bluuebunny/crossref_metadata_2025 huggingface dataset:
Vasopressin stimulation of Ca2+ mobilization, two bivalent cation entry pathways and Ca2+ efflux in A7r5 rat smooth muscle cells.
Authors: Byron K , Taylor C W
Published: December 2014

1. Arg8‐vasopressin (AVP)‐regulated Ca2+ transport were investigated in fura‐2‐loaded A7r5 cells using both single cell and population measurements. 2. AVP evokes an initial concentration‐dependent rise in cytosolic free Ca2+ concentration ([Ca2+ ]i) to a peak which is independent of extracellular Ca2+, and a sustained Ca2+ signal that results from a balance between stimulation of Ca2+ entry and efflux. 3. Depletion of intracellular Ca2+ stores with thapsigargin, ionomycin, or prior treatment with AVP in Ca2(+)‐free medium activates ‘capacitative’ entry of Ca2+, Ba2+ or Mn2+. Capacitative Mn2+ entry is inhibited by refilling stores with Ca2+; neither Sr2+ nor Ba2+ substitute for Ca2+ to give this effect. 4. In cells with empty stores, AVP stimulates further bivalent cation entry, and the effect persists when extracellular Na+ is replaced by N‐methyl‐D‐glucamine or under depolarizing condition (extracellular KCl concentration ([KCl]o), 135 mM). This effect of AVP is not therefore merely a consequence of AVP causing membrane hyperpolarization or stimulation of Na(+)‐Ca2+ exchange, but results from opening of a bivalent cation influx pathway. 5. Several lines of evidence indicate that AVP‐stimulated bivalent cation entry is not a consequence of more complete emptying of the intracellular stores and consequent further activation of the capacitative pathway. AVP stimulates Ba2+ entry when the intracellular Ca2+ stores have been both emptied by ionomycin and prevented from refilling by thapsigargin. Mn2+ permeates the capacitative pathway, but AVP does not further increase Mn2+ entry, confirming that AVP does not further activate the capacitative pathway and that the two pathways differ in their permeability to Mn2+. When the extracellular [Sr2+] is low, empty stores do not stimulate detectable Sr2+ entry, but addition of AVP causes substantial Sr2+ entry. 6. A decrease in [Ca2+]i occurs when 50 nM AVP is added during a sustained elevation of [Ca2+]i evoked by thapsigargin. Since AVP does not inhibit the capacitative pathway, this result suggests that AVP stimulates Ca2+ extrusion. 7. We conclude that stimulation of Ca2+ mobilization, two modes of bivalent cation entry, and Ca2+ efflux all contribute to the complex concentration‐dependent effects of AVP in A7r5 smooth muscle cells.
Nitric oxide hyperpolarizes rabbit mesenteric arteries via ATP‐sensitive potassium channels.
Authors: Murphy M E , Brayden J E
Published: December 2014

1. Nitric oxide (NO) relaxes vascular smooth muscle (VSM) by mechanisms which are not fully understood. One possibility is that NO hyperpolarizes membranes, thereby diminishing Ca2+ entry through voltage‐dependent Ca2+ channels. In the current study, the effects of NO on membrane potential of rabbit mesenteric arteries were recorded using intracellular microelectrodes. 2. NO, released by 3‐morpholinosydnonimine (SIN‐1, 3 microM), reversibly hyperpolarized arteries by ‐9.5 +/‐ 4.0 mV (means +/‐ S.D., n = 97) from a resting membrane potential of ‐53.1 +/‐ 5.7 mV. The hyperpolarization was blocked by oxyhaemoglobin (20 microM), and only occurred in arteries pre‐treated with N omega‐nitro‐L‐arginine (100 microM) or denuded of endothelium. 3. The effect of SIN‐1 was concentration dependent (EC50 approximately 0.4 microM) and its dose response was shifted to the left by zaprinast (100 microM), an inhibitor of cGMP‐specific phosphodiesterases. 4. The hyperpolarization due to SIN‐1 was modified by changes in extracellular K+ concentration, but not by changes in Ca2+, Na+ or Cl‐. The hyperpolarization was blocked by glibenclamide (IC50 approximately 0.15 microM), but not by apamin (3‐300 nM), barium (5‐150 microM), tetraethylammonium (0.1‐10 mM), or 4‐aminopyridine (5‐500 microM). The hyperpolarization due to lemakalim (0.03‐3 microM), an activator of ATP‐sensitive potassium channels (KATP), displayed the same sensitivities to these K+ channel blocking agents, whereas the endothelium‐derived hyperpolarizing factor, triggered by the addition of acetylcholine (3 microM), caused a hyperpolarization (‐15.3 +/‐ 6.2 mV) that was blocked by apamin, but not by any other agent. 5. These results suggest that NO hyperpolarizes VSM in rabbit mesenteric arteries by activating KATP channels, with the accumulation of cGMP as an intermediate step.
ATP‐activated inward current and calcium‐permeable channels in rat macrophage plasma membranes.
Authors: Naumov A P , Kaznacheyeva E V , Kiselyov K I , Kuryshev Y A , Mamin A G , Mozhayeva G N
Published: December 2014

