bluuebunny/crossref_metadata_2025 huggingface dataset:
The effects of Na+ replacement on intracellular pH and [Ca2+] in rabbit salivary gland acinar cells.
Authors: Elliott A C , Lau K R , Brown P D
Published: December 2014

1. The role of Na(+)‐dependent mechanisms in regulating the intracellular pH (pHi) and free calcium concentration ([Ca2+]i) in acinar cells of the rabbit mandibular salivary gland was examined. The fluorescent dyes BCECF and Fura‐2 were used to measure pHi and [Ca2+]i respectively in suspensions of isolated acini. 2. Replacement of all the extracellular Na+ with N‐methyl‐D‐glucamine (NMDG) decreased resting pHi from a control value of 7.1‐7.2 to 6.8‐6.9. Re‐addition of Na+ or Li+ caused a recovery of pHi towards control values. This recovery was blocked by 10‐50 microM‐ethylisopropylamiloride (EIPA), suggesting that it was mediated by Na(+)‐H+ exchange. The rate of recovery of pHi when Na+ was re‐introduced increased with Na+ concentration with an apparent Km for Na+ of around 30 mM. 3. Replacement of all of the extracellular Na+ with Li+ caused only a small decrease in resting pHi. 4. Stimulation of acini with 1 microM‐acetylcholine (ACh) evoked an intracellular acidosis both under control conditions and when acini were bathed in Na(+)‐free media. Following the acidosis pHi recovered in acini bathed in either control medium or Na(+)‐free (Li+) medium, but not in acini bathed in Na(+)‐free (NMDG) medium or in control medium containing EIPA. 5. Stimulation of acini bathed in Na(+)‐free, HCO(3‐)‐free medium with ACh did not cause any change in pHi. 6. Re‐addition of Na+ to acini bathed in Na(+)‐free, HCO(3‐)‐free medium evoked the same rate of alkalinization whether or not the acini had been stimulated with ACh, suggesting that receptor stimulation per se did not lead to an activation of acid extrusion. 7. Resting [Ca2+]i was elevated in acini bathed in Na(+)‐free (NMDG) medium, but not in acini bathed in Na(+)‐free (Li+) medium. 8. ACh evoked a maintained rise in [Ca2+]i in acini bathed in control medium and in Na(+)‐free media with either NMDG or Li+ as the Na+ substitute. 9. Experiments in which external Ca2+ was reduced to low levels (by the addition of EGTA) just prior to addition of ACh showed that ACh released intracellular Ca2+ stores under both control and Na(+)‐free conditions. 10. In acini bathed in Na(+)‐free (NMDG) solution and stimulated with ACh, re‐addition of either Na+ or Li+ reduced [Ca2+]i. The reduction of [Ca2+]i on Na+ re‐addition was blocked by EIPA. [Ca2+]i could also be reduced under these conditions by alkalinizing the cytosol using the weak base trimethylamine.(ABSTRACT TRUNCATED AT 400 WORDS)
Hyperpolarization as a mechanism for endothelium‐dependent relaxations in the porcine coronary artery.
Authors: Nagao T , Vanhoutte P M
Published: December 2014

