bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel tetrodotoxin‐resistant sodium current from an immortalized neuroepithelial cell line.
Authors: Zhang X , Phelan K D , Geller H M
Published: December 2014

1. Voltage‐gated ionic currents were recorded from cells of an immortalized neuroepithelial cell line named V1. The cell line was produced by insertion of the temperature‐sensitive tsA58 allele of the SV40 large T‐antigen into embryonic day 14 mouse hypothalamic cells. V1 cells display a mixed immature neural‐glial phenotype and have two phenotypes, round and flat. 2. Recordings from round V1 cells demonstrate voltage‐gated Na+ and K+ currents (n = 297), while no voltage‐gated currents were observed in flat V1 cells (n = 45). Voltage‐gated currents were recorded from cells cultured at both permissive and restrictive temperatures. 3. Internal Cs+ and external tetraethylammonium (TEA) were used to suppress outward currents. The remaining inward current has rapid activation and inactivation time constants which decreased as the test potential increased. In different cells, the amplitude of the peak inward current varied from about 50 pA to as large as 4500 pA (in 120 mM external Na+). The reversal potential for the inward current was close to the predicted Nernst equilibrium potential for Na+. Both the magnitude and reversal potential of the inward current were dependent on the external Na+ concentration. It is therefore considered to be a Na+ current, INa. 4. INa was found to be TTX resistant. About 5% of the INa was blocked by 200 nM TTX and 20 microM TTX fully suppressed the Na+ current. The apparent Kd for TTX blockade was estimated to be 1.49 microM. 5. The activation kinetics of INa could be described by a Hodgkin‐Huxley model with an m3 variable. The time constants of activation and inactivation of INa are fast, similar to those of the TTX‐resistant and TTX‐sensitive Na+ currents in central nervous system neurons and glial cells. 6. The divalent and trivalent cations Cd2+, Co2+, Ni2+, Zn2+ and La3+ shifted the activation of INa to more positive potentials and decreased the maximal conductance in a dose‐dependent manner. The apparent Kd values for blockade of the INa by Cd2+, Co2+, Ni2+, Zn2+ and La3+ were 430, 3500, 1900, 83 and 202 microM, respectively. 7. The addition of phorbol myristate acetate, an activator of protein kinase C, consistently produced a reduction in the amplitudeof INa without affecting the time course of activation or inactivation. 8. INa in V1 cells expresses a unique combination of voltage and time kinetics and sensitivity to blockade by TTX and cations. We hypothesize that this Na+ current may be expressed transiently during development of the central nervous system at the stage of development represented by the V1 cell line.
Modulation of the hyperpolarization‐activated Cl‐ current in human intestinal T84 epithelial cells by phosphorylation.
Authors: Fritsch J , Edelman A
Published: December 2014

1. Hyperpolarization‐activated Cl‐ currents (ICl,hyp) were investigated in the T84 human adenocarcinoma cell line, using the patch‐clamp whole‐cell configuration. 2. During whole‐cell recording with high‐chloride and ATP‐containing internal solutions, hyperpolarizing jumps from a holding potential of 0 mV elicited slow inward current relaxations, carried by Cl‐ and detected at membrane potentials more negative than ‐40 mV. Analysis of the relative permeabilities to monovalent anions gave the following sequence: Cl‐ > Br‐ > I‐ > glutamate. 3. ICl,hyp was partially inhibited by 1 mM diphenylamine‐2‐carboxylic acid or 0.1 mM 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate, and was completely blocked by Cd2+ (> 300 microM). It was insensitive to 1 mM external 4,4'‐diisothiocyanatostilbene‐2,2'‐disulphonic acid or 1 mM Ba2+. 4. ICl,hyp was inhibited by external application of 500 microM cptcAMP (8‐(4‐chlorophenylthio)‐adenosine 3':5'‐cyclic monophosphate) or 500 nM of the protein kinase C activator, phorbol 12‐myristate, 13‐acetate. 5. (i) Omission of ATP from the pipette solution, (ii) ATP replacement by the non‐hydrolysable ATP analogue 5'‐adenylylimidodiphosphate, and (iii) inhibition of protein kinase C by staurosporine or calphostin C accelerated the activation kinetics of the current and increased its amplitude, but did not alter its pharmacological properties. 6. We conclude that hyperpolarization‐activated Cl‐ channels similar to those of ClC‐2 channels (mammalian homologue of Torpedo chloride channel ClC‐0) are present in T84 cells, and that their gating properties are modulated by phosphorylation.
Functional N‐methyl‐D‐aspartate receptors in clonal rat phaeochromocytoma cells.
Authors: Casado M , López-Guajardo A , Mellström B , Naranjo J R , Lerma J
Published: December 2014

1. To characterize from a molecular and functional point of view the endogenous NMDA receptors expressed by phaeochromocytoma (PC12) cells, experiments involving polymerase chain reaction (PCR) amplification, Western blotting and patch‐clamp analysis of undifferentiated and nerve growth factor (NGF)‐differentiated PC12 cells were performed. 2. Analysis of PC12 mRNA demonstrated the presence of NMDAR1 and NMDAR2C transcripts. The NMDAR1 subunits lack the amino terminal insert of twenty‐one amino acid residues, where as transcripts with and without deletions I and II at the 3' end of the coding region were detected. Thus, NMDA receptors of the PC12 cells might include NMDAR1A, NMDAR1E, NMDAR1C and NMDAR1D subunits. 3. Differentiation by NGF treatment of PC12 cells did not alter mRNA expression for NMDA receptor subunits significantly but induced an increase in both the NMDAR1 protein and the total amount of functional receptors that correlated well with a parallel increase in membrane area. 4. NMDA receptors in differentiated PC12 cells had a high affinity for both glutamate and glycine. These were estimated kinetically as 0.59 microM and 74 nM, respectively. Responses to glutamate or NMDA were non‐desensitizing in the presence of saturating glycine, but slowly desensitized with low concentrations of glycine. Currents were completely blocked by D‐aminophosphonovalerate (APV), 7‐Cl‐kynurenate and phencyclidine, and showed a voltage‐dependent magnesium blockade. Spermine did not potentiate but inhibited NMDA receptor‐mediated responses in a voltage‐independent manner. 5. With 0.5 mM Ca2+, single‐channel analysis revealed very brief openings (mean open time (t(o)) = 0.42 ms), with at least two conductive states, 55 and 33 pS, both having markedly low open probability. At 2 mM Ca2+, conductances were reduced to 39 and 19 pS, without an effect in open probability or mean open time. 6. The functional properties of NMDA receptors in PC12 cells were very similar to those described for NMDAR1A‐NMDAR2C heteromers recombinantly expressed. The PC12 cell line provides a simple and reproducible system to analyse some specific NMDA receptor properties.
Postsynaptic modulation of NMDA synaptic currents in rat hippocampal microcultures by paired‐pulse stimulation.
Authors: Mennerick S , Zorumski C F
Published: December 2014

