bluuebunny/crossref_metadata_2025 huggingface dataset:
Inactivation of the slow calcium current in twitch skeletal muscle fibres of the frog.
Authors: Francini F , Pizza L , Traina G
Published: December 2014

1. We investigated inactivation of the slow calcium current (ICa) at very positive potentials (over 30‐40 mV) and recovery from inactivation in cut twitch skeletal muscle fibres of the frog, using the double‐vaseline‐gap technique. External solutions were buffered against changes in [Ca2+] (Ca(2+)‐buffered) with malate. Internal solutions were Ca(2+)‐buffered with high concentrations of either EGTA (60 mM) or BAPTA (30 mM). 2. ICa decayed to a steady‐state level somewhat less than zero. Inactivation was most rapid at a potential 10 mV more negative than that which elicited the maximal ICa. 3. Involvement of current‐dependent processes (i.e. tubular Ca2+ depletion and Ca2+ entry‐dependent inactivation) in determining the decay of ICa was excluded, since inactivation was not affected by replacing Ca2+ with Ba2+ or when the size of ICa was reduced by decreasing the [Ca2+]o. Partial block of Ca2+ channels with nifedipine slowed inactivation. This was, however, independent of the size of ICa. Furthermore, neither the peak of ICa nor its time constant of decay nor the time course of ICa recovery from inactivation were affected by changing the [Ca2+]i from pCa 10 to 6. 4. ICa was potentiated during a post‐pulse preceded by a pre‐pulse at potentials ranging from ‐60 to ‐30 mV, whereas a U‐shaped inactivation curve was observed at pre‐pulse potentials more positive than ‐30 mV. This curve was asymmetric, since the ascending branch stabilized at a level less than unity. The U‐shaped form of the curve depended on post‐pulse voltage: it became more pronounced when the post‐pulse depolarization increased. Moreover, the activation and inactivation kinetics of ICa during the post‐pulse differed from control values. Similar results were found when Ca2+ was replaced with Ba2+. 5. The ICa recovery from inactivation was voltage dependent from ‐50 to ‐80 mV; it was voltage independent at more negative potentials, proving that recovery includes a voltage‐independent step. 6. The asymmetric U‐shaped inactivation curve can be reproduced by a four‐state cyclic model without assuming a Ca(2+)‐dependent step. Taking into account that recovery from inactivation includes a voltage‐independent step which becomes rate limiting at extreme negative potentials, and that during the post‐pulse the activation kinetics is faster, we propose a model which has six states, two closed, one open and three inactivated.
Endothelin activates voltage‐dependent Ca2+ current by a G protein‐dependent mechanism in rabbit cardiac myocytes.
Authors: Lauer M R , Gunn M D , Clusin W T
Published: December 2014

1. Endothelin is a vasoactive peptide released from vascular endothelial cells which has potent cardiac inotropic effects. We examined the effect of endothelin on the verapamil‐sensitive Ca2+ current (ICa) in enzymatically dispersed rabbit ventricular myocytes. 2. Using the whole‐cell voltage clamp technique with a standard dialysing pipette solution, the application of extracellular endothelin (20 nM) did not increase the peak ICa, but in fact caused a small reversible decline (903 +/‐ 109 pA without endothelin, 727 +/‐ 95 pA with endothelin (means +/‐ S.E.M., n = 14, P less than 0.05)). 3. If GTP (100 microM) was added to the pipette solution, the extracellular application of endothelin (0.2 or 20 nM) caused a large, reproducible increase in peak ICa (871 +/‐ 85 pA without endothelin, 1230 +/‐ 110 pA with 20 nM‐endothelin (n = 10, P less than 0.05). The endothelin enhancement of ICa occurred after a delay of approximately 3‐4 min at room temperature. 4. The GTP requirement for the endothelin effect on ICa suggests that its effect may be mediated through a G protein‐dependent pathway. To investigate this further, experiments were performed with pipette solutions containing guanosine‐5'‐O‐(2‐thiodiphosphate) (GDP beta S), a GDP analogue which inhibits G protein cycling. With the addition of GDP beta S (0.5‐5.0 mM) to the pipette solution (along with 100 microM‐GTP), the effect of endothelin on peak ICa was blocked (1062 +/‐ 86 pA without endothelin, 1170 +/‐ 134 pA with endothelin (n = 11, P greater than 0.05)). 5. Incubation of myocytes with pertussis toxin (500 ng/ml) prevented the partial ACh‐induced reversal of the isoprenolol enhancement of ICa. However, this identical treatment failed to block the endothelin enhancement of the voltage‐dependent Ca2+ current (n = 4). 6. Taken together, these results confirm that while the effect of endothelin in rabbit cardiac ventricular myocytes is mediated through a G protein‐dependent pathway, the G protein involved is pertussis toxin‐insensitive.
Pain, hyperalgesia and activity in nociceptive C units in humans after intradermal injection of capsaicin.
Authors: LaMotte R H , Lundberg L E , Torebjörk H E
Published: December 2014

