bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of the effects of acetylcholine on the contractile properties and Ca2+ transients in ferret ventricular muscles.
Authors: Hongo K , Tanaka E , Kurihara S
Published: December 2014

1. We investigated the mechanism of signal transduction during the effect of muscarinic receptor stimulation on Ca2+ transients, tension, Ca2+ sensitivity and the cross‐bridge cycling rate (CCR). 2. Membrane‐permeable derivatives of cyclic GMP (8‐bromo‐cyclic GMP and dibutyryl cyclic GMP) did not cause any significant changes in the peaks of Ca2+ transients and tension and the time courses of either signal modulated by isoprenaline (Iso) (0.1 microM). 3. Nitroprusside (0.1‐1 mM) likewise did not change the peaks or the time courses of Ca2+ transients and tension in the Iso‐treated preparations. 4. In papillary muscles excised from ferrets treated with pertussis toxin (islet‐activating protein, IAP), which is known to abolish the function of GTP‐binding proteins (Gi, Go and Gt), similar changes in Ca2+ transients and tension produced by treatment with Iso (0.1 microM) were noted as in non‐IAP‐treated preparations. However, no effects of acetylcholine (ACh; 1 microM) on either signal were observed. 5. The relation between [Ca2+]i and tension measured during the steady state of tetanic contraction was shifted to the right by Iso (0.1 microM), and cyclic GMP derivatives (1 mM) did not change the altered relation. In the IAP‐treated preparations, ACh (1 microM) did not influence the relation altered by Iso (0.1 microM). 6. Cyclic GMP derivatives (1 mM) did not alter the Iso (0.1 microM)‐increased CCR measured by perturbation analysis. ACh (1 microM) did not restore the Iso‐increased CCR in the IAP‐treated preparations. 7. These results suggest that signal transduction in muscarinic receptor stimulation is primarily mediated by inhibition of adenylate cyclase via IAP‐sensitive GTP‐binding proteins, and that cyclic GMP does not play an important role in the effect of muscarinic receptor stimulation on Ca2+ transients, tension, Ca2+ sensitivity or CCR.
Permeability of disrupted cerebral microvessels in the frog.
Authors: Fraser P A , Dallas A D
Published: December 2014

1. This study reports the results of varying the hydrostatic pressure on measurements of permeability coefficient to the low molecular weight impermeant dye carboxyfluorescein (MW = 376) in single leaky cerebral microvessels. A mathematical model, that solved the convective diffusion equations used to analyse the measurements, showed that the measurements were consistent with leakiness being due to 22 nm wide parallel‐sided slits between endothelial cells. 2. Microvessels on the surface of the frog's brain were cannulated with a micropipette and perfused with an artificial cerebrospinal fluid containing the dye. Vessels were occluded with a glass microneedle and the rate of change in dye concentration in a 12 microns length section was measured using video‐intensified microscopy. 3. It was found that the rate of dye loss at all points along the occluded microvessel segment could be accounted for by a model for convection and diffusion, and that changes in dye concentration at a point remote from the segment entrance can give a good measure of diffusive permeability. 4. When series of measurements were carried out on a single vessel, permeability rose over the course of 20 min. Mean permeability for all measurements was 3.01 x 10(‐5) cm sec‐1, n = 64 (mode, 2.0; range, 0.48‐9.6). The hydrostatic pressure applied during the perfusion had no effect on the measured permeability. 5. The dye concentration along the vessel axis was measured at the steady state and was shown to respond to changes in hydrostatic perfusion pressure in a way predicted by the model. This indicates that hydrostatically driven bulk flow can be important, and thus convection may account for effects previously ascribed to vesicular transcytosis. 6. The possible anatomical basis for the porous pathway is discussed in the light of recent observations on the presence of 0.5 microns perijunctional gaps, the possibility of transendothelial channels, and the unzipping of tight junctions to leave a 22 nm wide slit.
Agonist‐independent effects of muscarinic antagonists on Ca2+ and K+ currents in frog and rat cardiac cells.
Authors: Hanf R , Li Y , Szabo G , Fischmeister R
Published: December 2014

1. The whole‐cell patch clamp and intracellular perfusion techniques were used for studying the effects of atropine and other muscarinic acetylcholine receptor (mAChR) antagonists on the L‐type calcium currents (ICa) in frog and rat ventricular myocytes, and on the mAChR‐activated K+ current (IK(ACh)) in frog atrial myocytes. 2. In frog ventricular myocytes, atropine (0.1 nM to 1 microM) reversed the inhibitory effect of acetylcholine (ACh, 1 nM) on ICa previously stimulated by isoprenaline (Iso, 2 microM), a beta‐adrenergic agonist. However, in the concomitant presence of Iso, ACh and atropine, ICa was > 50% larger than in Iso alone. 3. The effects of atropine were then examined in the absence of mAChR agonists. After a preliminary stimulation of ICa with Iso (0.1 or 2 microM), atropine induced a dose‐dependent stimulation of ICa. EC50 (i.e. the concentration of atropine at which the response was 50% of the maximum) and Emax (i.e. maximal stimulation of ICa expressed as percentage increase in ICa with respect to the level in Iso alone) were respectively 0.6 nM and 35%. The stimulatory effect of atropine on ICa was not voltage dependent. 4. Atropine (1 microM) had no effect on frog ICa (i) under basal conditions, (ii) upon stimulation of ICa by the dihydropyridine agonist (‐)‐Bay K 8644 (1 microM), or (iii) when ICa had been previously stimulated by intracellular perfusion with cyclic AMP (3 microM). However, atropine increased ICa after a stimulation by forskolin (0.3 microM). Therefore, an increased adenylyl cyclase activity was required for atropine to produce its stimulatory effect on ICa. 5. The order of potency of mAChR antagonists to reverse the inhibitory effect of ACh on Iso elevated ICa in frog ventricle was atropine > AF‐DX 116 >> pirenzepine. In the absence of ACh, mAChR antagonists produced their stimulatory effect on Iso elevated ICa with the same order of potency. 6. Intracellular substitution of Gpp(NH)p (5'‐guanylylimidiphosphate) for GTP (420 microM) induced a strong inhibition of frog ICa in the presence of Iso (2 microM). This effect was attributed earlier to the spontaneous and irreversible activation of the GTP‐binding regulatory protein (G protein), Gi, responsible for adenylyl cyclase inhibition. Atropine (1 microM) slowed down by a factor of 2 the rate of ICa inhibition induced by Gpp(NH)p. 7. In frog atrial myocytes, intracellular perfusion with 1 mM Gpp(NH)p induces spontaneous activation of IK(ACh). This effect was attributed earlier to the spontaneous and irreversible activation of the G protein, GK.(ABSTRACT TRUNCATED AT 400 WORDS)
Evaluation of spontaneous baroreflex sensitivity in conscious dogs.
Authors: Frankel R A , Metting P J , Britton S L
Published: December 2014