1. Paired‐pulse synaptic stimulation of hippocampal neurons in microcultures resulted in depression of synaptic currents mediated by both NMDA and alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptors. However, NMDA EPSCs were more severely depressed than AMPA EPSCs. 2. Partial NMDA receptor blockade reduced paired‐pulse depression of NMDA but not of AMPA synaptic currents while partial AMPA receptor blockade had no effect on paired‐pulse depression of AMPA EPSCs. These results suggest that ion flux through NMDA receptors is important in paired‐pulse depression of NMDA responses but has no effect on AMPA responses. 3. Low extracellular Ca2+ concentrations or positive postsynaptic holding potentials reduced paired‐pulse depression of NMDA EPSCs to near that of AMPA responses. 4. Brief paired applications of exogenous glutamate to neurons produced Ca(2+)‐dependent depression similar to the depression of NMDA synaptic responses and synaptic stimulation depressed responses to exogenously applied NMDA. 5. Physiological concentrations of Mg2+ prevented expression of the postsynaptic modulation of NMDA EPSCs at ‐70 mV, but partial relief of Mg2+ block of the NMDA channel with depolarization increased paired‐pulse depression of NMDA EPSCs.
Enhancement of delayed rectifier K+ current by P2‐purinoceptor stimulation in guinea‐pig atrial cells.
Authors: Matsuura H , Tsuruhara Y , Sakaguchi M , Ehara T
Published: December 2014

1. We studied the effects of P2‐purinoceptor stimulation on the delayed rectifier K+ current (IK) in guinea‐pig atrial myocytes using a whole‐cell voltage‐clamp technique. 2. External application of ATP increased IK, evoked by a 500 ms depolarizing pulse from a holding potential of ‐40 mV, under conditions in which the L‐type Ca2+ channel was blocked; the effect was dose dependent with a half‐maximal concentration (K1/2) of 0.95 microM. ATP (50 microM) produced a maximal increase of IK of about a factor of 2. 3. External ADP also enhanced IK in a dose‐dependent manner with a K1/2 of 3.65 microM, whereas adenosine (100 microM) failed to evoke this response. Theophylline (500 microM), a blocker of the Pi‐purinoceptor, did not antagonize the stimulating action of ATP on IK. These results indicate that IK was enhanced via P2‐purinoceptors. 4. External ATP or ADP did not produce a significant change in the current kinetics of IK. 5. Pre‐incubation of the atrial myocytes with pertussis toxin (PTX, 5 micrograms ml‐1) did not affect the stimulating action of ATP on IK, indicating that PTX‐sensitive G proteins did not mediate the ATP action. 6. The enhancement of IK by ATP developed slowly; the effects usually reached a maximum approximately 30‐60 s after the application of ATP. This suggests the involvement of a diffusible cytosolic second messenger(s) in the response. ATP could further increase IK after maximal enhancement by isoprenaline (0.5‐1.0 microM), suggesting that the intermediate steps were independent of cyclic AMP‐dependent protein kinase (protein kinase A). 7. Potentiation of IK by ATP was not attenuated by either (i) pretreatment of the cells with 5 microM 1‐(5‐isoquinolinylsulphonyl)‐2‐methylpiperazine dihydrochloride (H‐7) or (ii) intracellular perfusion of 20 mM 1,2‐bis(O‐aminophenoxy)ethane‐N,N,N',N'‐tetraacetic acid (BAPTA), suggesting that protein kinase C and intracellular Ca2+ did not mediate the response. 8. It is concluded that the activation of P2‐purinoceptors increases IK through intracellular mechanisms independent of protein kinase A, protein kinase C or intracellular free Ca2+ in guinea‐pig atrial myocytes.
Electrophysiological consequences of ligand binding to the desensitized 5‐HT3 receptor in mammalian NG108‐15 cells.
Authors: Bartrup J T , Newberry N R
Published: December 2014