1. Capsaicin, the potent algesic substance in chilli peppers, was applied topically to, or injected intradermally into or outside, the receptive fields of 14 C mechanoheat (polymodal) nociceptor units in awake humans. The nociceptor discharges were recorded using microelectrodes inserted into the peroneal nerve. Simultaneously, the subjects estimated the magnitude of pain as a function of time during the first 1.5‐3 min after injection. Magnitude estimates of pain produced by heat and/or mechanical stimuli were also obtained before and after capsaicin in order to assess the magnitude of cutaneous hyperalgesia. 2. An injection within or adjacent to, but not greater than 4 mm outside, the receptive fields of C nociceptor units evoked discharges. The magnitude of pain and the mean discharge rate of the units were both maximal on injection, declining rapidly over the next 1‐3 min, which indicates that these nociceptors contribute to the magnitude and duration of pain evoked by capsaicin injection. 3. Reduced or abolished excitability in C nociceptors after capsaicin injection within the receptive fields correlated with analgesia at the injection site. 4. Capsaicin injection produced a wide surround area of mechanical hyperalgesia, i.e. pain on gently stroking the skin or abnormally intense pain on punctate stimulation. Nevertheless, the injections did not lower the thresholds or enhance the responses to such mechanical stimuli of C nociceptor units with their receptive fields in this hyperalgesic area. 5. Topical application of capsaicin evoked on‐going discharges in four units tested. Both nociceptor response thresholds and pain thresholds were lowered for heat from 45 to 35 degrees C. A newly developed weak response to stroking the skin in two units after capsaicin was accompanied by faint pain. 6. On‐going activity in sensitized C nociceptors and concomitant pain were effectively reduced by cooling the skin in the receptive area. 7. It is concluded that activity in C mechanoheat (polymodal) nociceptors contributes to the magnitude and duration of pain evoked by intradermal injection of capsaicin. The after‐effects of capsaicin on C nociceptor excitability depend on concentration: high concentration (by injection) leads to desensitization, whereas low concentration (by topical application) leads to sensitization. On‐going discharges and lowered response thresholds to heat in these units after topical application of capsaicin correlates with background pain as well as lowered pain thresholds to heat of the affected skin (primary hyperalgesia). The unchanged responsiveness of C nociceptors in the skin well outside the injection area indicates that central rather than peripheral sensitization accounts for the observed mechanical hyperalgesia in this region (secondary hyperalgesia).
Mitochondrial function in type I cells isolated from rabbit arterial chemoreceptors.
Authors: Duchen M R , Biscoe T J
Published: December 2014

1. In this, and the accompanying paper (Duchen & Biscoe, 1992), we test the hypothesis that the oxygen sensitivity of mitochondrial electron transport forms a basis for transduction in the carotid body, the primary peripheral arterial oxygen sensor. We here describe for isolated type I cells the changes in autofluorescence of mitochondrial NAD(P)H that accompany changes in PO2. 2. NAD(P)H autofluorescence (excitation, 340‐360 nm; emission peak, 450 nm) increased with anoxia, reflecting a rise in the NAD(P)H/NAD(P) ratio. Graded increases in autofluorescence were seen in response to graded decreases in PO2, suggesting that mitochondrial function is progressively altered below a PO2 of about 60 mmHg. 3. A mitochondrial origin for the NAD(P)H autofluorescence was suggested by the mutual exclusion of the responses to anoxia and cyanide. 4. Oxidized flavoproteins fluoresce when excited at 450 nm with an emission peak at 550 nm. The small signals obtained under these conditions increased with uncoupler and showed a graded decrease with falling PO2 reflecting a rise in the FADH/FAD ratio. 5. Hypoxia raises [Ca2+]i. The hypoxia‐induced changes in mitochondrial function were not secondary to this rise. A brief K(+)‐induced depolarization leads to a transient increase in [Ca2+]i. At the same time there is a rapid decrease in NAD(P)H autofluorescence followed by an increase that far outlasts the rise in [Ca2+]i. This delayed increase in autofluorescence was smaller than was the increase with anoxia, even though K(+)‐induced depolarization raised [Ca2+]i more than does anoxia. In Ca(2+)‐free solutions the depolarization‐induced changes were abolished, while those associated with hypoxia were maintained. 6. The changes of autofluorescence with K(+)‐induced depolarization appear to reflect (i) oxidation of NAD(P)H by stimulation of respiration following mitochondrial Ca2+ uptake and (ii) reduction of NAD(P) by the Ca(2+)‐dependent activation of mitochondrial dehydrogenases. This activation could last several minutes following only 100 ms depolarization, while the changes accompanying hypoxia closely followed the time course of the change in PO2. 7. In similarly isolated rat or mouse chromaffin cells and mouse dorsal root ganglion neurons under identical conditions, no measurable change in autofluorescence or in [Ca2+]i was seen until the PO2 fell below about 5 mmHg. 8. Carbonyl cyanide p‐trifluoromethoxy‐phenylhydrazone (FCCP) increases O2 consumption, oxidizing mitochondrial NADH and hence decreasing autofluorescence, (delta FFCCP). Blockade of electron transport by anoxia or CN‐ decreases O2 consumption, increasing mitochondrial NADH/NAD and autofluorescence (delta FCN). The fractional change in autofluorescence with FCCP, delta FFCCP/delta FFCCP+FCN), is thus a measure of resting O2 consumption.(ABSTRACT TRUNCATED AT 400 WORDS)
Mediation by nitric oxide of neurogenic relaxation of the urinary bladder neck muscle in sheep.
Authors: Thornbury K D , Hollywood M A , McHale N G
Published: December 2014

1. Mechanical recordings were made in vitro from circularly oriented strips of the bladder neck muscle of sheep. In the absence of drugs, electrical field stimulation at frequencies of 0.2‐1 Hz evoked clear‐cut relaxations throughout 1 min stimulation periods, while higher stimulus frequencies (2‐8 Hz) evoked variable responses consisting of relaxation, contraction or a mixture of both. All of the responses were abolished by tetrodotoxin (10(‐6) M). 2. The contractions were reduced by guanethidine (10(‐6) M) and atropine (10(‐6) M), so that in the presence of these drugs clear‐cut relaxations were obtained at 0.2‐8 Hz stimulation, indicating that the relaxations were mediated by non‐adrenergic, non‐cholinergic (NANC) nerves. 3. The NANC relaxations were blocked by L‐NG nitro arginine methyl ester (L‐NAME), an inhibitor of nitric oxide synthesis. The antagonism by L‐NAME was reversed by L‐arginine. 4. Another feature of the NANC relaxation was ‘rebound contraction’ which occurred when the stimulus was switched off. The rebound contraction was also blocked by L‐NAME and restored by L‐arginine. 5. The relaxations and rebound contractions were unaffected by either alpha,beta‐methylene ATP (10(‐5) M) or 2‐methylthio ATP (10(‐5) M). 6. S‐Nitroso‐L‐cysteine, a substance which spontaneously releases NO at physiological pH, mimicked the relaxation and rebound contraction produced by nerve stimulation. 7. It is concluded that nerve‐evoked relaxation of the bladder neck is mediated by NO, or a closely related substance such as S‐nitroso‐L‐cysteine.
Calcium current activation and charge movement in denervated mammalian skeletal muscle fibres.
Authors: Delbono O
Published: December 2014