1. The nature of endothelium‐dependent relaxations resistant to nitro‐L‐arginine was investigated in porcine coronary arteries by measuring isometric force and membrane potential in the presence of indomethacin. 2. Bradykinin induced concentration‐ and endothelium‐dependent relaxations and hyperpolarization in tissues contracted with prostaglandin F2 alpha. Nitro‐L‐arginine did not affect either the relaxations or the hyperpolarization induced by bradykinin. The threshold concentration of bradykinin was the same for the nitro‐L‐arginine‐resistant relaxations and the membrane hyperpolarization. 3. Nitro‐L‐arginine‐resistant relaxations were evoked by several agents (A23187, thrombin and UK 14304) in addition to bradykinin. The amplitude of membrane hyperpolarizations observed with all agents was proportional to that of nitro‐L‐arginine‐resistant relaxations. 4. Thrombin caused more transient relaxations and hyperpolarizations than bradykinin in the presence of nitro‐L‐arginine. 5. In tissues contracted with high K+ or tetrabutylammonium (a non‐selective K(+)‐channel blocker), bradykinin inhibited the contractions in a concentration‐dependent manner, whereas membrane hyperpolarization was not observed. The relaxations evoked by the kinin were abolished by nitro‐L‐arginine. 6. These results suggest that endothelium‐dependent relaxations which are resistant to nitro‐L‐arginine are mediated by membrane hyperpolarization in the porcine coronary artery.
Conventional versus slug CO2 loading and the control of breathing in anaesthetized cats.
Authors: van der Grinten C P , Schoute E , de Vries W R , Luijendijk S C
Published: December 2014

1. Conventional inspiratory CO2 loading (CCL) is accomplished by having the subject breathe CO2‐enriched air. An alternative method of CO2 loading is to inject a bolus of CO2 at the start of each inspiration into the inspired air: slug CO2 loading (SCL). During SCL PCO2 in the conducting airways declines quickly towards 0 kPa in the course of inspiration, whereas PCO2 remains at a constant value equal to the inspiratory PCO2 during CCL. Therefore, CCL and SCL may stimulate the respiratory controller differently. 2. We compared the ventilatory responses to SCL and CCL in fourteen anaesthetized, spontaneously breathing cats. In each experimental animal we applied, in a fixed randomized order, five CCL experiments (fractional inspiratory CO2, FI,CO2 = 0.01‐0.05), six SCL experiments (slugs of 50% CO2 ranging from 0.5 to 6 ml) and three control experiments in which no CO2 was loaded. Partial pressure of CO2 in arterial blood was determined from small blood samples (0.14 ml). In three cats we repeated the experiments after bilateral transection of the cervical vagi to evaluate the contribution of vagal receptors to the responses observed. 3. The average slope of the CO2 response curves for SCL was 2 times steeper than that for CCL (P less than 0.01). The larger minute ventilation for SCL for a particular value of arterial PCO2 (Pa,CO2) could not be attributed exclusively to the increased breathing frequency or the increased tidal volume (P greater than 0.10). Further, mean inspiratory flow (VI) was larger for SCL than for CCL at the same Pa,CO2 (P less than 0.01), also because the ratio TI/TE (inspiratory duration/expiratory duration) was smaller (P less than 0.01). After vagotomy the difference between SCL and CCL response curves was much reduced. 4. It is concluded that SCL and CCL affect the respiratory controller in a different way causing differences in breathing pattern and CO2 sensitivity between the two methods. Evidently, a mechanism based on CO2 sensitivity of pulmonary receptors is involved in the responses observed.
Potassium channels and regulation of proliferation of human melanoma cells.
Authors: Nilius B , Wohlrab W
Published: December 2014