1. Using the whole‐cell variation of the patch‐clamp technique to record from mammalian NG108‐15 cells, we have studied the ligand‐gated ion channel current activated by a high concentration (100 microM) of local pressure‐applied 5‐hydroxytryptamine (5‐HT). The response was induced at intervals of at least 90‐120 s, which allowed the receptor to fully recover between activations. 2. The rapid inward current induced by pressure‐applied 5‐HT was reproducibly inhibited by the superfusion of low concentrations of 5‐HT which evoked little or no detectable inward current alone (0.01‐0.3 microM). This inhibitory effect was most likely to be due to a direct action on the 5‐HT3 receptor as it could be recorded using intracellular solutions with or without adenosine triphosphate (ATP) and guanosine triphosphate (GTP). 3. The maximum inhibitory effect of a given concentration of 5‐HT was not dependent on its superfusion time but on the number of activations of the receptor by pressure‐applied 5‐HT. This activation dependence was clearly evident, since the first inward current in the presence of 0.1 microM 5‐HT was often unaffected in amplitude. 4. The inhibitory effect of 5‐HT was evident at holding potentials of +60 and ‐60 mV; with the calcium chelator BAPTA in the recording pipette and with the nominal removal of extracellular calcium and magnesium ions. 5. The inhibitory effect was concentration dependent, with 50% inhibition of the inward current amplitude occurring at approximately 50 nM 5‐HT. The slope factor of the inhibition curve was 1.3. The effect was mimicked by two other 5‐HT3 receptor agonists, 2‐methyl‐5‐HT and m‐chlorophenylbiguanide (mCPBG) which gave 50% inhibition at approximately 600 nM and approximately 20 nM, respectively. These values are similar to the affinity values for these ligands determined in radioligand binding assays. 6. The 5‐HT3 receptor ‘antagonists’ (+)‐tubocurarine and quipazine (both at 3 nM) reduced the inward current amplitude by approximately 50%. The rate of onset of the inhibitory effect of bath‐applied 5‐HT was slowed in the presence of (+)‐tubocurarine but not in the presence of quipazine. This difference might be explained by the agonist properties seen only with quipazine. 7. The inhibition of the 5‐HT3 receptor mediated inward current by low concentrations of bath‐applied 5‐HT3 receptor agonists is compatible with the cyclic model of receptor activation and desensitization. We conclude that we have been studying the high‐affinity binding of agonists to the desensitized form of the 5‐HT3 receptor.
Evidence against a contribution by Na(+)‐Cl‐ cotransport to chloride accumulation in rat arterial smooth muscle.
Authors: Davis J P
Published: December 2014

1. Chloride accumulation into rat saphenous arterial smooth muscle has been examined using chloride‐sensitive microelectrodes, to assess the contribution of Na(+)‐Cl‐cotransport. 2. Bumetanide (10 microM) produced a fall in intracellular chloride ([Cl‐]i), and a hyperpolarization of membrane potential (Em). However, [Cl‐]i remained above the equilibrium level predicted from the membrane potential, indicating a residual accumulation. 3. Replacement of extracellular sodium with N‐methyl‐D‐glucamine or choline caused a fall in [Cl‐]i similar to that observed with bumetanide, but the hyperpolarization of Em was larger. In Na(+)‐free media, bumetanide had no effect. [Cl‐]i remained significantly above equilibrium. 4. In the presence of bumetanide, chlorothiazide produced a further dose‐dependent fall in [Cl‐]i, and hyperpolarization of Em. However, although [Cl‐]i fell more than with bumetanide alone, it remained significantly above equilibrium. Metolazone was without effect at 100 microM. 5. In the presence of bumetanide, ethacrynic acid and N‐ethyl maleimide caused a dose‐dependent hyperpolarization of Em and a fall in [Cl‐]i to equilibrium. 6. The third inward chloride pump in rat saphenous arterial smooth muscle appears not to be a form of Na(+)‐Cl‐ cotransport. The potency series of thiazide diuretic action (acetazolamide > chlorothiazide > metolazone) differed significantly from that published for Na(+)‐Cl‐ cotransport, and there is no sodium dependence.
Reopening of single L‐type Ca2+ channels in mouse cerebellar granule cells: dependence on voltage and ion concentration.
Authors: Slesinger P A , Lansman J B
Published: December 2014

1. We recorded the activity of single L‐type Ca2+ channels from cell‐attached patches on mouse cerebellar granule cells. The experiments investigated the mechanism of channel reopening at negative membrane potentials following a strong depolarization. 2. L‐type channels that reopened following a strong depolarization showed a wide distribution of single‐channel conductances, which ranged from 16 to 28 pS in the presence of 90 mM Ba2+. 3. The distribution of the latencies before reopening was fitted as the sum of two exponential components with time constants tau f approximately 1 and tau s approximately 12 ms at ‐70 mV. Hyperpolarization reduced the time constant of the slower component approximately e‐fold per 43 mV, but had no effect on the faster component. 4. Raising the concentration of external Ba2+ reduced the time constant of the slower component of the reopening latency without altering the fast component. The time constant of the slow component was approximately 27 ms in 10 mM Ba2+ and decreased to 12 ms in 90 mM Ba2+ at ‐70 mV. The relation between the time constant and external Ba2+ saturated with an apparent KD of approximately 20 mM. 5. The distribution of reopening times was best fitted as the sum of two exponential components with time constants tau f approximately 0.5 ms and tau s approximately 4.5 ms at ‐70 mV. The conditional latencies before reopening into either the short or long open state were indistinguishable. 6. The results are consistent with the idea that a positively charged blocker occludes the pore during depolarization and channels reopen as the blocker dissociates following repolarization to negative potentials.
The behaviour of muscle microcirculation in chronically hypoxic rats: the role of adenosine.
Authors: Mian R , Marshall J M
Published: December 2014