1. Calcium current (ICa) activation was studied in denervated extensor digitorum longus muscle fibres of the rat. Denervation was performed by surgically removing 6‐8 mm of the sciatic nerve at the sciatic notch. Controls were normal fibres from non‐operated rats. Electrical recordings were carried out using the double Vaseline‐gap technique. 2. Current‐voltage (I‐V) curves showed that the ICa amplitude increased during the first 4‐6 days after denervation and subsequently decreased during the second week. Between days 4 and 6 after denervation, the peak ICa amplitude (at 0 mV) was ‐5.9 +/‐ 0.5 microA/microF (mean +/‐ S.E.M.) as compared with ‐4.8 +/‐ 0.3 microA/microF in normal fibres. Between days 14 and 15 after denervation, the ICa amplitude was ‐2.9 +/‐ 0.4 microA/microF. 3. The time constant of ICa activation (tau a) was significantly increased by denervation. At 0 mV, tau a in normal fibres was 44.8 +/‐ 1.4 ms. Between 4 and 6 days after denervation tau a was 58.1 +/‐ 4.8 ms, and between 14 and 15 days after denervation, 55.8 +/‐ 3.8 ms. 4. The time constant of deactivation (tau d) decreased after denervation. At ‐10 mV, the tau d in normal fibres was 103.4 +/‐ 14 ms. The value decreased to 74.5 +/‐ 8.6 and 74.0 +/‐ 17 ms between days 4 and 6 and days 14 and 15 of denervation respectively. 5. Charge movement (Qon) was reduced progressively without major changes in the steepness (k) and position on the voltage axis of the Qon‐Vm relationship. The fitted parameters under control were Qmax = 15.4 nC/microF, mid‐point potential Vq1/2 = ‐25.2 mV and k = 11.9 mV. Between days 14 and 15 of denervation, the values for Qmax, Vq1/2 and k were 6.7 nC/microF, ‐36.8 mV and 11.3 mV respectively. 6. Calcium permeability (PCa) in normal and denervated fibres at stages during denervation was calculated according to the Hodgkin‐Huxley model. At 0 mV PCa was 1.24 x 10(‐5) cm/s in normal fibres, and 7.43 x 10(‐6) cm/s after 2 weeks of denervation. 7. The m infinity‐Vm relationship was shifted to more positive potentials after denervation without significant changes in the steepness factor k. The V1/2 value in normal fibres was ‐4.4 mV, and 5.8 mV after two weeks of denervation. 8. The ICa sensitivity to nifedipine was not modified in the different groups of denervated fibres studied. With 10 microM‐nifedipine, the 1‐(ICa in nifedipine/ICa control) relationships were 0.74 +/‐ 0.03 in normal fibres and 0.76 +/‐ 0.12, 14 days after denervation.
Effects of carbon dioxide on tetanic contraction of frog skeletal muscles studied by phosphorus nuclear magnetic resonance.
Authors: Nakamura T , Yamada K
Published: December 2014

1. Bullfrog skeletal muscles were stimulated tetanically for 2 s at a sarcomere length of 2.4 microns in Ringer solution saturated with a gas mixture of O2 and CO2 of various proportions, at 4 degrees C. Using 31P nuclear magnetic resonance (31P‐NMR), concentrations of inorganic phosphate (P(i)) and phosphocreatine (PCr) and intracellular pH (pHi) in whole muscle were measured in order to correlate the changes in these metabolites with the mechanical responses. 2. Resting muscles were first equilibrated to 5% CO2, then the CO2 concentration was increased up to 30%. This resulted in a rapid increase in [H+] (pH 6.72 in 30%, and 7.21 in 5% CO2) with little change in P(i) or PCr concentrations. When these muscles were given a single tetanic stimulation force fell in proportion to the decrease in pHi. 3. When a series of tetanic stimulations, with a long recovery period between tetani, was applied to the muscles in high CO2, force declined until a steady level was attained. A considerable increase in [P(i)] accompanied this whereas the decrease in pHi was relatively small. The force decline was much greater than could be predicted by the decrease in pHi alone. The concentration of ATP did not change significantly. 4. By subtracting the direct effect of [H+] on force a relationship between force and total P(i) concentration in the steady state of repeated contraction‐recovery cycles was obtained. Force was suppressed linearly by increasing P(i) up to 30 mmol (1 fibre water)‐1, while this relationship suggested an interaction between the effects of [P(i)] and [H+]. 5. These results have also shown that the inhibitory effects of CO2 on muscle contraction are dual. While elevation of [H+] directly suppresses contraction, this also accelerates P(i) accumulation in actively contracting muscles, which further suppresses contraction.
Classification of preganglionic neurones projecting into the cat cervical sympathetic trunk.
Authors: Boczek‐Funcke A , Dembowsky K , Häbler H J , Jänig W , McAllen R M , Michaelis M
Published: December 2014