1. To study mechanisms of receptor‐operated Ca2+ influx in non‐excitable cells, membrane currents of rat peritoneal macrophages were recorded using whole‐cell cell‐attached and outside‐out configurations of the patch clamp technique. Under whole‐cell recording conditions, ATP applied in micromolar concentrations elicited an inward current response when the bath solution contained Ba2+, Ca2+ or Na+ as the only permeant cations. 2. Increasing the Mg2+ concentration had an inhibitory effect on the ATP‐induced inward current indicating that the active form of ATP responsible for the cation entry is ATP4‐. The nucleotide potency order was ATP > ATP gamma S > ADP. UTP was completely ineffective (n = 19). The data obtained are consistent with the ATP receptor being of the P2Z type. 3. The macrophage plasma membrane was impermeable to Tris+ during the ATP‐induced current at ATP4‐ concentrations varying from 0.07 to 500 microM. At higher concentrations, ATP produced a large inward steady‐state current, which could be attributed to membrane permeabilization. 4. Activity of single channels was recorded when ATP was applied to the external surface of the patch membrane both in cell‐attached and outside‐out experiments. A specific property of the channels appeared to be the existence of at least four conductance sublevels. With 105 mM Ba2+ as the permeant cation, the conductance sublevels were 3.5, 7, 10 and 15 pS. With 10 mM Ca2+ the sublevel conductances were equal to 4, 9, 13 and 17 pS. 5. The unitary conductance estimated from the whole‐cell current noise analysis (3.5‐4.5 pS for 105 mM Ba2+) was significantly lower than that obtained from single channel measurements at the main (3rd) current level, but it was very close to the conductance of the minimum (1st) level. 6. Extrapolated reversal potential values estimated from current‐voltage curves for predominant conductance levels were equal to +40 and +26 mV for 105 mM Ba2+ and 10 mM Ca2+, respectively. The permeability ratios fell in the sequence: PCa:PBa:PK = 71.:29:1. Thus, ATP‐activated channels in the macrophage membrane are rather selective for divalent vs. monovalent cations, with the predominant permeability being for Ca2+.
Beta‐adrenergic and cholinergic modulation of inward rectifier K+ channel function and phosphorylation in guinea‐pig ventricle.
Authors: Koumi S , Wasserstrom J A , Ten Eick R E
Published: December 2014

1. To clarify the nature of the inhibition of whole‐cell inwardly rectifying K+ current (IK1) by isoprenaline (Iso) and its antagonism by acetylcholine (ACh), we studied the effects of Iso and ACh and their surrogates on single channel currents (iK1) carried by inwardly rectifying K+ channels in cell‐attached and excised inside‐out patches obtained from guinea‐pig ventricular myocytes. 2. Bath application of Iso suppressed iK1 channel activity in cell‐attached patches. This was inhibited by propranolol. Bath‐applied forskolin or dibutyryl cAMP mimicked the effect of bath‐applied Iso. 3. Exposure of the cytosolic face of inside‐out patches to purified catalytic subunit of the cAMP‐dependent protein kinase (PKA) also suppressed iK1 channel activity, mimicking the effect of bath‐applied Iso on iK1 recorded from cell‐attached patches. 4. When applied directly to cell‐attached patches via the patch pipette solution, ACh antagonized Iso‐induced (1 microM applied via the bath) suppression of iK1 channels. In contrast, bath‐applied ACh (10 microM) partially antagonized the effect of low concentrations of Iso (e.g. < 50 nM) on iK1 channels in cell‐attached patches but had no detectable effect when 1 microM or more Iso was used. 5. In myocytes pretreated with pertussis toxin (PTX), ACh failed to antagonize Iso‐induced suppression of iK1 channels. When inside‐out patches were used, bath‐applied preactivated exogenous inhibitory G protein subunit, G1 alpha, antagonized the suppression of iK1 channels induced by bath‐applied catalytic subunit of PKA (PKA‐CS), suggesting that a PTX‐sensitive G1 alpha mediates ACh‐induced antagonism of Iso‐induced suppression of iK1. 6. Neither GTP gamma S nor G1 alpha antagonized the suppression of iK1 produced by bath‐applied PKA‐CS in inside‐out patches when okadaic acid was present in the bath. In addition, bath application of alkaline phosphatase also reactivated iK1 channels suppressed by PKA‐CS. 7. Findings in guinea‐pig ventricular myocytes suggest that iK1 can be suppressed by a PKA‐mediated phosphorylation of the iK1 channel occurring in response to Iso‐induced beta‐adrenergic receptor activation and that ACh can antagonize the suppression by mechanisms that involve both intracellular and membrane‐delimited pathways. The membrane‐delimited pathway appears to involve M2‐cholinergic receptors, their associated G protein, G1, and a protein phosphatase, all located in the sarcolemma in close proximity to the involved iK1 channels.
Slowing of shortening velocity of rat cardiac myocytes by adenosine receptor stimulation regardless of beta‐adrenergic stimulation.
Authors: Strang K T , Mentzer R M , Moss R L
Published: December 2014

1. Single ventricular myocytes were enzymatically isolated, incubated with the A1‐purinergic and beta‐adrenergic receptor‐specific agonists N6‐cyclopentyladenosine (CPA) and isoprenaline (Iso), and then rapidly skinned. Ca2+ sensitivity of isometric tension and unloaded shortening velocity (Vo) were measured, and protein kinase A (PKA)‐specific phosphorylations of troponin I (TnI) and C‐protein were assessed by back‐phosphorylation of cell suspensions with [gamma‐32P]‐ATP. 2. Isoprenaline treatment decreased the Ca2+ sensitivity of isometric tension relative to propranolol‐treated controls, as did simultaneous stimulation with Iso and CPA (Iso + CPA). CPA alone had no effect on Ca2+ sensitivity. Vo was greater in Iso‐treated cells than in paired controls, while Vo was significantly less than control in both Iso + CPA‐treated and CPA‐treated cells. 3. Phosphorylation of TnI and C‐protein was increased by Iso treatment and also when Iso and CPA were simultaneously applied. CPA alone caused a significant decrease in the phosphorylation state of these two proteins. 4. From these results we conclude that A1‐purinergic receptor stimulation does not inhibit beta‐adrenergic receptor‐mediated phosphorylation of myofilament proteins, nor does it alter the Ca2+ sensitivity of isometric tension at the level of the myofilaments. However, A1‐receptor stimulation does decrease Vo at the level of the myofilaments by a mechanism that is independent of beta‐adrenergically mediated phosphorylation of TnI and C‐protein.
Activation of a Cl‐ conductance by SCN‐ in single proximal tubule cells isolated from Rana temporaria.
Authors: Robson L , Tarran R , Hunter M
Published: December 2014