1. In rats housed in a hypoxic chamber at 12% O2 for 3‐5 weeks (CH) and in normal rats housed in air (N), we directly observed responses of arterial and venous vessels of the spinotrapezius muscle to changes in O2 concentration in the inspirate. Both CH and N rats were anaesthetized with Saffan. They had haematocrits of 55.0 +/‐ 0.9% (mean +/‐ S.E.M.) and 41.9 +/‐ 0.5%, respectively. 2. In CH rats breathing 12% O2 and N rats breathing air, arterial and venous vessels from comparable anatomical positions in the vascular tree were of similar internal diameter. They also showed similar maximum dilator responses to topical adenosine (10(‐3) M); 14.1 +/‐ 1.1 and 16.3 +/‐ 1.7% in all arterioles, 15.5 +/‐ 1.2 and 11.5 +/‐ 0.6% in all venules in CH and N rats, respectively. 3. In CH rats, the change from 12% O2 to air for 3 min induced constriction in all arterioles and venules (‐12.9 +/‐ 1.0 and ‐14.3 +/‐ 1.7%, respectively), whereas in N rats, the change from air to 12% O2 for 3 min induced net dilatation (3.9 +/‐ 1.8% in arterioles and 4.7 +/‐ 0.8% in venules). Topical application of the adenosine receptor antagonist 8‐sulphophenyltheophylline (8‐SPT, 10(‐3) M) had no effect on control diameters in CH or N rats, nor on constrictor responses to air in CH, but reversed or reduced dilator responses to 12% O2 in N rats (to ‐2.4 +/‐ 1.3% in arterioles and 2.0 +/‐ 0.9% in venules). 4. In CH rats, the change from 12 to 8% O2 produced net dilatation as great as that induced in N rats by the larger change from air to 8% O2: 8.5 +/‐ 2.6 and 5.0 +/‐ 3.7% in arterioles and 10.3 +/‐ 1.8 and 6.4 +/‐ 1.9% in venules, respectively. These responses were similarly reduced by 8‐SPT to ‐4.3 +/‐ 1.9 and ‐5.2 +/‐ 2.7% in arterioles and to ‐6.9 +/‐ 2.0 and ‐1.5 +/‐ 2.0% in venules, respectively. 5. These results indicate that CH rats were acclimated to 12% O2 such that the resting tone of arterial and venous vessels of muscle was comparable to that of N rats breathing air. They also suggest that adenosine had little tonic dilator influence in CH rats breathing 12% O2 despite its contribution to the dilatation induced in N rats by acute exposure to 12% O2. This may reflect the greater haematocrit in CH rats which normalized the O2 supply to muscle. However, CH rats were more sensitive than N rats to the dilator influence of acute systemic hypoxia and this was largely mediated by adenosine.
Cardiovascular responses to static exercise in conscious cats: effects of intracerebroventricular injection of clonidine.
Authors: Ally A , Hand G A , Mitchell J H
Published: December 2014

1. Static exercise elicits increases in arterial blood pressure and heart rate (HR) in humans and conscious animals. In this study, the effects of intracerebroventricular (I.C.V.) administration of clonidine, an alpha 2‐adrenergic agonist, on these cardiovascular responses were investigated using conscious cats. Four cats were operantly trained to extend a forelimb and press a bar (200‐650 g) for 15‐60 s. A stainless‐steel cannula was inserted into the right lateral ventricle for I.C.V. injection of drugs, and a common carotid artery was catheterized to measure mean arterial pressure (MAP) and HR. The number of exercise trials and changes in MAP, HR and force were pooled for 30 min periods. After the cats exercised for 30 min, either artificial cerebrospinal fluid (CSF) or clonidine (2 or 5 micrograms) were administered intracerebroventricularly. 2. Before clonidine injection, fifty‐two exercise trials increased MAP and HR by 15 +/‐ 3 mmHg and 41 +/‐ 5 beats min‐1, respectively. Administration of clonidine (2 micrograms) did not alter the resting MAP and HR, but attenuated the increases in MAP and HR in response to exercise (0‐30 min post‐clonidine: n = 81; delta MAP, 6 +/‐ 3 mmHg; delta HR, 20 +/‐ 6 beats min‐1; 30‐60 min post‐clonidine: n = 71; delta MAP, 4 +/‐ 4 mmHg; delta HR, 17 +/‐ 8 beats min‐1). Administration of artificial CSF I.C.V. had no effect on the cardiovascular responses to static exercise. 3. An increased dose of clonidine (5 micrograms) decreased resting MAP and HR by 31 +/‐ 7 mmHg and 37 +/‐ 7 beats min‐1, respectively, and markedly blunted the cardiovascular responses to exercise (pre‐clonidine: n = 52; delta MAP, 17 +/‐ 3 mmHg; delta HR, 38 +/‐ 5 beats min‐1; post‐clonidine 0‐30 min: n = 66; delta MAP, 4 +/‐ 2 mmHg; delta HR, 15 +/‐ 5 beats min‐1; post‐clonidine 30‐60 min: n = 60; delta MAP, 4 +/‐ 2 mmHg; delta HR, 14 +/‐ 6 beats min‐1). Pretreatment with the alpha 2‐adrenergic antagonist, yohimbine (8 micrograms, I.C.V.), blocked the attenuating effects of I.C.V. administration of clonidine (5 micrograms). 4. These results show that stimulation of central alpha 2‐adrenoceptors by clonidine attenuates the cardiovascular responses to static exercise in conscious cats. In addition, this study suggests that alpha 2‐adrenoceptors blocked by yohimbine injected I.C.V. do not appear to have a tonic influence on HR and blood pressure.
Distribution of chloride permeabilities in normal human red cells.
Authors: Raftos J E , Bookchin R M , Lew V L
Published: December 2014

1. The rate of dehydration of K+ permeabilized red cells is influenced by their Cl‐ permeability (PCl). In instances of pathological K+ permeabilization, cell‐to‐cell differences in PCl may determine which red cells dehydrate most. The present study was designed to investigate whether PCl differed significantly among red cells from a single blood sample. 2. Previously available methods measure only the mean PCl of red cell populations. We describe a ‘profile migration’ method in which dilute red cell suspensions in low‐K+ media were permeabilized to K+ with a high concentration of valinomycin, rendering PCl the main rate‐limiting factor for cell dehydration. As the cells dehydrated, samples were processed to obtain full haemolysis curves at precise times. Variations in PCl among cells would have appeared as progressive changes in the profile of their haemolysis curves, as the curves migrated towards lower tonicities. 3. Red cells from five normal volunteers showed no change in profile of the migrating haemolysis curves, suggesting that their PCl distributions were fairly uniform. Quantitative analysis demonstrated that intercell variation in PCl was less than 7.5%. 4. Results obtained with this technique were analysed using the Lew‐Bookchin red cell model. The calculated PCl was within the normal range described in earlier studies.
Functional interaction between K(ATP) channels and the Na(+)‐K(+) pump in metabolically inhibited heart cells of the guinea‐pig.
Authors: Priebe L , Friedrich M , Benndorf K
Published: December 2014

