, there was no detectable outward current in the voltage range 0 to about +70 mV. Above +70 mV an outward current became clearly detectable. Rapid depolarizing jumps in the holding potential failed to reveal any rapidly decaying outward current. 3. The rectification was not alleviated by changing the main permeant cation, by removal of divalent cations from the intracellular or extracellular solutions or by altering the pH buffer in the extracellular solution from HEPES to Tris. 4. Intracellular magnesium ions can block the channel. This effect increases with depolarization, but dissociation outwards (i.e. permeation by Mg2+) appears to relieve the block at more extreme positive potentials. This effect alone, or in combination with the voltage dependence of the burst length, is unlikely to be able to account for the whole‐cell rectification in intact cells, much less that seen in cells perfused with Mg2(+)‐free intracellular medium. 5. When the reversal potential was shifted to approximately ‐50 mV (by the use of impermeant cations) nicotinic agonists produced small outward currents in the membrane potential range ‐20 to +10 mV while shifting it to about +40 mV produced small inward currents in the potential range 0 to +20 mV. The rectification therefore appears to be independent of the direction of current flow and is maximum at a potential positive to 0 mV. 6. At positive potentials the receptors desensitized much less than at negative potentials in the continued presence of agonist. Thus, exposure of the cells to a steady application of 30 microM‐ACh produced no detectable response if the cell was at a positive potential, but when the cell was stepped to a negative potential in the continued presence of ACh (at a time when much of the ACh current would be expected to have desensitized), ACh induced a large inward current. The onset of the ACh current had a time constant of 10 ms. It then decayed with a time constant of 790 ms as desensitization developed.(ABSTRACT TRUNCATED AT 400 WORDS)
Responses of type I cells dissociated from the rabbit carotid body to hypoxia.
Authors: Biscoe T J , Duchen M R
Published: December 2014

1. The carotid body chemoreceptors are stimulated in situ by hypoxia. We have studied type I cells freshly dissociated from the carotid body of the rabbit. We have used microfluorimetric and patch clamp techniques to examine the responses to hypoxia, to anoxia, and to metabolic inhibition. 2. NADH autofluorescence measured at both 400 and 500 nm increased rapidly and reversibly in response to anoxia or to cyanide (CN‐), reflecting a change in mitochondrial metabolism. 3. Indo‐1 was used to measure changes in intracellular calcium, [Ca2+]i. Anoxia reversibly increased [Ca2+]i from approximately 50‐100 to approximately 200‐450 nM in all cells tested. The response showed a striking temperature sensitivity. Responses to hypoxic stimuli were barely detectable at 17‐20 degrees C, and were dramatically increased on warming to 36 degrees C. In contrast, responses to K(+)‐induced depolarization were only slightly increased in rate of onset and recovery by warming. 4. The rise in [Ca2+]i originated largely from an intracellular store which was slowly depleted by exposure to nominally Ca2(+)‐free solutions. Responses were unaffected by blockade of Ca2+ channels with organic (D600, verapamil) or inorganic (Co2+) blockers, by blockade of Na+ channels with tetrodotoxin (TTX), or by increasing action potential duration with tetraethylammonium (TEA). Responses to anoxia were increased by the increased [Ca2+]i loading that follows prior exposure to Ca2(+)‐free solutions. 5. Responses to anoxia, to blockade of electron transport by CN‐, and to the mitochondrial uncoupler, carbonyl cyanide p‐trifluoromethoxy‐phenylhydrazone (FCCP), were equivalent in amplitude. The response to anoxia was occluded by concurrent application of FCCP, suggesting that the Ca2+ originates from the same pool in each case. 6. At 35‐36 degrees C, responses to graded levels of PO2 were also graded. Thresholds varied between cells, but were typically 30‐50 mmHg. Stimulus‐responses curves were essentially hyperbolic, increasing dramatically as the PO2 approached 0 mmHg. 7. The sensitivity of cells to hypoxic solutions was increased by acidification of the superfusate over the pH range from 7.3 to 6.85. 8. Cell‐attached patch clamp recordings showed depression of spontaneous action potentials associated with a rise in [Ca2+]i during exposure to anoxic solutions. Whole‐cell recordings showed that anoxia increased a voltage‐gated gK as described previously for CN‐, while producing no change in resting conductance. 9. These data suggest that the rise in [Ca2+]i originates largely from Ca2+ efflux from a mitochondrial pool.(ABSTRACT TRUNCATED AT 400 WORDS)
Oxytocin excites gastric‐related neurones in rat dorsal vagal complex.
Authors: McCann M J , Rogers R C
Published: December 2014