1. We evaluated a method of measuring cardiac baroreflex sensitivity (BRS) derived from spontaneous changes in systolic pressure (SP). SP was measured from the ECG signal in seven conscious, resting dogs. 2. Beat‐to‐beat changes in PI (dPI) were positively correlated with beat‐to‐beat changes in SP (dSP) in all dogs tested, suggesting spontaneous baroreflex function. The slope of the regression of dPI on dSP was used as an index of spontaneous BRS. 3. The spontaneous BRS was abolished by hexamethonium, atropine and bilateral carotid sinus denervation. Low dose atropine sulphate produced a paradoxical increase in spontaneous BRS, which has been observed in other studies. The spontaneous BRS was positively correlated with the average pulse interval in resting dogs. 4. Random modulation of heart rate after vagotomy failed to reproduce the strong positive correlation between dSP and dPI; this demonstrated that the correlation was not the result of mechanical coupling between heart rate and arterial blood pressure. 5. The BRS was measured pharmacologically in six dogs using a bolus injection of a vasoconstrictor. The pharmacological BRS was positively correlated with the spontaneous BRS measured after the bolus injection. 6. Finally, the spontaneous BRS was negatively correlated with the average arterial pressure in resting dogs. We conclude that the spontaneous BRS is a useful quantitative indicator of baroreflex function in conscious resting dogs.
Calcium released by photolysis of DM‐nitrophen triggers transmitter release at the crayfish neuromuscular junction.
Authors: Mulkey R M , Zucker R S
Published: December 2014

1. Spontaneous and evoked transmitter release at the crayfish neuromuscular junction were potentiated in response to photolytic release of calcium from the ‘caged’ calcium compound DM‐nitrophen, which had previously been injected into presynaptic terminals. 2. The amount of calcium released from DM‐nitrophen photolysis depends on the concentration of DM‐nitrophen, its photoproducts, Ca2+, Mg2+, H+, ATP and the cell's native buffer. Since none of these are known in the crayfish terminal, the study was conducted in a qualitative fashion. 3. Photolytic release of calcium from DM‐nitrophen increased excitatory junctional potentials (EJPs) by a range of 2‐31 times over control values and the miniature excitatory junctional potential (MEJP) frequency increased from resting values of 1‐10 quanta/s to 3000‐11,000 quanta/s. 4. Extracellular calcium was not required for the light‐evoked asynchronous release of transmitter. Calcium‐bound DM‐nitrophen previously pressure injected into crayfish presynaptic terminals increased the MEJP frequency from resting values of 1‐8 quanta/s to 800‐10,000 quanta/s during photolysis in a calcium‐free cobalt Ringer solution. 5. Iontophoresis of calcium‐free DM‐nitrophen into presynaptic terminals released transmitter upon photolysis, but only in a calcium‐containing Ringer solution. This suggests that DM‐nitrophen is capable of binding calcium once injected into terminals, but this is dependent on the presence of external calcium. 6. Photolysis of DM‐nitrophen at lower light intensities produced a slower rate of transmitter release. 7. Brief light exposures, i.e. those which photolysed 5‐20% of the DM‐nitrophen, resulted in a rapid decay of postsynaptic responses on extinguishing the light, due to rebinding of photolytically released calcium to unphotolysed DM‐nitrophen. Longer light exposures which completely photolysed DM‐nitrophen, leaving only the low affinity photoproducts, produced a slow decay of transmitter release after the light pulse, presumably due to the active extrusion of calcium from the presynaptic terminals. 8. During photolysis of DM‐nitrophen, the time courses of changes in EJP amplitude and MEJP frequency were different, indicating that the two measures of transmitter release were not linearly related. 9. MEJP frequency and EJP amplitudes during DM‐nitrophen photolysis were fitted to a ‘non‐linear summation model’ in which photolytically released calcium sums with calcium entering during an action potential to evoke transmitter release with a calcium co‐operativity of five.
Mechanism of baroreceptor adaptation in dogs: attenuation of adaptation by the K+ channel blocker 4‐aminopyridine.
Authors: Chapleau M W , Lu J , Hajduczok G , Abboud F M
Published: December 2014