1. This study investigated the effect of the anion, SCN‐, on the conductive properties of single proximal tubule cells isolated from frog kidney. Ionic currents were measured using the conventional whole‐cell patch clamp technique. 2. Addition of SCN‐ to the bath solution alone had a biphasic effect; there was an initial rapid rise, followed by a slower secondary increase, in both outward and inward conductances. However, when SCN‐ was added to the bath in the presence of pipette SCN‐, such that the concentration gradient for movement of SCN‐ into the cell was abolished, only the fast changes in conductance were observed. 3. Cells did not discriminate between cations and anions under nominally K(+)‐free control conditions. However, in the presence of intracellular SCN‐ cells became anion selective. Taken together with the increased conductance, this suggests activation of a Cl‐ channel (gSCN). 4. Niflumic acid, a Cl‐ channel blocker, inhibited gSCN in a dose‐dependent manner, with a half‐maximal blocking concentration (Ki) of 28 +/‐ 2.8 microM and a Hill coefficient of 1.2 +/‐ 0.3 (n = 6). 5. The activation of gSCN by intracellular SCN‐ was dose dependent and showed positive co‐operativity, with a Ki of 62.3 microM and Hill coefficient of 4.0. 6. Plasma and urine levels of SCN‐ range between 10 and 70 microM, thus the conductance described here may play a role in the regulation of Cl‐ handling by the kidney.
Modulation of Na(+)‐H+ exchange by altered cell volume in perfused rat mandibular salivary gland.
Authors: Seo J T , Larcombe‐McDouall J B , Case R M , Steward M C
Published: December 2014

1. Intracellular pH (pHi) was measured by spectrofluorometry in perfused mandibular salivary glands isolated from the rat and loaded with the pH‐sensitive fluoroprobe 2',7'‐bis(2‐carboxyethyl)‐5(6)‐carboxyfluorescein (BCECF). Cell volume changes were estimated from changes in intracellular water content measured by proton NMR spectroscopy. 2. Stimulation with 1 microM acetylcholine (ACh) led to a 15 +/‐ 2% decrease in cell volume. A transient decrease in pHi was followed by a sustained increase (0.17 +/‐ 0.03 pH units) that has previously been attributed to the upregulation of the Na(+)‐H+ exchanger. 3. Increasing perfusate osmolarity by addition of 60 mM sucrose caused a 19 +/‐ 2% decrease in cell volume and a sustained increase in pHi (0.12 +/‐ 0.01 pH units) that was abolished by 1 mM amiloride. Acid loading experiments indicated that the increase in pHi was due to an alkaline shift in the pH dependence of the Na(+)‐H+ exchanger. 4. A 20% reduction in perfusate osmolarity prevented the cell shrinkage normally associated with ACh stimulation and largely abolished the ACh‐induced increase in pHi. 5. Steady‐state Na(+)‐H+ exchanger activity, estimated from the initial rate of change in pHi following addition of amiloride, increased 9‐fold during stimulation with ACh. When cell shrinkage was prevented by simultaneous exposure to the hypotonic solution, the activity of the exchanger still increased 7‐fold in response to ACh. 6. We conclude that, although cell shrinkage leads to upregulation of the Na(+)‐H+ exchanger, this factor alone is insufficient to account for the marked increase in exchanger activity that follows muscarinic stimulation.
Role of airway receptors in the reflex responses of human inspiratory muscles to airway occlusion.
Authors: Butler J E , McKenzie D K , Crawford M R , Gandevia S C
Published: December 2014

1. This study was designed to investigate the role of airway receptors in the responses of a range of inspiratory muscles to airway occlusion. The occlusion had a rapid onset (< 10 ms), lasted 250 ms and produced only a slight impediment to inspiration. 2. Based on analysis of single trials and averaged rectified electromyographic responses (EMG) in six subjects, there was a major inhibition (IR) with an onset at 34 +/‐ 2 ms and a trough at 65 +/‐ 2 ms, and an excitation (ER) with a peak at 105 +/‐ 2 ms. These two responses are reflex given that voluntary reaction times to a tap on the chest wall occurred at latencies longer than the peak of ER. 3. The responses to airway occlusion did not appear in limb muscles which contracted phasically with inspiration. 4. Anaesthesia of the surface receptors of the upper airway did not attenuate the responses to occlusion. Because this procedure does not eliminate the inputs from muscle and deep laryngeal pressure receptors, two subjects were tested when intubated with a cuffed endotracheal tube so that the occlusion was delivered only to structures below the level of the trachea. Responses to airway occlusion were preserved when all upper airway receptors were ‘bypassed’. 5. Responses to airway occlusion also remained after prolonged inhalation of nebulized lidocaine (lignocaine) sufficient to block the cough reflex. 6. The receptors mediating the responses to airway occlusion are therefore likely to reside in inspiratory muscles acting on the chest wall. If so, the short‐latency inhibition contrasts with the excitatory stretch reflex responses observed in limb muscles.
A mini Cl‐ channel sensitive to external pH in the basolateral membrane of guinea‐pig parietal cells.
Authors: Kajita H , Morishima S , Shirakata Y , Kotera T , Ueda S , Okuma M , Okada Y
Published: December 2014