1. Ion channels and their possible relation to cell proliferation have been studied in a human melanoma cell line (IGR 1). Membrane currents were recorded by the patch‐clamp technique using the cell‐attached, cell‐free and whole‐cell mode. Cell growth was monitored by counting the number of cells at different days after seeding and [3H]thymidine incorporation. 2. A voltage‐dependent 10 pS non‐inactivating potassium channel (delayed rectifier) is the most commonly observed ion channel in this type of human cell. The channel is active at the normal resting potential and can be blocked by tetraethylammonium chloride (TEA) and also by a membrane‐permeable cyclic adenosine monophosphate (8‐(4‐chlorophenylthio)adenosine 3',5'‐cyclic monophosphate, cyclic AMP). A second type of potassium channel shows properties similar to voltage‐dependent A‐type potassium channels with complete inactivation. 3. A voltage‐independent, non‐selective cation channel with a single‐channel conductance of approximately 20 pS could be seen in only 8% of the patches. Its properties of modulation are still unknown. 4. The incidence of the 10 pS, non‐inactivated potassium channel was maximal at the fourth day after seeding (in 89% of the patches) and was significantly reduced at the seventh day (in 35% of the patches). 5. [3H]thymidine incorporation is maximal at the third day after seeding and is reduced when cells are grown in the presence of TEA or cyclic AMP. This peak of maximal [3H]thymidine incorporation correlated with the incidence of non‐inactivated potassium channels. 6. In the presence of TEA or cyclic AMP, growth of the cells is inhibited. We suppose that due to block of potassium channels, most of the melanoma cells are not able to enter the S‐phase in the cell division cycle. 7. It is concluded that delayed rectifier potassium channels are involved in the control of melanoma cell proliferation. A similar finding has been reported for K+ channels in T‐lymphocytes and human breast carcinoma cells. It is suggested that potassium channels may be involved in controlling the driving force for a calcium influx thereby interacting with Ca(2+)‐dependent cell cycle control proteins.
Stilbene disulphonates inhibit apparently separate chloride transporters in skeletal muscle of Rana temporaria.
Authors: Hansen M , Skydsgaard J M
Published: December 2014

1. Inhibition of 36Cl‐ efflux in frog muscle by the stilbene disulphonates (SD) SITS (4‐acetamido‐4'‐isothiocyanato‐stilbene‐2,2'‐disulphonate) and DNDS (4,4'‐dinitro‐stilbene‐2,2'‐disulphonate) depends on the external pH, while the blocking of Cl‐ conductance with SITS is independent of pH. The 36Cl‐ efflux inhibition with DNDS has been studied in thin depolarized muscle fibre bundles in order to examine whether this difference is consistent with one transporter or reflects the existence of two mutually independent conductive and non‐conductive SD‐sensitive Cl‐ pathways. 2. The 36Cl‐ efflux response to a sudden inward KCl gradient was studied. At high pH the efflux decreased as predicted for dominant conductive Cl‐ single‐filing, and at low pH the efflux increased in agreement with dominant non‐conductive Cl‐ antiport. DNDS inhibition resulted in the same response, an efflux decrease, at both high and low pH, suggesting a selective reduction of the non‐conductive contribution at low pH. 3. The inhibition of 36Cl‐ efflux as a function of the DNDS concentration at an external pH of 6.9 showed complex kinetics, which could be described as the sum of two Michaelis‐Menten functions with different half‐inhibition concentrations of DNDS. 4. The results support the hypothesis that the stilbene disulphonate‐sensitive Cl‐ transport is mediated by two separate transporters rather than one.
Liquid flow across the epithelium of the artificially perfused lung of fetal and postnatal sheep.
Authors: Ramsden C A , Markiewicz M , Walters D V , Gabella G , Parker K A , Barker P M , Neil H L
Published: December 2014