1. Increased arterial pressure increases baroreceptor activity but activity declines (i.e. baroreceptors adapt) as the pressure is maintained at the higher level. The purpose of this study was to investigate the role of a 4‐aminopyridine (4‐AP)‐sensitive K+ current in causing baroreceptor adaptation. 2. Multi‐ and single fibre recordings of baroreceptor activity were obtained from the vascularly isolated carotid sinus in anaesthetized dogs during step increases in carotid sinus pressure sustained for periods up to 5 min. 3. Baroreceptor activity increased with the rise in pressure, declined markedly over the first minute, and continued to decline at a slower rate during the remainder of the 5 min period of elevated pressure. Exposure of the isolated carotid sinus to 4‐AP (10(‐5) and 10(‐4) M) attenuated adaptation in a dose‐dependent and reversible manner (P < 0.05). 4‐AP attenuated the gradual decline in single fibre activity and also prevented derecruitment or dropout of fibres that occurred over time. 4‐AP did not alter peak nerve activity measured within the first 2 s of the pressure step. 4. Ouabain (5 x 10(‐7)‐10(‐6) M), an inhibitor of Na+,K(+)‐ATPase, increased baroreceptor activity but did not attenuate baroreceptor adaptation. 5. Neither 4‐AP nor ouabain altered the distensibility of the carotid sinus as measured with sonomicrometer crystals suggesting that the agents act directly on the nerve endings. 6. The results suggest that activation of a 4‐AP‐sensitive K+ current contributes significantly to baroreceptor adaptation with little or no contribution of Na+,K(+)‐ATPase.
Hyperpolarization‐activated currents in isolated superior colliculus‐projecting neurons from rat visual cortex.
Authors: Solomon J S , Nerbonne J M
Published: December 2014

1. In vivo injections of rhodamine beads into the superior colliculus of 4‐9 postnatal day rat pups label a population of layer 5 cells in the primary visual cortex that can be identified in tissue sections or dissociated cell cultures. 2. Under voltage clamp, hyperpolarizations of isolated superior colliculus‐projecting (SCP) neurons from rest elicit an instantaneous inward current (Iinst) with nearly linear current‐voltage properties that is not blocked by extracellular application of 3 mM CsCl. 3. Voltage clamp steps to potentials more negative than ‐60 mV evoke a slowly activating, non‐inactivating inward current that is not blocked by 1 microM TTX, 1 mM 4‐aminopyridine (4‐AP), 5 mM Co2+, or 25 mM TEA, but is potently blocked by extracellular application of 3 mM CsCl. This current is similar to Ih described in other systems. 4. Ih, the time‐dependent inward current in SCP neurons, begins to activate near the resting membrane potential and reaches full activation at ‐110 mV. The voltage dependence of activation is well fitted by a Boltzmann distribution with the membrane potential at half‐maximal activation (V1/2) = ‐81.0 mV and s (steepness of the curve parameter) = 7.2 mV. Thus, Ih may contribute to setting the resting membrane potential and resting input resistance of SCP neurons. 5. The inward rectification of the whole‐cell current vs. voltage relation is accounted for by the voltage dependence of Ih activation. Current through the activated h‐conductance shows slight outward rectification that is accounted for by constant field considerations. 6. The h‐conductance is substantially permeable only to sodium and potassium and, under normal physiological conditions, is expected to reverse at approximately ‐22 mV at 20 degrees C. For [K+]o < or = 20 mM and 120 mM > or = [Na+]o > or = 70 mM, Ih obeys independence with a PNa/PK (ratio of Na+ to K+ permeability) of 0.40. 7. Extracellular potassium increases gh. If this effect is modelled as the result of potassium binding to an extracellular conductance‐permitting site, potassium has an apparent dissociation constant (Kapp) of 25.7 mM and the ability to maximally increase gh in the order of 10‐fold over basal levels. 8. Ih underlies the depolarizing 'sag’ and ‘overshoot’ observed in SCP neurons following the onset and offset, respectively, of hyperpolarizing current injections. In addition, Ih appears to control the duration and the frequency of repetitive firing following the cessation of sustained hyperpolarizing current injections.(ABSTRACT TRUNCATED AT 400 WORDS)
pH recovery from intracellular alkalinization in Retzius neurones of the leech central nervous system.
Authors: Frey G , Schlue W R
Published: December 2014

1. Neutral‐carrier pH‐sensitive microelectrodes were used to investigate intracellular pH (pHi) recovery from alkalinization in leech Retzius neurones in Hepes‐ and in CO2‐HCO3(‐)‐buffered solution. The Retzius neurones were alkaline loaded by the addition and subsequent removal of 16 mM acetate, by changing from 5% CO2‐27 mM HCO3‐ to 2% CO2‐11 mM HCO3‐ or by changing from CO2‐HCO3(‐)‐ to Hepes‐buffered solution. 2. In Hepes‐buffered solution (pH 7.4) the mean pHi was 7.29 +/‐ 0.11 and the mean membrane potential ‐44.7 +/‐ 5.9 mV (mean +/‐ S.D.; n = 83). 3. The rate of pHi recovery from alkalinization increased with decreasing pH of the bathing medium (pHb). pHi changed about 0.30 pH units for a pHb unit change. 4. A decrease of extracellular buffer concentration (Hepes concentration lowered from 20 to 5 mM) caused an acidification of extracellular and intracellular pH and an acceleration of pHi recovery from alkalinization. 5. A depolarization of the Retzius cell membrane‐induced by increasing the K+ concentration of the bathing medium from 4 to 20 mM (delta Em = 16.5 +/‐ 5.5 mV) or from 4 to 40 mM (delta Em = 24.8 +/‐ 3.5 mV)‐‐evoked a decrease of pHi and an acceleration of pHi recovery from alkalinization. 6. The H+ current blocker Zn2+ (0.5 mM) inhibited pHi recovery from alkalinization at resting membrane potential as well as during depolarization. The inhibition was more pronounced during depolarization. 7. In Cl(‐)‐free, CO2‐HCO3(‐)‐buffered solution pHi recovery from an alkaline load by changing from 5% CO2‐27 mM HCO3‐ to 2% CO2‐11 mM HCO3‐ was slowed by 48‐71%. The rate of pHi recovery from an alkaline load induced by changing from CO2‐HCO3‐ to Hepes buffer was reduced by 33‐56% in Cl(‐)‐free solution. The removal of external Cl‐ did not affect pHi recovery in Hepes‐buffered solution. 8. The pHi recovery from alkalinization was DIDS‐insensitive in CO2‐HCO3(‐)‐ as in Hepes‐buffered solutions and was not slowed in the absence of external Na+. 9. It is concluded that in Retzius neurones pHi recovery from alkalinization is mediated by a passive voltage‐dependent H+ influx along the electrochemical proton gradient. In the presence of CO2‐HCO3‐ buffer a DIDS‐insensitive Cl(‐)‐HCO3‐ exchanger additionally regulates pHi after an intracellular alkaline load. It cannot be excluded that intracellular processes (e.g. H+ release from organelles, metabolic H+ production) are also involved in pHi recovery from alkalinization.
Electrophysiological properties of axotomized facial motoneurones that are destined to die in neonatal rats.
Authors: Umemiya M , Araki I , Kuno M
Published: December 2014