1. Dorsal medullary injections of oxytocin (OT) influence gastric motor and secretory function via a vagally mediated mechanism. Thus, it was hypothesized that OT altered the firing rate of brain stem vagal neurones that were specifically related to gastric function. 2. To study this, glass microelectrode/injection pipette arrays were used to record the activity of gastric‐related neurones in the dorsal vagal complex (DVC), which includes vagal sensory neurones in the nucleus tractus solitarius (NTS) and motor neurones in the dorsal motor nucleus (DMN). After identifying such a neurone, spontaneous activity was monitored before and after micropressure injection of OT and vehicle solutions from the pipettes. 3. Two methods were used to identify neurones that were related to gastric function. One method employed a gastric balloon to identify DVC neurones that were responsive to gastric inflation. The second method employed a gastric vagal stimulating electrode, which permitted the identification of gastric‐related NTS and DMN cells via orthodromic or antidromic activation, respectively. 4. Twenty‐four of forty‐two gastric‐inflation‐related neurones responded to administration of OT (100‐400 fmol in 100‐400 pl). The majority of those responding to OT were activated by this peptide (21/24). All the cells tested (n = 13) remained sensitive to gastric inflation after administration of OT. Also, OT was found to excite the majority of cells that were identified as gastric‐related NTS (nine excited; one no effect) or DMN cells (eleven excited; two no effect). 5. These studies support the hypothesis that central oxytocinergic neurones influence gastric motility and secretion by increasing the excitability of central vagal neurones in the NTS and DMN that are related to gastric function.
The actions of suxamethonium (succinyldicholine) as an agonist and channel blocker at the nicotinic receptor of frog muscle.
Authors: Marshall C G , Ogden D C , Colquhoun D
Published: December 2014

1. Patch clamp methods were used to study the equilibrium and kinetic properties of the acetylcholine analogue, succinyldicholine (suxamethonium), which is used clinically as a neuromuscular blocking agent. 2. The equilibrium concentration‐response curve, corrected for desensitization was estimated by measuring as the response the probability of being open of single ion channels during clusters of activity that occur between long desensitized periods. Suxamethonium (Sux) was about 7.6‐fold less potent than acetylcholine (ACh) (at low concentrations), partly because of 2.9‐fold lower affinity for the resting receptor, and partly because of a lower ability to activate the receptor once bound. 3. Sux was a more potent blocker of the open ion channel than ACh (equilibrium constant about 200 microM); this limited the maximum open probability to about 0.36 (at 12 degrees C and ‐120 mV). Individual channel blockages lasted about 65 microseconds on average. They appeared to get longer at high agonist concentration; however, a simulation method was used to show that this effect could be accounted for by the fact that at higher concentrations there are more openings that are too brief to be detected. Over the concentration range tested the effects were described by a simple open channel block mechanism. 4. No component of brief shut times could be detected other than those resulting from channel blockages. However, the results suggest that multiple channel openings (the nachschlag phenomenon) should be rare, so this is not inconsistent with previous results with other agonists. 5. Sux differed from ACh and carbachol in that it had a somewhat lower efficacy and a greater channel blocking action. However, in clinical practice channel block is unlikely to contribute to neuromuscular block to any significant extent; the main mechanism of paralysis, at least in the early stages, is probably a result of prolonged depolarization of the region of membrane surrounding the motor endplate leading to inactivation of the sodium channels therein.
Concomitant activation of two types of glutamate receptor mediates excitation of salamander retinal ganglion cells.
Authors: Mittman S , Taylor W R , Copenhagen D R
Published: December 2014