1. Voltage‐independent whole‐cell Cl‐ currents were recorded from both single, isolated parietal cells and parietal cells within gastric glands obtained from the fundus of guinea‐pig stomach. 2. The Cl‐ currents were rapidly suppressed by a Cl‐ channel blocker, NPPB (5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate), added to the (basolateral) bathing solution in a concentration‐dependent manner with a half‐maximal inhibition concentration of 12 microM. 3. The selectivity sequence among anions was I‐ > Br‐ > Cl‐ > F‐, corresponding to Eisenman's sequence I. 4. The Cl‐ currents were independent of cytosolic Ca2+, cyclic AMP, cyclic GMP, GTP‐gamma‐S and cell volume, and were not affected by application of acid secretagogues, omeprazol, arachidonic acid or prostaglandin E2. 5. Reduction of pH in the (basolateral) bathing solution immediately inhibited the Cl‐ current with a pK (‐log of KD) of 6.3, whereas changes in intracellular pH had no effect. 6. The single‐channel conductance was estimated to be 0.46‐0.6 pS by variance noise analysis during inhibition of whole‐cell Cl‐ currents by NPPB or acidic pH. 7. It is concluded that pH‐sensitive ‘mini’ Cl‐ channels, with a sub‐picosiemens unitary conductance, exist in the basolateral membrane of guinea‐pig parietal cells.
Extracellular osmotic pressure modulates sodium‐calcium exchange in isolated guinea‐pig ventricular myocytes.
Authors: Wright A R , Rees S A , Vandenberg J I , Twist V W , Powell T
Published: December 2014

1. The sensitivity of the cardiac Na(+)‐Ca2+ exchange current to changes in osmotic pressure was investigated in guinea‐pig ventricular myocytes, using the whole‐cell patch‐clamp technique. 2. A hyposmotic challenge applied by removal of sucrose from the standard bathing solution reduced exchanger current, measured as the Ni(2+)‐sensitive component of whole‐cell transsarcolemmal current. These changes were fully reversible. 3. No response of whole‐cell current to hyposmosis was observed when Ca2+ was removed from the bathing solution by chelation with 1 mM EGTA. 4. Inclusion of 25 microM exchanger inhibitory peptide (XIP) in the pipette solution caused a marked reduction in the Ni(2+)‐sensitive component of membrane current, but the percentage change in Ni(2+)‐sensitive membrane slope conductance evoked by hyposmosis was the same as when XIP was omitted from the pipette solution. 5. Exposure of cells to hyperosmotic solutions produced variable responses. In a majority of cells, solutions 30% hyperosmotic compared with control evoked a persistent increase in exchanger current, whereas for solutions 50% hyperosmotic, a larger but transient increase in current was observed. 6. Over a wide range of osmolalities (50‐130% of isosmotic) the changes in Ni(2+)‐sensitive membrane slope conductance were linearly related to the changes in extracellular osmotic pressure. 7. We propose that one consequence of exposing ventricular myocytes to anisosmotic solutions is modulation of Na(+)‐Ca2+ exchange current.
Injury to muscle fibres after single stretches of passive and maximally stimulated muscles in mice.
Authors: Brooks S V , Zerba E , Faulkner J A
Published: December 2014

1. Our purpose was to investigate the initial mechanisms responsible for contraction‐induced injury. Most studies of mechanisms of contraction‐induced injury have been based on observations made either shortly after many repeated contractions at the peak of fatigue, or days after, at the peak of delayed onset injury. As a result, conclusions based on these studies are complicated by interactions of mechanical and biochemical events, as well as the passage of time. We studied the initial mechanical events associated with contraction‐induced injury immediately following single stretches of whole skeletal muscles of mice in situ. 2. We tested the hypothesis that immediately following a single stretch, the severity of contraction‐induced injury is a function of both strain and average force. Consequently, the work done to stretch the muscle would be the best predictor of the magnitude of injury. Extensor digitorum longus muscles were adjusted to optimum length for force (L(o)). Passive (not stimulated) and maximally activated muscles were exposed to single stretches of 10, 20, 30, 50 or 60% strain, relative to muscle fibre length (Lf), at a rate of 2 Lf s‐1. 3. The magnitude of injury was represented by the force deficit 1 min after the stretch expressed as a percentage of the maximum force prior to the stretch. The occurrence of injury was confirmed directly by electron microscopic analysis of the ultrastructure of muscle fibres that were fixed immediately following single stretches. 4. For active muscles, a single stretch of only 30% strain produced a significant force deficit, whereas for passive muscles, a larger strain was required. Stretches of greater than 50% strain resulted in greater force deficits for passive than for maximally activated muscles. For either condition, the work done to stretch the muscle was the best predictor of the magnitude of injury, accounting for 76% of the variability in the force deficit for maximally activated muscles, and 85% for passive muscles.
Calbindin‐D28k: role in determining intrinsically generated firing patterns in rat supraoptic neurones.
Authors: Li Z , Decavel C , Hatton G I
Published: December 2014

1. Physiological activation of rat supraoptic nucleus (SON) neurones leads to phasic firing in vasopressin neurones and fast, continuous firing in oxytocin neurones. Using whole‐cell patch clamp methods in brain slices, we investigated the role of endogenous calbindin‐D28k (calbindin) in determining these intrinsically generated patterns of firing. 2. Direct introduction of calbindin (0.1‐0.2 mM) into twelve of twelve phasically firing neurones suppressed Ca(2+)‐dependent depolarizing after‐potentials (DAPs) and changed activity from phasic to continuous firing. Bovine calcium binding protein (0.3 mM), an analogue of calbindin, had similar effects on both DAPs and firing patterns in five of five cells tested. 3. Introduction of anti‐calbindin antiserum (1:2000‐5000) into thirteen of thirteen continuously firing neurones unmasked DAPs and converted continuous into phasic firing. Such effects could not be mimicked either by diffusion of normal rabbit serum or antibodies directed against glial fibrillary acidic protein or against neurophysin. 4. Immunocytochemical staining with antisera directed against calbindin revealed more intense staining in the dorsal, oxytocin‐rich and less intense staining in the ventral, vasopressin‐rich areas of the SON. 5. Elevated intracellular Ca2+ concentration ([Ca2+]i; 0.1 mM) induced DAPs and phasic firing in all twenty‐nine SON cells recorded. During chelation of intracellular Ca2+ with (1.1‐11 mM) BAPTA, fifty‐eight of fifty‐eight neurones recorded displayed regular continuous activity and had no DAPs. 6. These data suggest that firing activities in SON cells are dependent on [Ca2+]i and that calbindin, acting as an endogenous Ca2+ buffer, is involved in regulation of intrinsic firing patterns. It is likely that calcium binding proteins have a similar influence on the firing patterns of many neuronal types throughout the nervous system.
Kinetic characterization of rat brain type IIA sodium channel alpha‐subunit stably expressed in a somatic cell line.
Authors: Sarkar S N , Adhikari A , Sikdar S K
Published: December 2014