1. Rat facial motoneurones axotomized on the day after birth were examined morphologically, and their electrical properties were characterized using the whole‐cell recording technique in thin slices of the brainstem. 2. About 40% of facial motoneurones were lost within 4 days of axotomy, and only about 20% of the neurones survived 9 days after axotomy. 3. The surviving facial motoneurones examined 4 or 6 days after axotomy were reduced in size, and this was associated with a decrease in their input capacitance. 4. Both the resting potential and the amplitude of action potentials remained unchanged in axotomized facial motoneurones. 5. Facial motoneurones examined 4 or 6 days after axotomy showed an increase in the spike duration. When the preparation was superfused with a Ca(2+)‐free solution, the spike duration of axotomized facial motoneurones was shortened, whereas the spike duration of control facial motoneurones was prolonged. 6. The voltage‐gated transient K+ current (IA) density was significantly reduced in axotomized motoneurones, whereas the Ca(2+)‐dependent transient K+ current (IK, Ca) density was not affected. 7. Voltage‐gated Ca2+ currents in facial motoneurones showed inactivation, displaying an initial transient phase followed by a sustained phase. The rate of inactivation of Ca2+ currents was significantly faster in axotomized neurones than in control neurones. 8. A small subpopulation of facial motoneurones examined 4 or 6 days after axotomy had a disproportionately high input resistance and a significantly longer after‐hyperpolarization. The probability of this occurring was correlated with the time course of cell death induced by axotomy. 9. It is concluded that facial motoneurones axotomized in neonatal rats comprise two subpopulations. The subpopulation characterized by a markedly high input resistance is suggested to represent the neurones which are at the ‘prelethal’ stage or in the process of cell death.
Two types of 4‐aminopyridine‐sensitive potassium current in rabbit Schwann cells.
Authors: Baker M , Howe J R , Ritchie J M
Published: December 2014

1. Delayed rectifier K+ currents were studied in Schwann cells cultured from neonatal rabbit sciatic nerves with the whole‐cell patch‐clamp technique. 2. Depolarizing voltage steps (40 ms duration) activated two types of K+ current: type I, whose apparent activation threshold was about ‐60 mV (half‐maximal conductance at ‐40 +/‐ 1 mV, n = 10); and type II, whose apparent activation threshold was about ‐25 mV (half‐maximal conductance at + 11 +/‐ 1 mV, n = 9). 3. Type I current was blocked by alpha‐dendrotoxin (alpha‐DTX) with an apparent equilibrium dissociation constant (KD) of 1.3 nM, whereas the type II current was unaffected by exposure to 500 nM toxin. The action of alpha‐DTX on the type I current was reversible. 4. Most cells exhibited both types of current, but occasionally some cells displayed just type I or just type II. 5. Type I current activated rapidly and then showed a much slower fade, which became more noticeable with larger depolarizations. Activation of type II current was slower than that of type I and depended less steeply on voltage. The time constants of activation for type I and type II currents were derived with a Hodgkin‐Huxley formalism (based on second‐power activation and deactivation kinetics). The longest activation time constant for type II gating was more than twice the corresponding time constant for type I; however, the time constants determined from tail current decays at potentials more negative than ‐60 mV were shorter for the type II currents than for the type I currents. 6. Both type I and type II currents were sensitive to micromolar concentrations of 4‐aminopyridine (4‐AP). The KD for 4‐AP blockade of type II current was 630 microM (pH 7.2, membrane potential (Em) = ‐10 mV), which is about 6 times higher than the corresponding value for 4‐AP blockade of type I current at negative membrane potentials. The differential sensitivity of the type I and type II currents to 4‐AP may account for the apparent voltage dependence of 4‐AP block of delayed rectifier K+ currents. 7. In addition to types I and II, a third type of outward K+ current (type III) was generated in most cells at positive membrane potentials. This latter current was insensitive to millimolar concentrations of 4‐AP. 8. Similarities between Schwann cell and neuronal potassium channels are discussed.
Materials fatigue initiates eccentric contraction‐induced injury in rat soleus muscle.
Authors: Warren G L , Hayes D A , Lowe D A , Prior B M , Armstrong R B
Published: December 2014