1. Cells in the ganglion cell layer of salamander retinal slices were voltage clamped using patch pipettes. Light elicited transient excitatory postsynaptic currents (EPSCs) in on‐off ganglion cells and sustained EPSCs in on ganglion cells. Light‐evoked inhibitory postsynaptic currents in these cells could be blocked by 100 microM‐bicuculline methobromide and 500 nM‐strychnine. 2. In the presence of external Cd2+, at a concentration that blocked light‐evoked synaptic inputs, N‐methyl‐D‐aspartate (NMDA) and the non‐NMDA‐receptor agonists, quisqualate and kainate, gated conductances in both on‐off and on ganglion cells. The current‐voltage (I‐V) curve for the conductance elicited by NMDA had a negative slope between ‐40 and ‐70 mV and a reversal potential near 0 mV. The I‐V curves for the non‐NMDA‐receptor‐mediated conductances were nearly linear and also had reversal potentials near 0 mV. 3. I‐V curves were measured at an early time point near the peak of transient EPSCs and at a later time point during the decay phase of the responses. The late I‐V curve had a negative slope below ‐40 mV. The early I‐V curve had a positive slope over the entire voltage range but the slope was greater at positive than at negative potentials. The evoked current reversed near 0 mV at both time points. 4. The region of negative slope of the late I‐V curve was eliminated when Mg2+ was removed from the external saline. A slowly decaying component of transient EPSCs was eliminated in 20 microM‐DL‐2‐amino‐7‐phosphonoheptanoate (AP7), an NMDA‐receptor antagonist. 5. Application of 1 microM‐6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), a non‐NMDA‐receptor antagonist at this concentration, blocked a fast component of transient EPSCs. 6. Our results demonstrate that the synaptic inputs to on‐off ganglion cells have two components: a slower NMDA‐receptor‐mediated component having a time‐to‐peak of 110 +/‐ 45 ms and an e‐fold decay time of 209 +/‐ 35 ms at ‐31 mV (mean +/‐ S.D., n = 5), and a faster non‐NMDA‐receptor‐mediated component having a time‐to‐peak of 28 +/‐ 10 ms and an e‐fold decay time of 43 +/‐ 20 ms at ‐31 mV (n = 8). 7. A similar analysis of sustained EPSCs of on ganglion cells showed that these currents resulted from sustained activation of both NMDA and non‐NMDA receptors.
Voltage‐activated calcium currents in presynaptic nerve terminals of the chicken ciliary ganglion.
Authors: Yawo H
Published: December 2014

1. Calcium currents (ICa) were recorded from presynaptic calyces of ciliary ganglia of the chick embryo under whole‐cell voltage clamp. 2. Only high‐threshold ICa was recorded without any evidence for the presence of low‐threshold Ca2+ channels. 3. High‐threshold (high‐voltage‐activated, HVA) ICa could be classified into non‐inactivating (HVAn) and inactivating (HVAi) components. The mean inactivation time constant of the HVAi component was 213 ms (at 0 mV). The threshold for activation by depolarizing pulses was more negative for the HVAn component than for the HVAi component. The HVAi component was inactivated by 19% at a holding potential of ‐60 mV, while the HVAn component was little affected under this condition. 4. The activation of HVAn component was faster than that of the HVAi component. 5. Both the HVAn and HVAi components were blocked by Cd2+ (50 microM) and La3+ (1 microM). Both components were only slightly affected by Ni2+ (100 microM). The order of potency in blocking was La3+ greater than Cd2+ greater than Ni2+ for both components. Both the HVAi and HVAn components were irreversibly blocked by omega‐conotoxin GVIA(omega‐CgTX, 10 microM). 6. The two components could pharmacologically be distinguished by selective blockade of the HVAn component with nifedipine (2 microM) and D600 (100‐250 microM). 7. HVAn and HVAi components are suggested to represent two different subpopulations of Ca2+ channels. The HVAn subpopulation may be responsible for persistent Ca2+ influx during subthreshold depolarization of the nerve terminal.
Effect of melittin on renin and prostaglandin E2 release from rat renal cortical slices.
Authors: Churchill P C , Rossi N F , Churchill M C , Ellis V R
Published: December 2014