1. Transmembrane current through ATP‐regulated K(+) channels (IK(ATP)) was measured in ventricular heart cells of the guinea‐pig in the whole‐cell and cell‐attached patch configurations under conditions of metabolic poisoning with the mitochondrial uncoupler 2,4‐dinitrophenol (DNP). 2. Maintained exposure of the cells to DNP resulted in a transient appearance of whole‐cell IK(ATP) When IK(ATP) had reached several nanoamps, blocking the forward‐running Na(+)‐K(+) pump with 0.5 mM strophanthidin decreased IK(ATP) after a delay. The time course of this decrease could be described by a single exponential function, which yielded a time constant(T)of 4.51+/‐ 1.89 s (n=8). 3. Hyperpolarization from 0 mV to ‐100 or ‐150 mV for 2 s caused IK(ATP) (measured at 0 mV) to decrease by 34.2 +/‐ 14.1 % (n = 8) and 37.6 +/‐ 9.4% (n = 8), respectively. After the hyperpolarizing pulse, IK(ATP) returned to its higher initial level within a couple of seconds. 4. Driving the pump backwards by removing the extracellular K(+) ions caused the permanent disappearance of DNP‐induced IK(ATP). 5. Application of 0.5 mM strophanthidin in the absence of external K(+) ions induced a transient increase in IK(ATP), as did washing out the glycoside (n = 5). 6. When pump action was inhibited by using Na(+), K(+)‐free Tyrode solution (see Methods) in the bath, strophanthidin did not have a comparable direct effect on IK(ATP). 7. In cell‐attached patches, strophanthidin applied via the bath caused a reduction in IK(ATP) with a similar time course to that in whole‐cell experiments. This suggests that the interaction between the pump molecules and the K(ATP) channels is not restricted to closely neighbouring molecules. 8. The data support the hypothesis that [ATP] at the cytosolic face of the membrane may drop to practically zero, thereby passing an ‘ATP window’ in which the channels first open and then close, and that the submembrane [ATP] is readily controlled by the cytosolic [ATP].
Extracellular K(+)‐induced hyperpolarizations and dilatations of rat coronary and cerebral arteries involve inward rectifier K(+) channels.
Authors: Knot H J , Zimmermann P A , Nelson M T
Published: December 2014

1. The hypothesis that inward rectifier K(+) channels are involved in the vasodilatation of small coronary and cerebral arteries (100‐200 microm diameter) in response to elevated [K+]o was tested. The diameters and membrane potentials of pressurized arteries from rat were measured using a video‐imaging system and conventional microelectrodes, respectively. 2. Elevation of [K+]o from 6 to 16 mM caused the membrane potential of pressurized (60 mmHg) arteries to hyperpolarize by 12‐14 mV. Extracellular Ba(2+) (Ba2+(o)) blocked K(+)‐induced membrane potential hyperpolarizations at concentrations (IC(50), 6 microM) that block inward rectifier K(+) currents in smooth muscle cells isolated from these arteries. 3. Elevation of [K+]o from 6 to 16 mM caused sustained dilatations of pressurized coronary and cerebral arteries with diameters increasing from 125 to 192 microm and 110 to 180 microm in coronary and cerebral arteries, respectively. Ba2+(o) blocked K(+)‐induced dilatations of pressurized coronary and cerebral arteries (IC50, 3‐8 microM). 4. Elevated [K+]o‐induced vasodilatation was not prevented by blockers of other types of K(+) channels (1 mM 4‐aminopyridine, 1 mM TEA+, and 10 mu M glibenclamide), and blockers of Na(+)‐K(+)‐ATPase. Elevated [K+]o‐induced vasodilatation was unaffected by removal of the endothelium. 5. These findings suggest that K+(o) dilates small rat coronary and cerebral arteries through activation of inward rectifier K(+) channels. Furthermore, these results support the hypothesis that inward rectifier K(+) channels may be involved in metabolic regulation of coronary and cerebral blood flow in response to changes in [K+]o.
Component characteristics of the vectorial transport system for taurine in isolated bovine retinal pigment epithelium.
Authors: Kundaiker S , Hussain A A , Marshall J
Published: December 2014

1. A wide range of substrate concentrations (5‐1600 microM) were used to screen for the presence of systems capable of transporting taurine into isolated and free‐floating samples of bovine retinal pigment epithelium (RPE). Both high and low affinity systems displaying Michaelis‐Menten saturation kinetics were identified. The high affinity system was characterized by a K(m) of 23 microM and a V(max) of 86.7 pmol (5 min)(‐1) (4 mm disc of tissue sample)(‐1). Similarly, the low affinity system was characterized by a K(m) of 507 microM and a V(max) of 344 pmol (5 min)(‐1)(4 mm disc)(‐1). 2. Ussing‐type incubation chambers and double‐label radiotracer techniques were used to assess the presence of specific taurine carriers on apical and basolateral surfaces of the RPE. High affinity carriers were shown to be present on both surfaces and the kinetic constants (K(m) and V(max)) for apical and basolateral systems were determined as 23.2 microM and 34.8 pmol (5 min)(‐1) (4 mm disc)(‐1) and 29 microM and 54.7 pmol (5 min)(‐1)(4 mm disc)(‐1), respectively. Both these high affinity systems were sodium dependent with a Hill coefficient of about 2.0 indicating that two sodium ions are required for the translocation of one molecule of taurine. The low affinity system was unevenly distributed over the two surfaces of the RPE, basolateral capacities being roughly twofold higher. The basolateral system was totally insensitive to sodium whereas the apical one with 50% sodium sensitivity suggested the presence of low affinity carrier heterogeneity. 3. A temperature‐dependent mechanism for the release of pre‐loaded taurine from bovine RPF was also demonstrated. 4. The effect of [K+]o trans‐RPF gradients on the vectorial transport of taurine across the isolated preparation was also investigated. The results demonstrated that the direction and magnitude of taurine transport could be controlled by physiological variations in the extracellular concentration of potassium. 5. The determined kinetic parameters of the carriers were used to construct a working model of the vectorial pathway for the translocation of taurine across bovine RPE. This estimated the level of free intracellular taurine to be in the micromolar range. However, direct measurements of total RPE taurine by high performance liquid chromatography showed levels of 19.5 +/‐ 3.6 mM indicating that the major taurine pool in bovine RPE exists in the bound form. The model also showed that the magnitude and direction of net transport was a function of the transepithelial taurine gradient.
Nucleotide binding by actomyosin as a determinant of relaxation kinetics of rabbit phasic and tonic smooth muscle.
Authors: Khromov A S , Somlyo A V , Somlyo A P
Published: December 2014