1. The initiation of exercise‐induced muscle injury is thought to be the result of high tensile stresses produced in the muscle during eccentric contractions. Materials science theory suggests that high tensile stresses could initiate the injury during the first eccentric contraction (normal stress theory) or after multiple eccentric contractions (materials fatigue). It was the objective of this study to investigate the two possibilities. 2. Rat soleus muscles (n = 66; 11 protocols with 6 muscles per protocol) were isolated, placed in an oxygenated Krebs‐Ringer buffer at 37 degrees C, and baseline measurements were made. The muscle then performed an injury protocol which consisted of between zero and ten eccentric contractions (muscle starting length = 0.90 soleus muscle length, L0; length change = 0.25 L0; velocity = 1.5 L0/s; peak force = 180% maximal isometric tetanic tension (P0); time between contractions = 4 min; total duration of the injury protocol = 40 min). At the end of the injury protocol, the muscle was incubated in buffer for 1 h; every 15 min, an isometric twitch and tetanus were performed and lactate dehydrogenase (LDH) release was measured. Total muscle [Ca2+] was measured at the end of the incubation. 3. Change‐point regression analysis indicates that at 0 min into the incubation, declines in P0, maximal rate of tension development (+dP/dt), maximal rate of relaxation (‐dP/dt), and muscle stiffness (dP/dx) became significantly greater after eight eccentric contractions (p < or = 0.05). No relation was found between the number of eccentric contractions performed and the LDH activity at 0 min into the incubation, although after 60 min of incubation, LDH activity in the buffer was linearly related to eccentric contraction number (p = 0.01). There was no relationship between total muscle [Ca2+] and eccentric contraction number. These findings support the materials fatigue hypothesis of exercise‐induced muscle injury.
Enhanced expression of Ca2+ channels by nerve growth factor and the v‐src oncogene in rat phaeochromocytoma cells.
Authors: Lewis D L , De Aizpurua H J , Rausch D M
Published: December 2014

1. Rat phaeochromocytoma (PC12) cells were used to investigate the expression of Ca2+ channel types during neuronal differentiation. Neuronal differentiation was induced by treatment with nerve growth factor (NGF) or by activation of a temperature‐sensitive tyrosine kinase (pp60v‐src) in genetically modified PC12 (PC12/v‐src) cells. PC12 cells differentiated morphologically in the presence of NGF. When grown at the permissive temperature of 37 degrees C which activates the kinase activity of pp60v‐src, PC12/v‐src cells differentiated morphologically with the extension of neurites. In contrast, PC12/v‐src cells grown at the non‐permissive temperature of 40 degrees C continued to divide and were morphologically indistinguishable from control PC12 cells. 2. Whole‐cell Ca2+ currents were measured in PC12 cells using Ba2+ as the charge carrier. Ba2+ currents measured at the peak of the current‐voltage curve from a holding potential of ‐80 mV were ‐0.28 +/‐ 0.04 nA (mean +/‐ S.E.M.) in control PC12 cells compared to ‐1.25 +/‐ 0.16 nA in NGF‐differentiated cells. The current density increased from 9.4 +/‐ 0.7 pA/pF in control PC12 cells to 22.8 +/‐ 2.4 pA/pF in NGF‐differentiated PC12 cells. Ba2+ currents were ‐0.24 +/‐ 0.04 nA in undifferentiated PC12/v‐src cells grown at the non‐permissive temperature of 40 degrees C compared to ‐0.95 +/‐ 0.16 nA in differentiated PC12/v‐src cells grown at the permissive temperature of 37 degrees C. The current density increased from 4.5 +/‐ 0.5 pA/pF in PC12/v‐src cells grown at the non‐permissive temperature of 40 degrees C to 13.3 +/‐ 2.4 pA/pF in PC12/v‐src cells grown at the permissive temperature of 37 degrees C. 3. The sensitivity of Ba2+ currents to omega‐conotoxin GVIA (omega‐CgTX) was determined for currents measured at the peak of the current‐voltage curve (0 mV in 10 mM Ba2+) from a holding potential of ‐80 mV. In NGF‐differentiated PC12 cells, 10 microM omega‐CgTx inhibited 68.1 +/‐ 3.2% of the total Ba2+ current compared to 35.9 +/‐ 4.1% in control cells. The density of the omega‐CgTX‐sensitive current increased from 3.3 +/‐ 0.4 pA/pF in control cells to 15.7 +/‐ 2.0 pA/pF in NGF‐differentiated cells. In differentiated PC12/v‐src cells grown at 37 degrees C, omega‐CgTX inhibited 52.2 +/‐ 4.2% of total Ba2+ current compared to 41.1 +/‐ 3.8% in PC12/v‐src cells grown at 40 degrees C. The density of the omega‐CgTX‐sensitive current increased from 1.9 +/‐ 0.3 to 7.4 +/‐ 2.0 pA/pF with v‐src‐mediated differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)
Effect of extravascular plasma protein on pressure‐flow relations across synovium in anaesthetized rabbits.
Authors: McDonald J N , Levick J R
Published: December 2014