1. The spontaneous and reflex activity patterns of 167 single preganglionic axons dissected from the cervical sympathetic trunk were examined in chloralose‐anaesthetized cats. Each neurone was classified into one of four major groups, on the basis of three principal criteria: the presence or absence of significant cardiac rhythmicity of the activity, the response to noxious stimulation of the skin, and the coupling of its activity to central inspiratory drive (phrenic nerve activity). Most neurones were also subjected to additional tests, which included carotid chemoreceptor stimulation, nasopharyngeal probing, systemic hypercapnia (ventilation with 8% CO2), hyperventilation, adrenaline‐induced blood pressure rises and retinal illumination. 2. Group I neurones (n = 69; 41%) showed significant cardiac rhythmicity, indicating strong baroreceptor control. Most (54/69) were excited by noxious stimuli, the rest being unaffected. Their activity showed variable degrees of excitatory coupling to the central inspiratory drive, and was enhanced by hypercapnia (35/39). Their responses to stimulation of arterial chemoreceptors (12/15) and nasopharyngeal receptors (24/35) were excitatory. 3. Group II neurones (n = 39; 23%) were inhibited by noxious stimulation of skin. With nine exceptions, they showed no significant cardiac rhythmicity, although they were weakly inhibited by an adrenaline‐induced blood pressure rise. Their coupling to central inspiratory drive was weak or absent, and their responses to hypercapnia and hyperventilation were variable. By contrast to other groups, they were inhibited by both chemoreceptor stimulation (9/10) and nasopharyngeal stimulation (17/18). 4. Group III neurones (n = 33; 20%) showed no significant cardiac rhythmicity, but their activity was closely coupled to central inspiratory drive. They were inhibited by hyperventilation (9/9) and excited by hypercapnia (20/21), but only fired during the central inspiratory phase and sometimes during late expiration. Their responses to noxious stimulation (28/33), chemoreceptor stimulation (8/11) and nasopharyngeal probing (24/24) were excitatory, but the induced activity was ‘gated’ by the respiratory cycle, occurring primarily during inspiration and avoiding the postinspiratory phase. 5. Group IV neurones (n = 26; 16%) showed no significant cardiac or respiratory related activity and were either excited (n = 22) or unaffected (n = 4) by noxious stimuli. One of the latter and three group II neurones were inhibited by retinal illumination; thirty‐one other neurones of all classes were unaffected. 6. Approximately 45% of thoracic sympathetic neurones were silent under the experimental conditions. About 25% of these could be recruited by systemic hypercapnia leaving 34% without spontaneous and reflex activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Maturation of carotid chemoreceptor sensitivity to hypoxia: in vitro studies in the newborn rat.
Authors: Kholwadwala D , Donnelly D F
Published: December 2014

1. A preparation was developed to record single‐fibre chemoreceptor afferent activity from carotid bodies of newborn and adult rats in vitro. The response to severe hypoxia was studied as a function of developmental age in four age groups: 1‐2, 4‐7, 10‐15 days and adult (25‐30 days). 2. During superfusion with HEPES‐saline at room PO2 and at 26 or 35 degrees C, afferent chemoreceptor activity could be recorded in all age groups. No significant difference was found among groups in baseline discharge frequency at 26 or 35 degrees C. 3. All chemoreceptors responded to anoxia (PO2 congruent to 0 Torr) with a rapid increase in discharge frequency. At 35 degrees C, peak discharge frequency of single chemoreceptor afferents was significantly greater in the adult (15.7 +/‐ 1.6 Hz, mean +/‐ S.E.M., n = 18) and 10‐15 days (11.2 +/‐ 4.2, n = 8) compared to rats of 1‐2 days (4.3 +/‐ 0.7, n = 14) and 4‐7 days of age (3.9 +/‐ 1.0, n = 7). 4. At 2 min into the anoxia period, all chemoreceptor activities were reduced from their peak discharge levels. At 35 degrees C, this decrease was significantly greater in the adult compared to the newborn. 5. During the period of decreased activity during anoxia, the chemoreceptor discharge could not be increased by inter‐stream injection of acetylcholine (100 micrograms) or dopamine (100 micrograms), although these drugs were effective in increasing discharge rate prior to hypoxia. 6. We conclude that: (1) postnatal maturation of chemoreceptor sensitivity to hypoxia is present in vitro, (2) maturation occurs between the first and second week after birth in the rat, and (3) the decrease in activity during prolonged anoxia is not greater in the newborn compared to the adult. Thus, maturational changes occur in the sensitivity of the glomus cell‐nerve ending complex to hypoxia.
The giant cardiac membrane patch method: stimulation of outward Na(+)‐Ca2+ exchange current by MgATP.
Authors: Collins A , Somlyo A V , Hilgemann D W
Published: December 2014

1. A giant patch method was used to study the stimulatory effect of cytoplasmic MgATP on outward Na(+)‐Ca2+ exchange current in inside‐out cardiac membrane patches (1‐10 G omega seals with 14‐24 microns pipette tip diameters) excised from guinea‐pig, rabbit and mouse myocytes. 2. To establish the validity of the method with respect to structure, bleb formation was examined with electron microscopy and with confocal fluorescence light microscopy. The blebs, which form as the sarcolemma detaches, excluded intracellular organelles and transverse tubules. The blebbed cells contained normal sarcomeres, sarcoplasmic reticulum, triads and diads. 3. To further establish the validity of the method for ion transport studies, measurements of Na(+)‐K+ pump currents and charge movements are described briefly which demonstrate (i) free access to the cytoplasmic membrane side, (ii) MgATP dependence comparable to reconstituted pump (Kd, 94 microns), (iii) fast, rigorous concentration control and (iv) Na(+)‐K+ pump densities in the range of whole‐cell densities. 4. Stimulation of outward Na(+)‐Ca2+ exchange current by MgATP attenuated exchange current decay during step increments of cytoplasmic sodium, shifted the secondary activation of outward exchange current by cytoplasmic calcium to lower free calcium concentrations and, particularly in mouse cardiac sarcolemma, induced cytoplasmic calcium‐independent current. 5. Upon removal of MgATP the stimulatory effect usually decayed with a t50 (half‐time) of about 3 min. However, the reversal took place much more rapidly (t50, 5‐20 s) in patches from individual guinea‐pig and rabbit myocyte batches. When decay was rapid, secondary activation by cytoplasmic calcium was shifted to higher free cytoplasmic calcium concentrations (Kd, 10‐65 microns‐free calcium). 6. With repeated applications of MgATP the rate and magnitude of the stimulatory effect progressively decreased. 7. The Kd for MgATP of the initial rate of stimulation of outward exchange current was 3 mM or greater. When decay was rapid, the steady‐state dependence of exchange current on MgATP also had a Kd of 3 mM or greater. 8. Stimulation of Na(+)‐Ca2+ exchange current by MgATP occurred in the absence of cytoplasmic calcium with 9 mM‐EGTA. 9. The stimulatory effect of 2 mM‐MgATP was not inhibited by up to 200 microM of the protein kinase inhibitor 1‐(5‐isoquinoline sulphonyl)‐2‐methylpiperazine (H7), or by peptide inhibitors of cyclic AMP‐dependent protein kinase, protein kinase C and calcium‐calmodulin‐dependent protein kinase II.(ABSTRACT TRUNCATED AT 400 WORDS)
Electrophysiological responses in the rat tail artery during reinnervation following lesions of the sympathetic supply.
Authors: Jobling P , McLachlan E M , Jänig W , Anderson C R
Published: December 2014