1. The apparent second‐order rate constants (k+T) of ATP‐induced cross‐bridge detachment from rigor in the absence of Ca2+ were determined with laser flash photolysis of caged ATP (cATP) in alpha‐toxin‐permeabilized tonic, rabbit femoral artery and phasic, rabbit bladder smooth muscles. The potential effect of cATP binding to actomyosin (AM) on cross‐bridge kinetics was examined by varying the initial concentration of cATP 2‐fold. For a given [ATP] released from either 10 or 5 mM cATP, the kinetics of relaxation were not significantly different; the estimated dissociation constant for cATP binding to smooth muscle AM was 1‐3 mM. 2. k+T was significantly higher ((9.5 +/‐ 1.3) x 10(4) M‐1 s‐1) in the phasic than in the tonic ((3.0 +/‐ 1.0) x 10(4) M‐1 s‐1) smooth muscle. 3. We conclude that the combination of the significantly lower (approximately 3 times) apparent second‐order rate constant of MgATP association with the approximately 5 times higher affinity of cross‐bridges for MgADP in tonic, than in phasic, smooth muscle is a major determinant of the slower kinetics of relaxation and, probably, shortening velocity of tonic smooth muscle.
Functional development of the sheep diaphragmatic ligament.
Authors: Griffiths R I , Berger P J
Published: December 2014

1. The diaphragmatic ligament, which lines the thoracic surface of the diaphragm, plays a major role in setting the passive compliance of the diaphragm in the adult. Since many elastic tissues are more compliant at younger ages, our aim was to determine whether this diaphragmatic ligament plays an important role during development of the diaphragm. 2. We examined the passive biaxial length‐tension properties of the isolated mid‐costal diaphragm from fetal, newborn and adult sheep; initially with the diaphragm intact and then with the diaphragmatic ligament removed to reveal its functional significance. 3. We found that unlike other elastic tissues the diaphragmatic ligament is stiffer in early development than in the adult. The diaphragmatic ligament supports about 75% of the passive tension in the diaphragm in the 121 day fetus (term at 147 days) declining to about 50% from 3 weeks of age. The fetal diaphragm muscle fibres are exposed to 1‐7 kPa of stress over the last 26 days of gestation. 4. We suggest that the diaphragmatic ligament provides a structural framework on which the muscle fibres of the diaphragm grow in fetal life, and that the ligament's elasticity is regulated to maintain a low and constant stress on the diaphragm muscle fibres during development.
Burst‐generating neurones in the dorsal horn in an in vitro preparation of the turtle spinal cord.
Authors: Russo R E , Hounsgaard J
Published: December 2014

1. In transverse slices of the spinal cord of the turtle, intracellular recordings were used to characterize and analyse the responses to injected current and activation of primary afferents in dorsal horn neurones. 2. A subpopulation of neurones, with cell bodies located centrally in the dorsal horn, was distinguished by the ability to generate a burst response following a hyperpolarization from rest or during a depolarization from a hyperpolarized holding potential. The burst response was inactivated at the resting membrane potential. 3. The burst response was mediated by a low threshold Ca2+ spike assumed to be mediated by T‐type Ca2+ channels since it resisted tetrodotoxin and was blocked by 3 mM Co2+ or 100‐300 microM Ni2+ and resembled the low threshold spike (LTS) described elsewhere. 4. Some burst‐generating cells also displayed plateau potentials mediated by L‐type Ca2+ channels. In these cells the burst following a hyperpolarizing current pulse, applied from the resting membrane potential, facilitated the activation of the plateau potential. Wind‐up of the plateau potential was produced when the hyperpolarizing pulse generating the burst was repeated at 0.1‐0.3 Hz or faster. 5. The burst response and the underlying low threshold Ca2+ spike were activated synaptically by primary afferent stimuli in a voltage range hyperpolarized from the resting membrane potential. 6. Cells with bursts were morphologically distinguishable from cells with bursts and plateau properties. 7. Our findings in this and the preceding paper show that the intrinsic response properties of particular subtypes of neurones in the dorsal horn have a profound influence on the amplitude and time course of the responses mediated by primary afferent fibres. We predict that these postsynaptic properties are probable targets for synaptic modulation.
Mechanisms of manganese transport in rabbit erythroid cells.
Authors: Chua A C , Stonell L M , Savigni D L , Morgan E H
Published: December 2014

1. The mechanisms of manganese transport into erythroid cells were investigated using rabbit reticulocytes and mature erythrocytes and 54Mn‐labelled MnCl2 and Mn2‐transferrin. In some experiments iron uptake was also studied. 2. Three saturable manganese transport mechanisms were identified, two for Mn2+ (high and low affinity processes) and one for transferrin‐bound manganese (Mn‐Tf). 3. High affinity Mn2+ transport occurred in reticulocytes but not erythrocytes, was active only in low ionic strength media such as isotonic sucrose and had a Km of 0.4 microM. It was inhibited by metabolic inhibitors and several metal ions. 4. Low affinity Mn2+ transport occurred in erythrocytes as well as in reticulocytes and had Km values of approximately 20 and 50 microM for the two types of cells, respectively. The rate of Mn2+ transport was maximal in isotonic KCl, RbCl or CsCl, and was inhibited by NaCl and by amiloride, valinomycin, diethylstilboestrol and other ion transport inhibitors. The direction of Mn2+ transport was reversible, resulting in Mn2+ efflux from the cells. 5. The uptake of transferrin‐bound manganese occurred only with reticulocytes and depended on receptor‐mediated endocytosis of Mn‐Tf. 6. The characteristics of the three saturable manganese transport mechanisms were similar to corresponding mechanisms of iron uptake by erythroid cells, suggesting that the two metals are transported by the same mechanisms. 7. It is proposed that high affinity manganese transport is a surface representation of the process responsible for the transport of manganese across the endosomal membrane after its release from transferrin. Low affinity transport probably occurs by the previously described Na(+)‐Mg2+ antiport, and may function in the regulation of intracellular manganese concentration by exporting manganese from the cells.
A patch‐clamp study of delayed rectifier currents in skeletal muscle of control and mdx mice.
Authors: Hocherman S D , Bezanilla F
Published: December 2014