1. The effect of extravascular plasma protein on fluid flux through interstitial matrix was investigated in vivo by studying the pressure‐flow relation across synovium during intra‐articular infusions of protein solutions (usually bovine serum albumin). Synovium is a sheet of non‐epithelial cells separated by interstitium‐filled gaps, beneath which are fenestrated capillaries: synovium regulates synovial fluid volume and composition. 2. Albumin solutions (10‐150 g l‐1) of measured oncotic pressure and viscosity were infused at known pressure into the synovial cavity of knees of anaesthetized rabbits. Flow across the synovial lining in the steady state (absorption rate Qs) was recorded at a series of joint pressures (Pj) to define the pressure‐flow relation. Krebs solution was infused into the opposite knee as a control (26 animals). 3. Infusion of a low albumin concentration (10 g l‐1, bovine or rabbit) or diluted rabbit serum revealed no specific effect of plasma protein on interstitial matrix permeability (cf. specific protein effect on capillary glycocalyx permeability). Physiological (22.5 g l‐1) and higher concentrations reduced trans‐synovial absorption rate. The slope of the pressure‐flow relation was reduced and the pressure intercept displaced to the right (i.e. Pj at zero flow was raised). 4. Slope dQs/dPj correlated negatively with intra‐articular viscosity (P = 0.001‐0.04), in keeping with viscous interstitial flow. The reduction in normalized slope, however, did not equal the reduction in fluidity (1/viscosity) quantitatively. It is proposed that apparent fluidity within the interstitial matrix is higher than in the bulk phase due to steric exclusion of albumin (radius 3.55 nm) by the interstitial glycosaminoglycans. The latter form spaces of estimated mean hydraulic radius 14‐18 nm in synovium. 5. The joint‐pressure intercept at zero net trans‐synovial flow was displaced 0.015 cmH2O per cmH2O intra‐articular oncotic pressure (pi j; S.E.M. +/‐ 0.006). Thus large trans‐synovial osmotic gradients were not maintained at physiological flow velocities. The 1.5% displacement of the Pj intercept by pi j was attributed principally to interstitial albumin exerting pericapillary oncotic pressure and enhancing net Starling filtration pressure. Indeed, net trans‐synovial flow at low joint pressure sometimes reversed from absorption to filtration into the joint cavity at high intra‐articular oncotic pressures. 6. The displacement of the trans‐synovial flow intercept per unit change in intra‐articular oncotic pressure, (dQs/d pi j)Pj = 0, was 18 +/‐ 3 nl min‐1 cmH2O‐1.(ABSTRACT TRUNCATED AT 400 WORDS)
The effects of inhibitors of sarcoplasmic reticulum function on the systolic Ca2+ transient in rat ventricular myocytes.
Authors: Negretti N , O'Neill S C , Eisner D A
Published: December 2014

1. The effects of thapsigargin, ryanodine and caffeine were examined on systolic Ca2+ transients in indo‐1‐loaded rat ventricular myocytes. 2. Thapsigargin (1‐10 microM) decreased the magnitude of the Ca2+ transient. This was accompanied by a decrease of the rate constant of decay of the transient. 3. Ryanodine (1‐10 microM) decreased the magnitude of the Ca2+ transient. Initially there was no change in the rate of decay but further reduction of the magnitude was accompanied by a slowing. 4. Caffeine (0.5‐10 mM) decreased the magnitude of the Ca2+ transient and its rate of decay. These effects were graded with caffeine concentration. 5. For a given submaximal reduction of the magnitude of the Ca2+ transient, the effect on the rate of decay was greatest for thapsigargin, least for ryanodine and intermediate for caffeine. 6. The above data are reproduced by a model in which all three agents decrease the magnitude of the Ca2+ transient by decreasing the calcium content of the sarcoplasmic reticulum (SR) (thapsigargin by inhibiting the Ca2+ pump and ryanodine and caffeine by increasing the leak of Ca2+ from the SR). The decreased contribution of the SR will thereby slow relaxation. The fact that thapsigargin inhibits the SR Ca2+ pump accounts for the observation that, for a given decrease of amplitude, it has more effect than the other agents on the rate of decay. The difference between caffeine and ryanodine is suggested to arise because caffeine potentiates Ca2+ release from the SR and thereby attenuates the effect of the decreased SR calcium content on the magnitude of the Ca2+ transient.
Anomalous permeation of Na+ through a putative K+ channel in rat superior cervical ganglion neurones.
Authors: Zhu Y , Ikeda S R
Published: December 2014

1. An unanticipated inward tail current was recorded from freshly isolated adult rat superior cervical ganglion (SCG) neurones using the whole‐cell variant of the patch‐clamp technique. The tail current was present when Na+ was substituted for tetraethylammonium (TEA) as the primary monovalent cation in external solutions designed to isolate Ca2+ channel currents (0.5 microM tetrodotoxin present and K+ omitted). 2. The tail current was observed following step potentials positive to ‐30 mV and reached half‐activation near ‐9.0 mV. The decay of the tail current was voltage dependent and could be described with two time constants. Between potentials of ‐120 and ‐70 mV, tau f, the fast component, varied from 3 to 8 ms and tau s, the slow component, changed from 12 to 30 ms, respectively. 3. The tail current was not carried by Ca2+, and did not appear to flow through a voltage‐gated Ca2+ channel or a Ca(2+)‐dependent channel as it persisted in the absence of external Ca2+ or in the presence of the Ca2+ channel blocker, Cd2+ (0.1 mM). 4. Varying the external [Cl‐] did not alter the reversal potential of the tail current indicating that Cl‐ was not the charge carrier. 5. The reversal potential of the tail current changed in accordance with the Nernst relationship when [Na+]i/[Na]o was altered. Our results suggested that this ‘unusual or unanticipated current’ (Iu) was carried primarily by Na+. 6. Iu was inhibited by the K+ channel‐blocking agents quinidine (0.1 mM), external Ba2+ (5 mM) and internal Cs+ (145 mM). TEA (20 mM either internally or externally) and dendrotoxin (10 microM) were not effective inhibitors of Iu. 7. The decay time constants of the tail current and parameters of activation and inactivation of Iu were similar to those of TEA‐insensitive delayed rectifier‐type K+ channel currents observed in the presence of 145 mM external K+. 8. Iu was reduced in the presence of either external or internal K+. The interaction of external K+ with Na+ on the Iu tail amplitude was reminiscent of anomalous mole‐fraction behaviour. 9. Ion permeability studies revealed that the channel producing Iu had a permeability sequence to monovalent cations of 3.5:2.5:2:1:0.5 for Rb+, K+, Cs+, Na+ and Li+, respectively. 10. These data suggest that in the absence of external K+, the ion selectivity of a TEA‐insensitive K+ channel in sympathetic neurones is profoundly diminished. Under these conditions, Na+ traversing a K+ channel can generate an unanticipated inward current.(ABSTRACT TRUNCATED AT 400 WORDS)
Convective fluid flow through the paracellular system of Necturus gall‐bladder epithelium as revealed by dextran probes.
Authors: Shachar‐Hill B , Hill A E
Published: December 2014