1. The rat brain type IIA Na+ channel alpha‐subunit was stably expressed in Chinese hamster ovary (CHO) cells. Current through the expressed Na+ channels was studied using the whole‐cell configuration of the patch clamp technique. The transient Na+ current was sensitive to TTX and showed a bell‐shaped peak current vs. membrane potential relation. 2. Na+ current inactivation was better described by the sum of two exponentials in the potential range ‐30 to + 40 mV, with a dominating fast component and a small slower component. 3. The steady‐state inactivation, h infinity, was related to potential by a Boltzmann distribution, underlying three states of the inactivation gate. 4. Recovery of the channels from inactivation at different potentials in the range ‐70 to ‐120 mV were characterized by an initial delay which decreased with hyperpolarization. The time course was well fitted by the sum of two exponentials. In this case the slower exponential was the major component, and both time constants decreased with hyperpolarization. 5. For a working description of the Na+ channel inactivation in this preparation, with a minimal deviation from the Hodgkin‐Huxley model, a three‐state scheme of the form O<‐‐>I1<‐‐>I2 was proposed, replacing the original two‐state scheme of the Hodgkin‐Huxley model, and the rate constants are reported. 6. The instantaneous current‐voltage relationship showed marked deviation from linearity and was satisfactorily fitted by the constant‐field equation. 7. The time course of activation was described by an m chi model. However, the best‐fitted value of chi varied with the membrane potential and had a mean value of 2. 8. Effective gating charge was determined to be 4.7e from the slope of the activation plot, plotted on a logarithmic scale. 9. The rate constants of activation, alpha m and beta m, were determined. Their functional dependence on the membrane potential was investigated.
Thyroid status and diabetes modulate regional differences in potassium currents in rat ventricle.
Authors: Shimoni Y , Severson D , Giles W
Published: December 2014

1. The rate dependence and recovery kinetics of the Ca(2+)‐independent transient (I(t)) and steady‐state or ‘pedestal’ (Iss) outward potassium (K+) currents were studied in single myocytes isolated from epicardial and endocardial regions of rat left ventricles. The whole‐cell, suction microelectrode method was used to measure baseline (fully reactivated) I(t), as well as its rate‐dependent attenuation. Results from a group of control animals were compared with data from three other groups having an experimentally altered hormonal status. 2. I(t) was significantly smaller in endocardial cells than in epicardial cells, in part due to a very large difference in the recovery kinetics of this current in endocardial cells. This was reflected in a pronounced rate‐dependent prolongation of endocardial action potentials. In contrast, the non‐inactivating ‘pedestal’ current, Iss, was very similar in magnitude and showed comparable rate dependence in cells from both epicardium and endocardium. 3. Changing the thyroid status had selective, differential actions on the amplitude and rate dependence of It in epicardial and endocardial cells. Under hypothyroid conditions there was a more pronounced reduction of baseline I(t) in epicardial than in endocardial cells. Moreover, a slowing of the recovery kinetics in epicardial cells resulted in an enhanced attenuation of this current at high rates. Changing thyroid status had no effect on the magnitude or rate dependence of Iss in cells from either region of the left ventricle. 4. Following establishment of hyperthyroid conditions, there was no significant change in I(t) magnitude at baseline. However, when compared with control data, the recovery of I(t) was considerably faster in endocardial cells, and marginally faster in epicardial cells. 5. Streptozotocin‐induced diabetic conditions resulted in a much greater attenuation of I(t) in epicardial cells than in endocardial cells. Epicardial action potentials in these conditions showed prominent rate‐dependent prolongation. Iss was reduced to a similar extent in cells from these two regions. 6. Our findings demonstrate that altered hormonal status can selectively change the amplitude and kinetics of It in the epi‐ and endocardium of rat left ventricle. These changes can reduce the epicardial‐endocardial gradients in the magnitude and recovery kinetics of It and hence diminish the intrinsic differences in both action potential duration and refractoriness.
Dependence of force on length at constant cross‐bridge phosphorylation in the swine carotid media.
Authors: Wingard C J , Browne A K , Murphy R A
Published: December 2014

1. The dependence of force (F) on length (L) in smooth muscle remains uncertain since (i) it is influenced by changes in activation (myosin light chain phosphorylation), (ii) no anatomical reference length for the contractile unit is available, (iii) the length at which optimum force is generated (L(o)) exhibits a broad, flat optimum, and (iv) the presence of an extensive connective tissue network makes it difficult to stretch tissues without damage. 2. A swine carotid medial ring preparation prepared by removal of the adventitia and endothelium could be stretched to 1.8 L(o) without decreasing active force generation on return to shorter lengths. 3. A highly reproducible mechanically defined reference length, L(o), was obtained by fitting force‐length data between 0.3 and 1.6 L(o) with a third‐order polynomial where L = L(o) when dF/dL = 0. 4. Activation as assessed by myosin regulatory light chain (MRLC) phosphorylation increased with length in 100 microM histamine‐stimulated tissues from 0.6 to 1.8 L(o). 5. Activation was constant in K(+)‐depolarized and field‐stimulated tissues from 1.0 to 1.8 L(o) allowing determination of the descending limb of the force‐length relation to be assessed independently of activation. 6. The slope of the descending limb of the force‐length relation was linear except at very long lengths, which often produced tissue damage. The slope was not statistically different from that estimated for sarcomeres in vertebrate skeletal muscle. 7. The medial ring preparation and the procedures used to define the reference length provide advantages for the measurement of length‐dependent variables.
Adenosine mediates metabolic and cardiovascular responses to hypoxia in fetal sheep.
Authors: Koos B J , Chau A , Ogunyemi D
Published: December 2014