1. Responses to perivascular stimuli have been recorded with intracellular microelectrodes from the smooth muscle of isolated segments of the main caudal artery of rats at various times between 7 and 128 days after all four collector nerve trunks had been lesioned near the base of the tail at 21 days of age. 2. In proximal segments (< 40 mm distal to the lesions), excitatory junction potentials (EJPs) and neurogenic alpha‐depolarizations (NADs) evoked by stimuli presented via a proximally located suction electrode were similar to those in the same segments of unoperated control animals of the same age. Supramaximal EJPs in these segments decreased in amplitude with age. 3. Stimuli just supramaximal for EJPs in innervated preparations failed to evoke responses in segments farther than 30‐40 mm distal to the lesions at any time after the nerves had been cut and 1 cm excised. Higher voltages evoked slow depolarizing potentials (SDPs) which were of longer time course than EJPs. Similar responses occurred in segments over 60 mm distal to the lesions at 20‐50 days after the nerves had been frozen, and in all segments sampled over 100 mm distal to nerve lesions. 4. Spontaneous transient depolarizations (STDs) were recorded at all depths of the media in denervated segments. These occurred at frequencies similar to those of spontaneous events (including attenuated spontaneous EJPs) in innervated segments. 5. The earliest signs of reinnervation (24‐42 days after freeze lesions) consisted of very small amplitude EJPs of normal time course which facilitated markedly during a short train of stimuli (5‐10 Hz); these were followed by NADs which were large relative to the amplitudes of the EJPs. Less commonly, small focal EJPs of brief time course (resembling spontaneous EJPs in superficial cells of innervated arteries) were evoked in very restricted regions of the vessel wall. 6. At later times (57‐128 days postoperative), six of eight segments located 40‐70 mm distal to freeze lesions showed EJPs of nearly control amplitude, but NADs that were larger than in equivalent segments from control animals. In the remaining two cases, reinnervation at this level was similar to that seen at the earliest postoperative times. High stimulus voltages prolonged the decay of EJPs in both control and reinnervated arteries. 7. Sensitivity to exogenous noradrenaline, assessed in terms of membrane depolarization, was increased in both denervated and reinnervated segments. 8. Catecholamine fluorescence disappeared from the arteries at a distance greater than 30‐40 mm distal to the site of the nerve lesions.(ABSTRACT TRUNCATED AT 400 WORDS)
Ca(2+)‐activated K+ channels modulate muscarinic secretion in cat chromaffin cells.
Authors: Uceda G , Artalejo A R , López M G , Abad F , Neher E , García A G
Published: December 2014

1. This study was aimed at testing the hypothesis that Ca(2+)‐dependent K+ channels regulate the release of catecholamines mediated by muscarinic stimulation of cat adrenal chromaffin cells. Two parameters were measured: the secretory response to brief pulses of methacholine (100 microM for 10 s) in intact cat adrenal glands perfused at a high rate with oxygenated Krebs solution; and the changes in cytosolic Ca2+ concentrations, [Ca2+]i, produced by puff applications of methacholine pulses (also 100 microM for 10 s) in isolated single cat adrenal chromaffin cells loaded with Fura‐2. 2. A pulse of methacholine released 805 +/‐ 164 ng of catecholamines (mean of thirty‐two pulses). d‐Tubocurarine (DTC) increased the secretory response in a concentration‐dependent manner. The maximum increase (around 1000 ng catecholamines over control values) was reached at 100 microM‐DTC and the EC50 was around 10 microM. 3. The secretory responses to methacholine alone, or to the combination of methacholine plus DTC, were strongly dependent on the extracellular Ca2+ concentration, [Ca2+]o. Thus Ca2+o removal from the perfusing solution for 5‐10 min abolished catecholamine release. 4. At 0.1 microM, isradipine (an L‐type Ca2+ channel blocker) inhibited by 71% the secretory response to DTC plus methacholine. At 1 microM, Bay K 8644 (an L‐type Ca2+ channel activator) increased 2‐fold the secretory response to DTC plus methacholine (2746 ng of catecholamines). 5. Apamin (1 microM) increased 3.5‐fold the secretory response to methacholine pulses (from 500 to 1800 ng of catecholamines). 6. Methacholine pulses enhanced [Ca2+]i from the resting level of 100 nM to a peak of 1000 nM which quickly declined to basal level. DTC (100 microM) enhanced by 20% the [Ca2+]i peak and substantially prolonged its duration. 7. Apamin (1 microM) increased by 60% the [Ca2+]i peak evoked by methacholine, and delayed the initiation of decline of the [Ca2+]i peak. 8. These results are compatible with the idea that muscarinic stimulation depolarizes the cat adrenal chromaffin cell through an unidentified mechanism. Depolarization is probably counteracted by activation of Ca2+i‐dependent K+ channels. Therefore, inhibition of these channels enhances depolarization and firing of action potentials which activate voltage‐dependent L‐type Ca2+ channels to increase further the Ca2+i signal and the secretory response. Thus Ca2+i‐dependent K+ channels, probably of the small‐conductance type (SK), seem to be involved in the modulation of muscarinic‐evoked catecholamine release responses in cat adrenal chromaffin cells.
The relationship between glucose‐induced K+ATP channel closure and the rise in [Ca2+]i in single mouse pancreatic beta‐cells.
Authors: Valdeolmillos M , Nadal A , Contreras D , Soria B
Published: December 2014