1. Bidirectional paracellular fluxes using radioactive dextrans as inert molecular probes have been measured across Necturus gall‐bladder epithelium during conditions of normal fluid absorption. There is a net flux at all radii analysed (0.4‐2.2 nm) in the direction of fluid absorption. 2. The net flux is substantial at all radii within the range. The data extraplate to 2 x 10(‐6) cm s‐1 at zero probe radius, which is very close to the rate of epithelial fluid absorption. 3. The unstirred layers at the epithelial faces during transport have been determined; their contribution to the net fluxes is negligible. 4. Two possible mechanisms for the net flow of probes are considered: (i) that the probes diffuse across the junctions and are then entrained in a local osmotic flow along the interspaces and subepithelium; (ii) that the probes are entrained in volume flow across the junctions and the emergent solution subsequently passes through the interspaces and subepithelium. Model calculations clearly rule out mechanism (i) in which the maximum net flow obtainable is less than 10% of that observed. In addition the presence of leak paths shunting the junctions is not compatible with the observed fluxes. With mechanism (ii) the net flows are correctly predicted with all the fluid flow being transjunctional. The fluid absorption is therefore entirely paracellular. 5. The slope of the net flow curve shows no apparent change in magnitude over the range of the probe radii, indicating that effectively only one population of convective channels is present with parallel walls separated by about 7.7 nm. This agrees with the width previously determined by electron microscopy. 6. If the fluid absorption is junctional then the cellular route offers little if any relative contribution. The hydraulic conductivity of the junctions is not high enough, or the osmotic permeability of the membranes low enough, to accommodate this by osmosis and therefore the junctional fluid absorption must be non‐osmotic.
Response of sensory units with unmyelinated fibres to mechanical, thermal and chemical stimulation of the cat's cornea.
Authors: Gallar J , Pozo M A , Tuckett R P , Belmonte C
Published: December 2014

1. In the cat anaesthetized with sodium pentobarbitone, electrical activity was recorded from single unmyelinated sensory fibres innervating the cornea. 2. Based on their response to mechanical (calibrated aesthesiometer), chemical (10 mM acetic acid or 616 mM NaCl) and thermal (cooling from 35 to 5 degrees C; heating to 51 degrees C) stimuli, corneal unmyelinated fibres were classified as polymodal (71%) or ‘cold’ nociceptors (29%). 3. Polymodal units responded to mechanical indentation of the cornea and developed fatigue after repeated stimulation. They were excited by temperatures over 37 degrees C and exhibited sensitization to repeated heating. 4. Corneal polymodal units were also activated by topical application of 10 mM acetic acid and hypertonic NaCl (616 mM). Capsaicin (0.33 mM) elicited a discharge of impulses that was followed by inactivation to mechanical, chemical and thermal stimuli. 5. ‘Cold’ nociceptors had small receptive fields, preferentially located at the periphery of the cornea. They were excited by small temperature decreases of the corneal surface in a range between 30 and 8 degrees C, but were not responsive to noxious heat. 6. ‘Cold’ nociceptors encoded temperature changes between 35 and 23 degrees C. The discharge was proportional to the velocity of the temperature drop; sustained temperatures were not signalled by changes in static frequency values. ‘Cold’ nociceptive fibres responded to hypertonic NaCl (616 mM) and weakly to 10 mM acetic acid. Capsaicin (0.33 mM) first excited and then inactivated ‘cold’ nociceptors. 7. Thermoreceptive fibres were found in the episclera. They fired in bursts and responded to small temperature decreases, but were insensitive to irritant chemical and capsaicin.
Changes of intracellular pH in rat mesenteric vascular smooth muscle with high‐K+ depolarization.
Authors: Austin C , Wray S
Published: December 2014

1. In mesenteric vascular smooth muscle cells changes in extracellular pH are rapidly transmitted to the cytoplasm. The mechanism involved is unknown, but it may be due to a high proton permeability of the surface membrane, in which case changes in membrane potential would alter the driving force for proton entry. We have therefore examined the voltage sensitivity of intracellular pH (pHi) in these cells. 2. Strips of mesenteric resistance vessels were loaded with SNARF‐1 to monitor pHi and tension was simultaneously measured. Tissues were superfused with oxygenated solutions at 37 degrees C and pH 7.4. Isosmotic substitution of K+ for Na+ was used to depolarize the preparations. 3. pHi was found to be sensitive to alteration of [K+]. Depolarization of the tissue with K+ caused contraction and produced transient increases in pHi. When pHi regulation was blocked there was no significant change in the size of the alkalinization induced by high K+, thus it is unlikely that the results can be explained by voltage sensitivity of pHi regulating mechanisms. 4. In nominally Ca(2+)‐free solution, the tissue does not contract and the alkalinization with high K+ was significantly greater than that occurring in 3 mM Ca2+. 5. There was a rapid acidification, when pHi regulation was blocked, which is consistent with a high proton permeability. 6. The effects of membrane potential on pHi have been modelled and show that they can be accounted for by effects of voltage on H+ influx through a proton channel. The effects of changing external pH on H+ influx also fit the model. Estimation of the proton permeability gave a high value (0.4 cm s‐1). 7. The results presented demonstrate (i) a voltage sensitivity of pHi in mesenteric vascular smooth muscle cells and (ii) a particularly high permeability of the membrane to protons. The physiological significance of these findings is discussed.
Mobile and immobile calcium buffers in bovine adrenal chromaffin cells.
Authors: Zhou Z , Neher E
Published: December 2014