1. The present experiments were designed to determine the effect of melittin on renin secretion. Melittin is a polypeptide component of bee venom which stimulates phospholipase A2 activity, thereby increasing arachidonic acid release and prostaglandin (PG) synthesis, and which inhibits protein kinase C activity. Either of these actions might be expected to stimulate renin secretion, since renin secretion is stimulated by arachidonic acid and by several PGs, and since renin secretion is inhibited by several activators of protein kinase C. 2. In rat renin cortical slices incubated at 37 degrees C in a buffered and oxygenated physiological saline solution, 0.1‐10 microM‐melittin produced a concentration‐dependent stimulation of both prostaglandin E2 (PGE2) synthesis and renin secretion. However, melittin‐stimulated renin secretion is independent of melittin‐stimulated phospholipase A2 activity, arachidonic acid release, and PG synthesis, since 20 microM‐quinacrine (a phospholipase A2 antagonist) and 50 microM‐meclofenamate (a cyclooxygenase antagonist) antagonized basal and melittin‐stimulated PGE2 synthesis but had no effects on basal or melittin‐stimulated renin secretion. 3. Furthermore, melittin‐stimulated renin secretion is not produced by inhibition of protein kinase C, since an activator of protein kinase C (12‐O‐tetradecanoylphorbol 13‐acetate, TPA), enhanced rather than antagonized melittin‐stimulated renin secretion. Ouabain partially antagonized, but did not completely block, melittin‐stimulated renin secretion. 4. Thus, melittin‐stimulated phospholipase A2 activity probably accounts for stimulated PGE2 production, but not for stimulated renin secretion. The mechanism of melittin‐stimulated renin secretion is unclear; an effect on protein kinase C does not appear to be involved, and in contrast to the stimulatory effects of a variety of other substances, melittin‐stimulated renin secretion is only partially antagonized by ouabain.
An ATP‐activated conductance in pheochromocytoma cells and its suppression by extracellular calcium.
Authors: Nakazawa K , Fujimori K , Takanaka A , Inoue K
Published: December 2014

1. ATP‐activated inward current in PC12 pheochromocytoma cells was characterized using the whole‐cell voltage‐clamp technique. 2. ATP (100 microM) applied extracellular elicited an inward rectifying current with a reversal potential of about +7 mV. The current was desensitized in seconds in spite of continued presence of ATP. 3. A comparison was made of the ability of ATP and its analogues. The order of potency in activating the inward current was ATP greater than ATP gamma S greater than ADP; AMP, adenosine and alpha, beta‐methylene ATP were inactive at concentrations up to mM. 4. The ATP‐activated current was also observed when external Na+ and Ca2+ were replaced with K+, TEA, Tris or glucosamine. The order of ion selectivity was Na+ greater than K+ greater than TEA not equal to Tris greater than glucosamine. 5. The ATP‐activated current was also recorded in extracellular solutions containing Ca2+ as the only external cation. The amplitude increased as the concentration of Ca2+ was increased in the range between 1.8 and 16.2 mM. However, the current amplitude decreased at higher Ca2+ concentrations and the current was not recorded in 110 mM‐Ca2+ solution. 6. In the presence of 140 mM‐Na+ in the external solution, the current amplitude also decreased as the external Ca2+ concentration was increased (from 1.8 to 16.2 mM). 7. The results indicate that Ca2+ as well as monovalent cations permeate through the ATP‐sensitive pathway and that Ca2+ blocks ion permeation, including its own permeation through the pathway. This regulation by extracellular Ca2+ is different to the ATP‐activated current in smooth muscle cells.
Modulation of N‐methyl‐D‐aspartic acid receptor desensitization by glycine in mouse cultured hippocampal neurones.
Authors: Vyklický L , Benveniste M , Mayer M L
Published: December 2014