1. Potassium currents were measured in the extensor digitorum longus muscle of normal and mdx mice, which lack the protein dystrophin, using the cell‐attached and inside‐out patch clamp techniques, in the presence of asymmetrical K+ concentrations (3 mM in the pipette, 160 mM in the bath). 2. In cell‐attached patches, the delayed rectifier was the most commonly found potassium channel, with a density of roughly 8 channels microns‐2. Outward macroscopic currents were activated in macropatches depolarized to potentials positive to ‐60 mV. The probability of opening reached half‐maximal values around ‐40 mV for control patches and ‐31 mV for patches from mdx mice. 3. Tail currents were linear in the range between ‐60 and +20 mV, reversing close to ‐100 mV. The single channel current at 0 mV, estimated from non‐stationary analysis of variance, was used in conjunction with the slope of the linear part of the tail current to calculate the single channel conductance, yielding a value of 19 +/‐ 1 pS. 4. At 0 mV, the delayed rectifier inactivated with two time constants, of 70 +/‐ 20 ms and 600 +/‐ 200 ms. Prepulses of 500 ms duration to different potentials produced incomplete inactivation with inactivation reaching 50% of its maximum at ‐50 mV. 5. Single channel activity was recorded using small pipettes. Both single channel conductance and kinetic behaviour were in agreement with the macroscopic current data. 6. In excised patches, the delayed rectifier current ran down, unmasking other K+ channels. A Ca(2+)‐dependent K+ channel of 186 pS (BK‐like channel) was found frequently in patches bathed in solutions containing appropriate concentrations of calcium, especially at stronger depolarizations. A K+ channel of 63 pS was unmasked in control excised patches bathed in solutions devoid of ATP. This channel was not observed in patches excised from mdx fibers.
Substance P hyperpolarizes vagal sensory neurones of the ferret.
Authors: Jafri M S , Weinreich D
Published: December 2014

1. Intracellular recordings were made in intact and in acutely dissociated vagal afferent neurones (nodose ganglion cells) of the ferret to investigate the effects of substance P(SP). 2. In current‐clamp recordings, SP (100 nM) applied by superfusion hyperpolarized the membrane potential (7 +/‐ 0.7 mV; mean +/‐ S.E.M.; n = 105) and decreased the input resistance in 80% of the neurones. With voltage‐clamp recording, SP produced an outward current of 3 +/‐ 0.2 nA (n = 10). 3. The SP current was concentration dependent with an estimated EC50 of 68 nM. The SP‐induced hyperpolarization or current was mimicked by the tachykinin receptor NK1 agonist Ac‐[Arg6, Sar9, Met(O2)11]SP(6‐11) (ASM‐SP; 100 nM; n = 10) and blocked by the NK1 antagonist CP‐96,345 (10 nM; n = 6), but not by the NK2 antagonist SR48968 (100 nM; n = 4). No measurable change in membrane potential or input resistance was observed with application of either [beta‐Ala8]neurokinin A or senktide, selective NK2 and NK3 receptor agonists, respectively (100 nM; n = 3 for each agonist). 4. The reversal potential (Erev) for the SP outward current was ‐85 +/‐ 2.5 mV (n = 4). The Erev for the SP response shifted in a Nernstian manner with changes in extracellular potassium concentration. Alterations in extracellular sodium or chloride concentrations had no significant effect on the Erev for the SP response (n = 3 for each ion). 5. Nominally Ca(2+)‐free external solution abolished the SP response. Removal of magnesium from the extracellular solution had no effect on the response. 6. Caesium (100 microM), barium (1 mM), tetraethylammonium (TEA; 5 mM), apamin (10 nM) and 4‐aminopyridine (4‐AP; 4 mM) each completely prevented the SP response (n > or = 3 for each). 7. These results indicate that SP, via an NK1 receptor, can induce a Ca(2+)‐dependent outward potassium current which hyperpolarizes the resting membrane potential of vagal afferent somata.
Involvement of protein kinases in the upregulation of acetylcholine release at endplates of alpha‐bungarotoxin‐treated rats.
Authors: Plomp J J , Molenaar P C
Published: December 2014

1. ACh release from motor nerve endings in diaphragms of rats treated chronically with alpha‐bungarotoxin (alpha‐BuTX) is upregulated at the level of the individual endplate. Involvement of protein kinases in this mechanism of synaptic adaptation was investigated. 2. Miniature endplate potentials (MEPPs) and endplate potentials (EPPs) were recorded after mu‐conotoxin treatment, which prevents muscle action potentials. The quantal content at endplates was calculated ‘directly', i.e. by dividing the EPP amplitude by the MEPP amplitude. 3. Incubation of muscles from control and alpha‐BuTX‐treated rats with H‐7, a protein kinase C (PKC) inhibitor, reduced MEPP amplitudes but had no clear effect on quantal contents. Polymyxin B, another PKC inhibitor, had a similar effect on muscles from alpha‐BuTX‐treated rats. 4. Incubation of muscles from alpha‐BuTX‐treated rats with K252a, a broad‐spectrum protein kinase inhibitor of, amongst others, PKC, Ca(2+)‐calmodulin‐dependent protein kinase II (CaMKII) and neurotrophin receptor tyrosine kinases, resulted in a 30% decrease of the quantal content. However, K252a did not change the quantal content of controls. Incubations with the closely related compound K252b, which has an exclusively extracellular action, had a similar effect. 5. KN62, a specific inhibitor of CaMKII, decreased the mean quantal content of muscles from alpha‐BuTX‐treated rats by 18%. 6. Tyrphostin 51, a selective tyrosine kinase inhibitor, had no effect on quantal contents of muscles from alpha‐BuTX‐treated and control rats. However, it increased the frequency and amplitude of MEPPs in muscles from alpha‐BuTX‐treated rats, leaving those of controls unchanged. 7. The extent of reduction of quantal content, caused by K252a, K252b and KN62, varied between endplates of individual muscles from alpha‐BuTX‐treated rats; quantal contents at endplates with small MEPPs were more sensitive than those at endplates with large MEPPs. 8. It is concluded that PKC does not play a role in the mechanism of upregulation of ACh release at endplates of alpha‐BuTX‐treated rats. Instead, CaMKII and some tyrosine kinases in the presynaptic membrane, as well as in the cytoplasm, might be involved.
Effect of low [ATP] on depolarization‐induced Ca2+ release in skeletal muscle fibres of the toad.
Authors: Owen V J , Lamb G D , Stephenson D G
Published: December 2014