1. The calcium binding capacity (kappa S) of bovine chromaffin cells preloaded with fura‐2 was measured during nystatin‐perforated‐patch recordings. 2. Subsequently, the perforated patch was ruptured to obtain a whole‐cell recording situation, and the time course of kappa S was monitored during periods of up to one hour. 3. No rapid change (within 10‐20 s) of kappa S was observed upon transition to whole‐cell recording, as would be expected, if highly mobile organic anions contributed significantly to calcium buffering. However, approximately half of the cells investigated displayed a drop in kappa S within 2‐5 min, indicative of the loss of soluble Ca2+ binding proteins in the range of 7‐20 kDa. 4. The average Ca2+ binding capacity (differential ratio of bound calcium over free calcium) was 9 +/‐ 7 (mean +/‐ S.E.M.) for the poorly mobile component and 31 +/‐ 10 for the fixed component. It was concluded that a contribution of 7 from highly mobile buffer would have been detected, if present. Thus, this value can be considered as an upper bound to highly mobile Ca2+ buffer. 5. Both mobile and fixed calcium binding capacity appeared to have relatively low Ca2+ affinity, since kappa S did not change in the range of Ca2+ concentrations between 0.1 and 3 microM. 6. It was found that cellular autofluorescence and contributions to fluorescence of non‐hydrolysed or compartmentalized dye contribute a serious error in estimation of kappa S. ‘Balanced loading’, a degree of fura‐2 loading such that the calcium binding capacity of fura‐2 equals cellular calcium binding capacity, minimizes these errors. Also, changes in kappa S at the transition from perforated‐patch to whole‐cell recording can be most faithfully recorded for similar degrees of loading in both situations. 7. Nystatin was found unable to make pores from inside of the plasma membrane of chromaffin cells. With careful preparation and storage the diluted nystatin solution maintained its high activity of membrane perforation for more than one week. 8. An equation for the effective diffusion constant for total cytoplasmic calcium, D'Ca, was derived, which takes into account fixed buffer and poorly mobile buffer as determined, as well as calcium bound to fura‐2 and some highly mobile buffers.(ABSTRACT TRUNCATED AT 400 WORDS)
Does endogenous peripheral arginine vasopressin have a role in the febrile responses of conscious rabbits?
Authors: Milton N G , Hillhouse E W , Milton A S
Published: December 2014

1. The actions of peripheral arginine vasopressin (AVP) on the febrile responses of conscious rabbits induced by peripherally administered polyinosinic:polycytidylic acid (poly(I).poly(C)) have been studied using an AVP V1 receptor antagonist ([deamino‐Pen1, O‐Me‐Tyr2, Arg8]‐vasopressin). 2. Temperature responses were monitored continuously using rectal thermistor probes. Test substances were administered intravenously (i.v.). Blood samples were taken at timed intervals from a marginal ear vein and plasma PGE2 and PGF2 alpha levels determined by radioimmunoassay. 3. Poly(I).poly(C) (2.5 micrograms/kg) stimulated a reproducible biphasic rise in body temperature with a lag phase of 45‐60 min and peaks at 90 and 225 min. The febrile response was accompanied by a 5‐fold rise in circulating immunoreactive (ir) PGE2, which peaked after 90 min and remained elevated up to 300 min. Poly(I).poly(C) also stimulated a 2.5‐fold rise in circulating irPGF2 alpha, which peaked after 150 min and was followed by a return to basal levels after 300 min. 4. The overall magnitude of the febrile response to poly(I).poly(C) (2.5 micrograms/kg, i.v.) was significantly antagonized by the AVP V1 receptor antagonist (250 micrograms/kg, i.v.) administered 5 min prior to the pyrogen. 5. The irPGE2 response to poly(I).poly(C) (2.5 micrograms/kg, i.v.) was significantly antagonized by the AVP V1 receptor antagonist (250 micrograms/kg, i.v.) administered 5 min prior to the pyrogen. The irPGF2 alpha response was only reduced at the peak 150 min time point measurement. 6. In conclusion, these results show a modulatory role for a peripherally administered AVP V1 antagonist in the febrile responses to poly(I).poly(C), suggesting a possible propyretic role for endogenous peripheral AVP. This modulatory role appears to be mediated via actions on prostaglandin E2.
The electrophysiological effects of endogenous GABA in the guinea‐pig inferior mesenteric ganglion.
Authors: Stapelfeldt W H , Parkman H P , Szurszewski J H
Published: December 2014

1. GABA receptor‐modulating drugs and intracellular recording techniques were used to determine the functional significance of peripheral afferent GABA‐containing nerves projecting from the distal colon to sympathetic neurones in the inferior mesenteric ganglion of the guinea‐pig. 2. GABAA receptor‐modulating drugs added selectively to the inferior mesenteric ganglion side of a two‐compartment bath had pronounced effects on on‐going colonic afferent cholinergic synaptic input. Bicuculline (20 microM) decreased the amplitude and frequency of fast excitatory postsynaptic potentials (EPSPs) by 40% whereas diazepam (5 microM) increased cholinergic input by 43%. Neither drug had any effect on the resting membrane potential or membrane input resistance of ganglion cells. 3. Bicuculline (20 microM) significantly reduced, whereas diazepam (5 microM) significantly enhanced, distension‐induced increases in nicotinic fast EPSPs and action potentials. 4. Slow EPSPs evoked by colonic distension were not affected by bicuculline or diazepam. 5. Manual voltage clamp of the postsynaptic depolarizing response to exogenous GABA revealed GABA‐induced presynaptic facilitation of colonic afferent but not central preganglionic efferent cholinergic synaptic input. 6. The data suggest that endogenously released GABA participates in on‐going colo‐colonic reflex activity by acting on presynaptic GABAA receptors to facilitate release of acetylcholine from colonic mechanosensory nerves.