1. In seven unanaesthetized fetal sheep (> 80% term), isocapnic hypoxia (arterial partial pressure of O2, Pa,O2, approximately 15 mmHg) was induced for 1 h by lowering maternal inspired PO2. Fetal hypoxia was also produced during intra‐arterial administration of the adenosine receptor antagonist 8‐(p‐sulphophenyl)‐theophylline (8‐SPT). The fetal 8‐SPT infusion was begun just prior to hypoxia and was stopped when fetal Pa,O2 was returned to normal. 2. Hypoxia induced a progressive fetal acidosis, a rise in mean arterial pressure, a transient fall in heart rate and a decrease in breathing movements. 8‐SPT significantly reduced the metabolic acidosis and abolished the hypertension and bradycardia without altering hypoxic inhibition of fetal breathing. Administration of the vehicle for 8‐SPT during hypoxia did not significantly affect the normal fetal metabolic and cardiovascular responses to acute O2 deprivation. 3. It is concluded that adenosine mediates the fetal bradycardia and hypertension produced by hypoxia, indicating that adenosine modulates fetal autonomic responses to acute oxygen deficiency. Secondly, adenosine contributes to fetal metabolic acidaemia, suggesting that adenosine also modulates fetal glycolytic responses to hypoxia.
Ca2+ current enhancement by alpha 2/delta and beta subunits in Xenopus oocytes: contribution of changes in channel gating and alpha 1 protein level.
Authors: Shistik E , Ivanina T , Puri T , Hosey M , Dascal N
Published: December 2014

1. A combined biochemical and electrophysiological approach was used to determine the mechanism by which the auxiliary subunits of Ca2+ channel enhance the macroscopic Ca2+ currents. Xenopus oocytes were injected with RNA of the main pore‐forming subunit (cardiac: alpha 1C), and various combinations of RNAs of the auxiliary subunits (alpha 2/delta and beta 2A). 2. The single channel open probability (Po; measured at 0 mV) was increased approximately 3‐, approximately 8‐ and approximately 35‐fold by alpha 2/delta, beta 2A and alpha 2/delta+beta 2A, respectively. The whole‐cell Ca2+ channel current was increased approximately 8‐ to 10‐fold by either alpha 2/delta or beta 2A, and synergistically > 100‐fold by alpha 2/delta+beta 2A. The amount of 35S‐labelled alpha 1 protein in the plasma membrane was not changed by coexpression of beta 2A, but was tripled by coexpression of alpha 2/delta (either with or without beta). 3. We conclude that the increase in macroscopic current by alpha 2/delta is equally due to changes in amount of alpha 1 in the plasma membrane and an increase in Po, whereas all of the effect of beta 2A is due to an increase in Po. The synergy between alpha 2/delta and beta in increasing the macroscopic current is due mainly to synergistic changes in channel gating.
Anterior pretectal nucleus facilitation of superficial dorsal horn neurones and modulation of deafferentation pain in the rat.
Authors: Rees H , Terenzi M G , Roberts M H
Published: December 2014

1. Functional relationships between the anterior pretectal nucleus (APTN) and nociceptive dorsal horn neurones were investigated electrophysiologically in the anaesthetized rat. The effects of APTN lesions were assessed behaviourally in a model of deafferentation pain. 2. Cells in the dorsal and rostral parts of the APTN were excited orthodromically by electrical stimulation of the ipsilateral dorsolateral funiculus or the contralateral dorsal columns, and by noxious and innocuous cutaneous stimuli. 3. Electrical stimulation of the APTN excited nociceptive lamina I spinal neurones. These cells all projected rostrally in the contralateral dorsolateral funiculus. Identical APTN stimulation also inhibited multireceptive spinal neurones which lay deep in the dorsal horn. These particular cells were shown to project to the brain in the ventrolateral funiculus. 4. It is proposed that noxious stimuli excite spinal lamina I projection neurones which send excitatory axons to the brain, including the APTN. The APTN inhibits deep multireceptive neurones, to reduce the perception of noxious stimuli. The discharge of spinal lamina I neurones, however, will be sustained by the noxious stimulus and by facilitation from the APTN. A sustained descending inhibition of this nature would reduce responses to prolonged injury. 5. The involvement of the APTN in responses to a chronic pain state was examined by comparing the behaviour of animals with bilateral lesions of the APTN with normal controls. Lesions of the APTN strongly enhanced the autotomy behaviour triggered by sectioning of the dorsal roots. 6. These observations support the suggestion that the APTN reduces the debilitating effects of prolonged injury.
Colocalization of ATP and nicotinic ACh receptors in the identified vagal preganglionic neurone of rat.
Authors: Nabekura J , Ueno S , Ogawa T , Akaike N
Published: December 2014