1. Responses to N‐methyl‐D‐aspartic acid (NMDA) were recorded from mouse embryonic hippocampal neurones in dissociated culture, using the tight‐seal, whole‐cell, patch‐clamp technique for voltage clamp. A rapid perfusion system, with an exchange time constant of less than 10 ms, was used to apply NMDA under conditions which minimized slow, calcium‐sensitive desensitization. With no added glycine, responses to 100 microM‐NMDA applied for 1.5 s declined by greater than 90%, due to an additional component of desensitization of time constant 250 ms. 2. Adding glycine to the extracellular solution, over the range 30 nM to 3 microM, both potentiated responses to NMDA and to L‐glutamate, and reduced fast desensitization. In the presence of 3 microM‐glycine responses to NMDA declined by only 10%. Similar potentiation and reduction of desensitization was obtained with 3 microM concentrations of the glycine analogues D‐alanine and D‐serine. 3. Analysis of dose‐response curves for the action of glycine on responses to 100 microM‐NMDA revealed a 3‐fold higher potency of glycine for potentiation of peak versus steady‐state responses, with concentrations for half‐activation of 134 and 382 nM, respectively. The competitive glycine antagonist 7‐chlorokynurenic acid produced a similar shift of both the peak and steady‐state dose‐response curves for glycine, consistent with an equilibrium dissociation constant of 280 nM for interaction of 7‐chlorokynurenic acid with the glycine binding site on NMDA receptors. 4. In the presence of 100 nM‐glycine, 10 microM‐7‐chlorokynurenic acid produced nearly complete block of the response to 3 nM‐NMDA, suggesting that glycine is absolutely required for activation of the NMDA receptor channel complex. 5. In some neurones responses to NMDA showed essentially no desensitization in the presence of 3 microM‐glycine. Under these conditions, 7‐chlorokynurenic acid produced a concentration‐dependent block of both the initial and equilibrium response to NMDA, with a 4‐fold greater sensitivity for block of the steady‐state current (IC50 = 2.25 microM) than for block of the peak current (IC50 = 8.96 microM). As a result, in the presence of 7‐chlorokynurenic acid, responses to NMDA showed strong desensitization, even in the presence of 3 microM‐glycine. 6. Our results show that glycine‐evoked potentiation of NMDA receptor activity is accompanied by reduced desensitization.(ABSTRACT TRUNCATED AT 400 WORDS)
A kinetic analysis of the modulation of N‐methyl‐D‐aspartic acid receptors by glycine in mouse cultured hippocampal neurones.
Authors: Benveniste M , Clements J , Vyklický L , Mayer M L
Published: December 2014

1. Responses to N‐methyl‐D‐aspartic acid (NMDA) were recorded from mouse embryonic hippocampal neurones in dissociated culture, using whole‐cell patch‐clamp recording. A fast perfusion system, with an exchange time constant of less than 10 ms, was used to study modulation of NMDA receptor desensitization by glycine. 2. The onset of NMDA receptor desensitization was well fitted by a single‐exponential function; with 30 nM‐glycine the time constant was 250 ms, corresponding to a rate of 4 s‐1. The rate of onset of desensitization became faster with increasing glycine concentration, with a slope of 0.87 x 10(7) M‐1 s‐1. Recovery from desensitization, studied with a twin‐pulse technique, was also well fitted by a single‐exponential function; with 30 nM‐glycine the time constant of recovery was 1.95 s‐1. The rate of recovery from desensitization became faster with increasing glycine concentration, with a slope of 0.76 x 10(7) M‐1 s‐1. These results are consistent with a model in which the effect of glycine occurs via an increase in the rate constant for recovery from desensitization, with little effect on the rate constant for onset of desensitization. Over the range 30‐300 nM‐glycine, the ratio of the rate constants calculated for recovery and onset of desensitization was a good predictor of the degree of desensitization recorded at equilibrium. 3. Concentration jump experiments with glycine were performed with 100 microM‐NMDA present continuously, and for a single binding site model gave estimates of the association (1.1 x 10(7) M‐1 s‐1) and dissociation (3.1 s‐1) rate constants for interaction of glycine with the NMDA receptor. In the presence of NMDA, concentration jumps from 3 microM‐glycine to lower concentrations gave relaxations which became slower with decreasing glycine concentration over the range 1 microM‐30 nM. A similar slowing of desensitization occurred when the glycine concentration was altered over the same range. 4. Glycine analogues of lower affinity produced desensitization with faster kinetics. D‐Alanine, 150 nM, produced desensitization with a time constant of 175 ms, faster than recorded with an equipotent concentration of glycine (50 nM, time constant 259 ms). Responses of similar peak amplitude, recorded with 60 microM‐L‐alanine, and 500 microM‐D,L‐homoserine, did not produce strong desensitization, consistent with desensitization too rapid to resolve in our experiments.(ABSTRACT TRUNCATED AT 400 WORDS)
Sympathetic‐parasympathetic interactions at the heart, possibly involving neuropeptide Y, in anaesthetized dogs.
Authors: Revington M L , McCloskey D I
Published: December 2014