1. Intracellular calcium [Ca2+]i and channel activity were simultaneously recorded in single, dissociated mouse beta‐cells kept in culture for 1‐3 days. [Ca2+]i was estimated from microfluorometric ratio methods using Indo‐1. Channel activity was measured using the cell‐attached configuration of the patch‐clamp technique. 2. At low glucose concentrations (0.3 mM), resting K+ATP channel activity was prevalent. Increasing glucose up to 16 mM, produced a gradual decrease in K+ATP channel activity over a time course of 90‐120 s (temperature = 23 degrees C) and an increase in [Ca2+]i. 3. In the majority of experiments, glucose elicited biphasic action currents (action potentials) which preceded the rise in [Ca2+]i. There was a close correlation between spike frequency and the levels of [Ca2+]i. 4. The sulphonylurea tolbutamide (1 mM) blocked K+ATP channels in 10‐20 s. K+ATP channel blockade was associated with a quick rise in [Ca2+]i. 5. When K+ATP channel activity was stimulated in the presence of diazoxide (100 microM), increasing the glucose concentration from 3 to 16 mM produced a decrease in [Ca2+]i. Only when diazoxide was removed did glucose produce an increase in [Ca2+]i. 6. In a small population of cells, glucose (16 mM) produced a small decrease in K+ATP channel activity but not an increase in [Ca2+]i. In such cells, tolbutamide blocked K+ATP channels and produced an increase in [Ca2+]i. 7. These results demonstrate a close correlation between K+ATP channel activity and [Ca2+]i in beta‐cells. The findings are consistent with the model in which glucose metabolism produces a rise in [Ca2+]i through the blockade of K+ATP channels, membrane depolarization and calcium current activation.
Electrophysiological studies of anion secretion in cultured human epididymal cells.
Authors: Huang S J , Leung A Y , Fu W O , Chung Y W , Zhou T S , Chan P S , Wong P Y
Published: December 2014

1. Primary monolayer cultures from adult human epididymis were grown on Petri dishes and previous supports. The epithelia so formed were used for whole‐cell patch clamp recording and short‐circuit current (ISC) measurement. 2. After 50 days of culture, the cells formed a tight epithelium with transepithelial potential of 5.5 +/‐ 1.3 mV (mean +/‐ S.E.M.., n = 16), apical side negative, and a basal ISC of 6.9 +/‐ 0.9 microA cm‐2 (mean +/‐ S.E.M., n = 16). 3. Adrenaline, when added to the basolateral side, at a concentration of 0.23 mumol l‐1 increased the ISC by 3.0 +/‐ 1.2 microA cm‐2 (mean +/‐ S.E.M., n = 4). This increase was blockable by diphenylamine‐2‐carboxylate (DPC, 1 mmol l‐1). Forskolin (10 mumol l‐1) also evoked a similar response to adrenaline. 4. In whole‐cell patch clamp experiment, the resting membrane potential of the cells after dialysis with pipette solution containing 135 mmol l‐1 KCl was found to be ‐30 +/‐ 14 mV (mean +/‐ S.E.M., n = 15). 5. About 90% of the cells successfully forming patches responded to 1 mumol l‐1 adrenaline by an increase in inward current at ‐70 mV holding potential (delta I = ‐1600 +/‐ 900 pA, mean +/‐ S.E.M., n = 15). This increase in current was accompanied by a shift in reversal potential to ‐2 +/‐ 1 mV (mean +/‐ S.E.M., n = 15). 6. The adrenaline‐induced inward current was found to be blockable by the Cl‐ channel blocker, DPC (0.25 mmol l‐1). Ion substitution experiments showed that the adrenaline‐evoked current was carried mainly by Cl‐. 7. The effect of adrenaline on the whole‐cell current was found to be mimicked by forskolin and could be abolished by including GDP beta S or a protein kinase A inhibitor in the pipette solution. Propranolol, but not phentolamine, completely abolished the effect of adrenaline. 8. Inclusion of 20 mmol l‐1 EGTA or 2 mmol l‐1 BAPTA + 100 mumol l‐1 TMB‐8 (to inhibit intracellular Ca2+ release) in the pipette did not seem to have any marked effect on adrenaline‐evoked whole‐cell current. Lowering the pipette Ca2+ concentration to 1 nmol l‐1 or raising it to 10 mumol l‐1 had no effect on the whole‐cell current response to adrenaline. 9. This study shows that adrenaline stimulates Cl‐ secretion in cultured human epididymal cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Multiple kinetic components of sodium channel inactivation in rabbit Schwann cells.
Authors: Howe J R , Ritchie J M
Published: December 2014