1. Effects of exogenous adenosine 5'‐triphosphate (ATP) and acetylcholine (ACh) were investigated on acutely dissociated preganglionic neurones in the dorsal motor nucleus of vagus (DMV) of rats using whole‐cell patch clamp recording methods. 2. The DMV neurones identified by retrograde transport of 1,1'‐dioctadecyl‐3,3,3',3'‐tetramethylindocarbocyanine perchlorate (DiI) fixed onto the cervical vagal nerve bundle were large in size (25‐35 microns diameter) and bipolar or tripolar in shape. 3. About 90% of DiI labelled DMV neurones responded to both ATP (10(‐4) M) and ACh (10(‐4) M) with inward currents at a holding potential (Vh) of ‐40 mV. 4. The ATP‐induced current (IATP) and the ACh‐induced current (IACh) reversed their polarities at membrane potentials between +5 and +15 mV, indicating that ATP and ACh increase the membrane permeability to cations. 5. The inhibitory potency of Reactive Blue on 5 x 10(‐4) M IATP is more effective (concentration for half‐inhibition (IC50), 4.4 x 10(‐7) M) than suramin (IC50, 6.0 x 10(‐6) M). In addition, alpha,beta‐methylene ATP up to 10(‐4) M could not induce any current. As intracellular application of guanosine 5'‐O‐(2‐thiodiphosphate) (GDP beta S) did not block the IATP, the IATP was mediated not by guanosine triphosphate (GTP) binding protein, but rather by ligand‐gated ionic channels, presumably via P2X receptors. 6. Currents produced by ACh were due to activation of nicotinic receptors because they were mimicked by nicotine and carbachol, and blocked by hexamethonium. In addition, muscarine evoked no response. 7. Only 25% of nucleus tractus solitarii (NTS) neurones and no hypoglossal neurones responded to the exogenous application of ATP. 8. These results suggest that vagal preganglionic neurones colocalize functionally nicotinic and P2X purinergic receptors.
The effects of age on the release of adenine nucleosides and nucleotides from rat caudal artery.
Authors: Hashimoto M , Shinozuka K , Bjur R A , Westfall D P , Hattori K , Masumura S
Published: December 2014

1. The spontaneous and alpha‐adrenoceptor‐induced release of ATP, ADP, AMP and adenosine were determined from arterial segments and from isolated endothelial cells from caudal arteries of young (5‐week‐old), adult (30‐week‐old) and old (100‐ to 110‐week‐old) Wistar rats. 2. The spontaneous (non‐evoked) release of the sum total of the four purines was significantly greater from artery segments of young rats than from adult and old rats. 3. The release of the adenine nucleotides and adenosine induced by methoxamine (10 microM), an alpha 1‐adrenoceptor agonist, was greater from artery segments from young rats than from old rats. 4. The spontaneous release of the sum total of the four purines was significantly greater from endothelial cells prepared from caudal arteries of young rats than of old rats. 5. The noradrenaline (10 microM)‐induced release of the sum total of the four purines was significantly greater from endothelial cells prepared from caudal arteries of young rats than of old rats. 6. The levels of adenine nucleotides and adenosine, determined in plasma from anaesthetized rats, were significantly higher in young rats compared with adult and old rats. 7. These findings suggest that the release of ATP from the vascular endothelial cells is reduced with advancing age.
Effect of noxious stimulation on sympathetic vasoconstrictor outflow to human muscles.
Authors: Nordin M , Fagius J
Published: December 2014

1. In fifteen healthy volunteers, muscle nerve sympathetic activity (MSA) was recorded from the peroneal nerve using microneurography. Blood pressure and electrocardiogram were also recorded. 2. Painful stimuli, adjusted to the subject's tolerance level, were delivered over 30‐60 s via (a) pressure to the nail‐bed of different digits, and to the trigeminal region, (b) electrical stimulation (5 Hz) of digital nerves and of the supraorbital nerve, or (c) instillation of soap solution into one eye. Non‐painful ocular pressure was also applied. 3. All procedures except electrical stimulation of digital nerves caused a marked increase in MSA (mean, 160‐248%) with preserved pulse synchrony and a rise in blood pressure. Stimulation of digits induced tachycardia, whereas stimulation of the trigeminal region tended to cause bradycardia. 4. Despite similar pain ratings, electrical stimulation of digital nerves caused a smaller MSA response than the other stimuli (mean increase, 40%). 5. It is concluded that sustained noxious stimulation in awake humans evokes a generalized MSA increase; the activity is still under baroreflex control, but the inhibitory level is reset. Both spinal and brainstem reflexes may contribute; a defence reaction is an unlikely explanation. It is suggested that the number of afferent C fibres activated by electrical stimulation of digital nerves was insufficient to induce any marked MSA response. 6. The non‐painful oculo‐cardiac reflex is associated with a strong increase in MSA.
Sodium‐‐potassium pump current in rabbit sino‐atrial node cells.
Authors: Sakai R , Hagiwara N , Matsuda N , Kassanuki H , Hosoda S
Published: December 2014

1. The Na(+)‐K+ pump current (Ip) was studied in sino‐atrial (SA) node cells of rabbits using the whole‐cell patch‐clamp technique. 2. With 50 mM Na+ in the pipette solution ([Na+]pip), changing the external K+ concentration (‐K+‐o) from 0 to 5.4 mM caused the holding current to shift in an outward direction and reach a new steady state. The current‐voltage relationships obtained by subtraction of current traces recorded at 0 mM Ko+ from those recorded at 5.4 mM Ko+ revealed time‐independent and voltage‐dependent characteristics. The external K(+)‐induced current was completely blocked by external application of 10 microM ouabain, indicating the existence of Ip in SA node cells of rabbit heart. 3. Ip increased as [K+]o increased. With 30 mM Na+pip, Ip at 0 mV was activated by [K+]o with non‐linear least‐squares fit parameters for the Hill equation of K0.5 of 1.4 mM and a Hill coefficient (nH) of 1.2 (n = 7). 4. The cation dependence of the K+ site of the Na(+)‐K+ pump was examined using various monovalent cations. The sequence was K+ > or = Rb+ > Cs+ > > > Li+. 5. Ip at 0 mV also increased as [Na+]pip was increased from 10 to 150 mM at 5.4 mM Ko+, with a K0.5 value of 14 mM and a nH of 1.3 (n = 54). 6. Ip at 0 mV was reduced by lowering the temperature from 37 to 25 degrees C with 30 mM Na+pip and 5.4 mM Ko+. The temperature coefficient (Q10) for Ip was 2.1 (n = 27). 7. With 10 mM Na+pip and 5.4 mM Ko+, the half‐activation voltage of Ip was ‐52 +/‐ 16 mV and the current at this voltage was 22.5 +/‐ 3.5 pA (n = 10), indicating that Ip contributes significantly to the background outward current during the normal pacemaker potential of SA node cells.
Characteristics of type I and type II K+ channels in rabbit cultured Schwann cells.
Authors: Baker M D , Ritchie J M
Published: December 2014