1. The effect of low [ATP] on depolarization‐induced Ca2+ release from the sarcoplasmic reticulum (SR) was examined using mechanically skinned skeletal muscle fibres of the toad. The amount of Ca2+ released was determined by examining the extent of SR Ca2+ depletion following a single depolarization in the presence of specified total [ATP] and free [Mg2+] and 1 mM EGTA (< 10 nM Ca2+). 2. Reducing the total [ATP] from 8 to 2 mM did not significantly affect the extent of depolarization‐induced Ca2+ release. When the total [ATP] was lowered to 0.5 mM, depolarization‐induced Ca2+ release was markedly reduced. The additional presence of 1.5 mM AMP did not reverse this effect of low [ATP]. 3. At each total [ATP], elevation of the free [Mg2+] from the normal resting level of 1 mM to 3 mM, caused a similar reduction in depolarization‐induced Ca2+ release. This effect was not due to the concomitant decrease in free [ATP] and occurred independently of the reduction in Ca2+ release observed at very low total [ATP]. 4. These results show myoplasmic [ATP] modulates depolarization‐induced Ca2+ release. Thus, if the total [ATP] near the Ca2+ release channels becomes locally depleted in severely fatigued muscle fibres, it may contribute, in combination with an accompanying increase in free [Mg2+], to the reported reduction in Ca2+ release.
In vitro relation between preganglionic sympathetic stimulation and activity of cutaneous glands in the bullfrog.
Authors: Jobling P , Horn J P
Published: December 2014

1. Activation of cutaneous glands was studied by measuring changes in transepithelial potentiation (TEP) after pre‐ and postganglionic sympathetic stimulation in the bullfrog, Rana catesbeiana. 2. In normal Ringer solution, TEP was 20‐90 mV with the basolateral (inside) surface positive. Single shocks to the preganglionic B pathway decreased TEP by up to 3 mV. Cutaneous depolarizations had a latency of 1.2 s, a rise time of 2.5 s, and decayed with an exponential time constant of 15 s. Similar depolarizations were evoked by postganglionic stimulation. 3. Cutaneous depolarizations summed during repetitive stimulation and > 0.05 Hz. For trains of three stimuli, peak amplitude increased with frequency and saturated at 2 Hz. In some preparations, longer trains evoked polyphasic changes in TEP. Preganglionically evoked cutaneous responses were abolished by (+)‐tubocurarine. Postganglionically evoked cutaneous depolarizations were antagonized by phentolamine, but not propranolol. 4. Repetitive preganglionic stimulation of the C pathway (> 100 at 20 Hz) evoked little change in TEP and did not modulate depolarizations evoked through the B pathway. In nicotine, peptidergic cotransmission was enhanced in the ganglia, and repetitive C pathway stimulation evoked cutaneous depolarizations whose time course mirrored that of the postganglionic peptidergic after‐discharge. The after‐discharge and associated cutaneous depolarization were blocked by a luteinizing hormone‐releasing hormone antagonist. 5. The results show cutaneous glands are selectively innervated by B neurones and respond to low levels of neural activity. Asynchronous postganglionic firing mediated by peptidergic cotransmission can provide a basis for heterosynaptic interactions between the B and C pathways.
Inward rectification of the IRK1 channel expressed in Xenopus oocytes: effects of intracellular pH reveal an intrinsic gating mechanism.
Authors: Shieh R C , John S A , Lee J K , Weiss J N
Published: December 2014

1. The effects of intracellular pH (pHi) were investigated in inside‐out giant patches from Xenopus oocytes expressing the inward rectifier K+ channel IRK1. 2. After excising patches into Mg2(+)‐ and polyamine‐free solution, a residual time‐dependent inactivation of outward current during depolarizing voltage‐clamp pulses persisted, reaching an apparent steady‐state by 5 min. Raising pHi from 7.2 to 9.0 increased the inactivation rate of the outward current. 3. In the presence of intracellular Mg2+ or polyamines, however, pHi 9.0 either decreased or did not change the inactivation rate of outward current. 4. These results suggest that the inactivation of outward current remaining after > 5 min in Mg2(+)‐ and polyamine‐free solution is not due to slow washout of these substances, but represents a third and probably intrinsic gating mechanism contributing to the inward rectifying property of IRK1. 5. The voltage dependence and kinetics of this gating mechanism were well described by a sequential two open‐ and one closed‐state model in which the rate constants for transitions between the open states were voltage dependent, and those between the open and closed state were pH dependent. 6. In the absence of intracellular Mg2+ and polyamines, reduced pHi blocked inward and outward current through IRK1 channels in a voltage‐independent manner without appreciably altering the kinetics. Half‐maximal block occurred at pH 6.2‐6.4 (Hill coefficient, 1.6). Block of IRK1 by intracellular protons may contribute to membrane depolarization in ischaemic tissue.