1. We have studied the kinetics of inactivation of sodium currents evoked in Schwann cells from neonatal and adult rabbits with patch‐clamp recording techniques. The decay both of whole‐cell currents and of ensemble currents obtained from outside‐out patches was reasonably well‐described by single exponential fits which gave values for the time constant, tau h, similar to those found for such currents in nerve (about 0.5 ms at 0 mV). Although inclusion of an additional exponential component usually improved the fits to the decay of these currents, the relative amplitude of the slower component (time constant 3‐6 ms) was always small. 2. The time course of recovery from steady‐state inactivation clearly consisted of two exponential components. At ‐120 mV, recovery from steady‐state inactivation at ‐50 mV consisted of a fast component with a time constant of 2.2 +/‐ 0.2 ms and a much slower component with a time constant of 1.2 +/‐ 0.2 s (n = 9). The relative amplitude of the slow component (expressed as a fraction of the sodium current when inactivation was removed completely) was 0.56 +/‐ 0.03. The corresponding amplitudes of the slow component when similar experiments were done from holding potentials of 0 and ‐70 mV were 0.80 +/‐ 0.04 and 0.36 +/‐ 0.03, respectively (n = 5 and 8). 3. The onset of steady‐state inactivation also followed a bi‐exponential time course. The time constant of the slower component was similar at each potential examined (0, ‐50 and ‐70 mV), being 6‐7 s. The relative amplitude of the slow component of onset depended on membrane potential, and it was similar (at each potential examined) to the corresponding amplitude of the slow component of recovery. 4. The inclusion of 40 mM‐iodate ions in the pipette solution slowed the decay of whole‐cell sodium currents, as did the extracellular application of venom (10 micrograms ml‐1) from the scorpion Leiurus quinquestriatus. Prolonged exposure of the cells to Leiurus venom appeared to increase the steady‐state amount of slow inactivation. 5. Records of single‐channel sodium currents tended to cluster into records which either did, or did not, contain openings. This apparently non‐random behaviour depended on membrane potential, and on the frequency at which the test steps were repeated, in the way expected if it resulted from slow inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)
Ca(2+)‐dependent heat production under basal and near‐basal conditions in the mouse soleus muscle.
Authors: Chinet A , Decrouy A , Even P C
Published: December 2014

1. The rate of energy expended for the clearance of sarcoplasmic Ca2+ by sarcoreticular Ca2+ uptake process(es), plus the concomitant metabolic reactions, was evaluated from measurements of resting heat production by mouse soleus muscle before and after indirect inhibition of Ca2+ uptake by sarcoplasmic reticulum (SR). 2. Direct inhibition of the Ca2+, Mg(2+)‐ATPase of SR membrane in intact muscle preparations exposed to the specific inhibitor 2,5‐di(tert‐butyl‐1,4‐benzohydroquinone (tBuBHQ) slowly increased the rate of heat production (E). Indirect inhibition of SR Ca2+ uptake was obtained by reducing sarcoplasmic Ca2+ concentration (Ca2+i) as a consequence of reducing Ca2+ release from the SR using dantrolene sodium. This promptly decreased E by 12%. Exposure of the preparations to an Mg(2+)‐enriched environment (high Mg2+) or to the chemical phosphatase 2,3‐butanedione monoxime (BDM), two other procedures aimed at decreasing SR Ca2+ release, also acutely decreased E, by 20 and 24%, respectively. 3. Subthreshold‐for‐contracture depolarization of the sarcolemma achieved by increasing extracellular K+ concentration to 11.8 mM induced a biphasic increase of E: an initial peak to 290% of basal E, followed by a plateau phase at 140% of basal E during which resting muscle tension was increased by less than 3%. Most, if not all, of the plateau‐phase metabolic response was quickly suppressed by dantrolene or high Mg2+ or BDM. Another means of increasing SR Ca2+ cycling was to partially remove the calmodulin‐dependent control of SR Ca2+ release using the calmodulin inhibitor W‐7. The progressive increase in E with 30 microM‐W‐7 was largely reduced by dantrolene or high Mg2+ or BDM. 4. In the presence of either dantrolene or BDM to prevent the effect of W‐7 on SR Ca2+ release, exposure of the muscle to W‐7 acutely suppressed about 3% of E. This and the above results confirm that the plasmalemmal, calmodulin‐dependent Ca(2+)‐ATPase, although a qualitatively essential part of the Ca2+i homeostatic system of the cell, can only be responsible for a very minor part of the energy expenditure devoted to the homeostasis of Ca2+i. Active Ca2+ uptake by SR which, at least in the submicromolar range of Ca2+i, is expected to be responsible for most of this Ca(2+)‐dependent energy expenditure, might dissipate up to 25‐40% of total metabolic energy in the intact mouse soleus under basal and near‐basal conditions.
Volume‐regulatory Cl‐ channel currents in cultured human epithelial cells.
Authors: Kubo M , Okada Y
Published: December 2014

1. During osmotic swelling, cultured human small intestinal epithelial cells (Intestine 407) exhibited activation of large Cl‐ currents under the patch‐clamp whole‐cell configuration. The volume‐sensitive Cl‐ conductance was independent of intracellular Ca2+ and cyclic AMP. 2. The anion permeability sequence of the current was SCN‐ > I‐ > Br‐ > Cl‐ > F‐ > gluconate‐, corresponding to Eisenman's sequence I. 3. Cl‐ currents were instantaneously activated by command pulses in a range of ‐120 to +45 mV. At potentials more positive than +50 mV the current showed a time‐dependent inactivation. This inactivation was accelerated by increased depolarization. The instantaneous current‐voltage relationship rectified in the outward direction. 4. A stilbene‐derivative Cl‐ channel blocker, 4‐acetamido‐4'‐isothiocyanostilbene (SITS), inhibited the Cl‐ current at micromolar concentrations. SITS facilitated inactivation at positive potentials. Outward currents were more prominently suppressed by SITS than inward currents. The concentrations required for 50% inhibition (IC50) of outward and inward currents were 1.5 and 6 microM, respectively. The outward and inward currents were equally inhibited by a carboxylate analogue Cl‐ channel blocker, 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate (NPPB) or diphenylamine‐2‐carboxylate (DPC) at higher doses (IC50 = 25 for NPPB or 350 microM for DPC). Inactivation kinetics at large depolarizations was not affected by NPPB or DPC. 5. The Cl‐ current was blocked by an unsaturated fatty acid, arachidonic acid (IC50 = 8 microM). Arachidonic acid was still effective in the presence of inhibitors of lipoxygenase (nordihydroguaiaretic acid, 10 microM), cyclo‐oxygenase (indomethacin, 10 microM) and protein kinase C (polymyxin B, 30 microM). The Cl‐ current was also sensitive to another cis unsaturated fatty acid, oleic acid, which is not a substrate for oxygenases. A trans isomer of oleate, elaidic acid, and a saturated fatty acid, palmitic acid, were ineffective. 6. Single Intestine 407 cells exposed to a hypotonic solution showed a regulatory volume decrease after initial osmotic swelling. The volume regulation was abolished by SITS, NPPB, arachidonate and oleate, but not by elaidate and palmitate. 7. It is concluded that outwardly rectifying Cl‐ channels, which are sensitive to arachidonic acid, are activated upon osmotic swelling and involved in the subsequent cell volume regulation.
Intracellular cations modulate noradrenaline‐stimulated calcium entry into smooth muscle cells of rat portal vein.
Authors: Pacaud P , Loirand G , Bolton T B , Mironneau C , Mironneau J
Published: December 2014