1. Voltage‐dependent K+ currents were studied in rabbit Schwann cells cultured from neonatal sciatic nerve and from the lumbar or sacral spinal roots of 10‐day‐old animals. 2. Whole‐cell K+ currents, evoked in response to depolarizing voltage‐clamp steps, were categorized as type I or type II on the basis of their apparent threshold and activation kinetics. In the presence of a quasi‐physiological [K+] gradient, the magnitude of the fully activated type I current varied linearly with membrane potential, whereas type II current always gave rise to a curved and outwardly rectifying current‐membrane potential (I‐E) relation. 3. Type II whole‐cell currents, obtained with long duration voltage‐clamp steps (> or = 1 s), have an apparent threshold for activation close to ‐40 mV. Type II current inactivated slowly, and apparently to completion. The current is more than 90% inactivated over 5 s at 0 mV (time consant of inactivation, tau h, approximately 2 s, 20‐22 degrees C). Type I current, which activates at close to ‐60 mV, inactivated at about half this rate at the same potential, assuming that inactivation also proceeds to completion. 4. Type I whole‐cell currents were reversibly blocked by superfused beta‐bungarotoxin (beta‐BuTX; apparent KD = 46 nM). beta‐BuTX did not appear to reduce type II whole‐cell currents at concentrations up to 500 nM. 5. In outside‐out patches, the type I channel had an almost linear I‐E relation over the potential range ‐60 to +60 mV with a quasi‐physiological [K+] gradient. A best linear fit gave a single‐channel conductance of 12 pS under these conditions. In symmetrical 170 mM K+, type I channels had a single‐channel conductance of 30 pS over the same potential range. 6. More slowly activating type II single‐channel currents were also recorded in inside‐out patches. With symmetrical 170 mM K+, the major conductance level was close to 9.0 pS. With a quasi‐physiological [K+] gradient, type II single channels exhibit outward rectification that is reasonably well described by the Goldman‐Hodgkin‐Katz current equation. 7. In the presence of 2 nM externally superfused alpha‐dendrotoxin (alpha‐DTX), or 50 nM superfused beta‐BuTX, unitary currents were recorded (outside‐out patches, ‐60 or ‐50 mV) that were smaller than control type I currents. Virtually all transitions in the presence of 50 nM beta‐BuTX were at one‐third of the control current level. The currents did not conform to the characteristics of type II. 8. The electrophysiological and pharmacological characteristics of the type I channel strongly suggest that it is a member of the mammalian K+ channel subfamily of Shaker homologues, most similar to the homomultimeric Kv1.1 translation product. The type II channel may be a member of the mammalian Shab subfamily. 9. Possible roles for Na+ channels and type I K+ channels in the Schwann cell are discussed.
Control of action potential propagation by intracellular Ca2+ in cultured rat dorsal root ganglion cells.
Authors: Lüscher C , Lipp P , Lüscher H R , Niggli E
Published: December 2014

1. To assess the role of intracellular Ca2+ in action potential (AP) propagation, whole‐cell recordings of cultured dorsal root ganglion (DRG) cells were carried out while Ca2+ was simultaneously measured with a laser‐scanning confocal microscope. 2. Flash photolytic liberation of a Ca2+ buffer during trains of APs which partly failed to invade the DRG cell body immediately lowered intracellular Ca2+ and restored safe AP propagation. Furthermore, the speed of the propagated AP was reduced considerably when intracellular Ca2+ was increased by flash photolysis of caged Ca2+. 3. Both results suggest that intracellular Ca2+ regulates the safety factor for AP propagation and may thus provide a control mechanism for synaptic integration, which acts pre‐ as well as postsynaptically.
Vasopressin‐induced intracellular Ca2+ increase in isolated rat supraoptic cells.
Authors: Dayanithi G , Widmer H , Richard P
Published: December 2014

1. The intracellular Ca2+ concentration ([Ca2+]1) was monitored in single magnocellular neurones freshly isolated from rat supraoptic nucleus. Application of 100 nM vasopressin increased [Ca2+]1. Two types of [Ca2+]1 responses were observed: (i) a transient response, displayed by 86% of the vasopressin‐sensitive neurones, and (ii) a sustained response displayed by 14% of the vasopressin‐sensitive neurones. 2. Among responding neurones, 52% were vasopressin sensitive, 44% were oxytocin sensitive and 4% were sensitive to both peptides. 3. Responses to vasopressin were dose dependent, showed a progressive desensitization after successive applications, were specifically blocked by the V1a vasopressin receptor antagonist, SR 49059, and were unaffected by the oxytocin receptor antagonist, d(CH2)5OVT. 4. Vasopressin responses were completely suppressed by the removal of external Ca2+. 5. The intracellular Ca2+ mobilizers, caffeine and tBuBHQ, did not affect resting or vasopressin‐induced [Ca2+]1 changes. Thapsigargin (200 nM) on its own evoked an increase in [Ca2+]1, and reduced the [Ca2+]1 increase evoked by vasopressin by 52%, suggesting that thapsigargin‐sensitive Ca2+ stores are partially involved in the vasopressin response. 6. Immunocytochemical identification revealed that vasopressin‐responding neurones synthesize vasopressin whereas oxytocin‐responding neurones synthesize oxytocin. 7. In conclusion, vasopressin‐ (partially external Ca2+ dependent) and oxytocin (totally external Ca2+ independent)‐induced [Ca2+]1 changes are mediated by specific receptors. In addition, vasopressin and oxytocin neurones are specifically autoregulated by their own peptides.