1. Stimulation of cardiac sympathetic nerves caused prolonged inhibition of vagal effects on heart rate, an effect which has been proposed on the basis of previous studies to be due to neuropeptide Y or a neuropeptide Y‐like substance, released from the sympathetic nerves. 2. This prolonged vagal inhibitory effect was attenuated or abolished when the sympathetic stimulation responsible was given together with continuous vagal stimulation. 3. Continuous vagal stimulation alone did not modify the ability of administered neuropeptide Y to cause inhibition of cardiac vagal action. 4. The results are consistent with the cardiac vagal nerves releasing a transmitter (probably acetylcholine) which acts on cardiac sympathetic nerve endings, inhibiting them from releasing neuropeptide Y or a neuropeptide Y‐like substance.
Activation of human respiratory muscles during different voluntary manoeuvres.
Authors: Gandevia S C , McKenzie D K , Plassman B L
Published: December 2014

1. This study used three techniques (bilateral phrenic nerve stimulation, motor cortex stimulation and quantitative electromyography) to assess the degree of activation of the diaphragm, intercostal‐accessory muscles and abdominal muscles during postural tasks and respiratory manoeuvres. They included maximal inspiratory and expulsive efforts. 2. Bilateral phrenic nerve stimuli at supramaximal levels produced an average change in transdiaphragmatic pressure (Pdi) of 28 cmH2O during relaxation. During maximal inspiratory or expulsive efforts, all subjects were able to activate the diaphragm fully at functional residual capacity as judged by the failure of stimuli delivered during the voluntary efforts to increase Pdi. Peak voluntary Pdi was about 30% less for inspiratory than expulsive manoeuvres. 3. By contrast, transcranial activation of motor cortical output to the diaphragm and abdominal muscles produced an increment in abdominal pressure of 25 +/‐ 7 cmH2O during maximal voluntary expulsive efforts. Given the lack of response to phrenic nerve stimulation at similar voluntary pressures, this suggests that abdominal muscles, and not the diaphragm, fail to generate their full contractile force during maximal voluntary expulsive manoeuvres. 4. Motor cortical stimulation during weak inspiratory efforts produced a small reduction in oesophageal pressure (i.e. increase in net inspiratory force) of 7‐14 cmH2O. This response could not be extinguished during maximal voluntary inspiratory efforts in two of three subjects. This occurred despite the cortical co‐activation of ‘antagonist’ muscles in the chest wall and abdomen, and passive transmission of pressure from the abdominal to thoracic compartments. 5. Integrated electromyographic activity (EMG) recorded from abdominal muscles (rectus abdominis, external oblique) was greater during trunk flexion than during maximal expulsive efforts. Similarly, integrated EMG of the intercostal‐accessory muscles (sternomastoid, scalenes, parasternal intercostals) was greater during tasks requiring head and/or neck flexion than during the maximal inspiratory efforts. 6. These data show that the diaphragm can be fully activated by the central nervous system during voluntary respiratory tasks but that other agonist ‘respiratory’ muscles need not be activated fully. Given the complex actions of ‘inseries’ respiratory muscles revealed here, it is argued that differences in the transdiaphragmatic pressure during various postural and respiratory tasks do not necessarily imply variation in the level of diaphragmatic neural drive.