1. The action of noradrenaline (NA, 10 microM) was studied in single patch‐clamped smooth muscle cells of rat portal vein where free internal Ca2+ concentration in the cell (Ca2+i) was estimated using the emission from the dye Indo‐1. 2. In the presence of 50 microM D600, a blocker of voltage‐dependent Ca2+ channels, NA applied to the cell evoked an initial peak in Ca2+i followed by a smaller sustained rise. The initial rise in Ca2+i was associated with the activation of Ca(2+)‐dependent Cl‐ channels. 3. The maintained rise in Ca2+i induced by NA was enhanced by increasing the external Ca2+ concentration and was abolished in Ca(2+)‐free solution. The transient rise was resistant to the absence of external Ca2+. 4. Following the transient rise in Ca2+i induced by NA, the mechanisms extruding and/or sequestering cytoplasmic Ca2+ were stimulated. This stimulation was measured during the sustained rise in Cai and was maintained for a few seconds after NA was removed. 5. Unlike the transient rise in Ca2+i, the sustained rise in Ca2+i produced by NA was affected by changing the cell membrane potential. 6. Changing the Na+ gradient showed that the Na(+)‐Ca2+ exchange was not involved in the sustained rise in Ca2+i. 7. The sustained increase in Ca2+i produced by NA was modulated by intracellular cations. This phase could be observed with 130 mM Na+ or 130 mM K+ in the pipette solution, but was severely reduced when the only cation in the intracellular solution was Cs+ and abolished with NMDG (N‐methyl‐D‐glucamine) or TEA. However, inclusion of only 10 mM Na+ or 50 mM K+ in the pipette solution was sufficient to obtain a sustained rise in Ca2+i of maximal amplitude, similar to that obtained with 130 mM Na+i or 130 mM K+i during NA application. 8. In portal vein smooth muscle cells, NA induced a two‐phase increase in Ca2+i similar to the two phases which have been previously observed upon muscarinic receptor activation by carbachol in intestinal smooth muscle. The transient rise was due to Ca2+ store release whereas the sustained rise was due to an increased Ca2+ entry into the cell down its electrochemical gradient, but not through voltage‐dependent Ca2+ channels. The Ca2+ permeability pathway involved in the sustained rise in Ca2+i induced by NA was modulated by the intracellular cations.
Multiple motor pathways to single smooth muscle cells in the ferret trachea.
Authors: Mitchell H W , Coburn R F
Published: December 2014

1. We investigated the distribution and characteristics of motor pathways to individual smooth muscle cells activated by electrical stimulation of either, single nerves which enter the tracheal plexus (inlet nerves), or a longitudinal nerve trunk (LNT) located near the entrance of an inlet nerve into the plexus. Excitatory junction potentials (EJPs) were recorded using intracellular microelectrodes as an index of smooth muscle cell activation. In all experiments EJPs were completely blocked by tetrodotoxin and by atropine. 2. In smooth muscle fields located in the caudal direction from the point of inlet or LNT nerve stimulation, neural input decreased as a function of distance. There was evidence of a demarcated area innervated by neurons entering the plexus in one inlet nerve. In smooth muscle fields located in the rostral or transverse direction from the site of nerve stimulation, no such demarcated area could be identified. 3. Of the smooth muscle cells located within the innervated fields studied, 83‐95% were activated following stimulation of a single inlet nerve or LNT. Evoked EJPs were similar in different innervated cells or units of electrically coupled cells located within the same 1 mm2 ‘field’. 4. There was overlapping cholinergic motor input to single smooth muscle cells originating from neurons present in different inlet nerves or different neurons present in the same inlet nerve or region of the LNT. Multiple small step increases in the voltage used to stimulate a LNT resulted in three or four step increases in EJP amplitudes. This gives a minimal value for the number of motor pathways that can be activated by neurons in a region of LNT leading to a single smooth muscle cell. 5. Motor pathways to smooth muscle cells located in caudal and rostral fields ran initially in the LNT and exited in proximity to the smooth muscle cell studied. 6. Motor pathways used in transmitting signals to smooth muscle cells to different areas of trachealis muscle varied in their sensitivity to hexamethonium or curare. EJPs evoked in fields located in the caudal direction from the stimulating electrode were abolished by these drugs. Muscle cells located in different rostral fields showed EJPs that were either sensitive or resistant to these drugs. 7. The rostral hexamethonium‐resistant pathway ran initially in the LNT but it exited from the LNT several millimetres before reaching the level of the smooth muscle field innervated. This pathway followed stimulation frequencies up to 25 Hz. The final neuron in this pathway released acetylcholine and evoked EJPs were entirely inhibited by atropine.(ABSTRACT TRUNCATED AT 400 WORDS)