1. The lungs of five fetal (133‐140 days gestation) and thirty‐four postnatal (2‐240 days) sheep were artificially perfused in situ with warmed and oxygenated sheep blood. In postnatal animals the airspace of the lung was filled with liquid similar in composition to fetal lung liquid. In fetal and postnatal animals luminal liquid volume was measured by the impermeant tracer technique. 2. Under resting conditions the pulmonary epithelium of fetal animals secreted liquid at a mean (+/‐ S.E.M.) rate of 2.0 (+/‐ 0.4) ml (kg body weight)‐1 h‐1, those of postnantal animals absorbed liquid at ‐1.8 (+/‐ 0.2) ml (kg body weight)‐1 h‐1. 3. Addition of 2,4‐dinitrophenol to achieve a concentration of 1.5 x 10(‐3) M in the perfusing blood in postnatal animals caused complete cessation of liquid absorption. 4. Light and electron microscopic examination of the lung after periods of up to 6 h of artificial perfusion showed no evidence of epithelial damage. From 3 h onwards, liquid accumulation was evident in the perivascular spaces. 5. Addition of adrenaline to the perfusate in fetal animals caused absorption of liquid to occur at a mean rate of ‐2.9 (+/‐ 1.3) ml (kg body weight)‐1 h‐1. In postnatal animals adrenaline caused the rate of liquid absorption to increase from a mean rate of ‐1.4 (+/‐ 0.2) to ‐2.2 (+/‐ 0.3) ml (kg body weight)‐1 h‐1. 6. In the fetus addition of amiloride (0.8 x 10(‐4) M) to the luminal fluid blocked adrenaline‐induced liquid absorption and caused secretion to occur at 1.3 (+/‐ 0.3) ml (kg body weight)‐1 h‐1. 7. In postnatal animals the response to amiloride was age dependent. In newborn lambs (2‐14 days) amiloride blocked liquid absorption and caused secretion of liquid to occur in seven out of eight animals at a mean rate of 0.9 (+/‐ 0.3) ml (kg body weight)‐1 h‐1 (n = 8). In older animals (15‐240 days) the characteristic response to amiloride was slowing of the rate of liquid absorption (mean rate of absorption,‐0.2 (+/‐ 0.09) ml (kg body weight)‐1 h‐1, n = 18) with liquid secretion being seen in only three of eighteen animals.(ABSTRACT TRUNCATED AT 400 WORDS)
Factors causing difference in force output among motor units in the rat medial gastrocnemius muscle.
Authors: Kanda K , Hashizume K
Published: December 2014

1. The isometric contractile properties and morphological characteristics of the muscle unit portion of motor units were investigated in the medial gastrocnemius (MG) muscle of Fischer 344 rats. Individual motor units were functionally isolated by stimulating single MG axons in finely dissected ventral root filaments. 2. To study the mechanical properties of the motor units in the rat MG muscle, ninety‐six motor units in five animals were classified into three categories (type FF, FR and S units) using two physiological criteria: presence or absence of the 'sag' property and fatigability. The relative distribution of the different motor unit types in the sample was 35.4% for type FF, 47.9% for type FR, and 16.7% for type S units. 3. There was little overlap in the distribution of twitch contraction time between type F (including types FF and FR) and type S units. The mean value was 17.1 ms for type FF, 15.7 ms for type FR, and 28.0 ms for type S units. Type FF units produced the largest tetanic tension (mean +/‐ S.D.; 201 +/‐ 75 mN). Tension output of type S units was the smallest (15 +/‐ 6 mN), and that of type FR units was intermediate (100 +/‐ 45 mN). These values were significantly different. 4. A muscle unit portion of twenty‐three motor units (8 FF, 6 FR, and 9 S units) was depleted of its glycogen through repetitive stimulation after characterization of its mechanical properties. Cross‐sectional areas of units fibres and innervation ratio were directly measured in sections stained for glycogen using a periodic and acid‐Schiff (PAS) reaction. Specific tension of unit fibres was calculated by dividing the maximum tetanic tension of a unit by its total fibre area. 5. The number of unit fibres ranged from 44 to 77 for type S, 116 to 198 for type FR, and 221 to 356 for type FF units, and differences among their means (66, 154 and 271, respectively) were significant. Tetanic tension was correlated with innervation ratio for all of the twenty‐three units, or units within a particular type. 6. Mean fibre area for type S units (1983 microns2) was significantly smaller than that for type FF units (3489 microns2). Fibres belonging to type FR units had an intermediate size (2648 microns2). Correlation between tetanic tension and fibre area was significant for either all units or units within a particular type. 7. Total cross‐sectional area was significantly different among the motor unit types, and was highly correlated to the maximum tetanic tension.(ABSTRACT TRUNCATED AT 400 WORDS)
Flows of liquid and electrical current through monolayers of cultured bovine arterial endothelium.
Authors: Turner M R
Published: December 2014

1. Monolayers of arterial endothelium on porous membranes were exposed to a constant pressure between 15 and 35 cmH2O. The rates of liquid flow per unit area (Jv/A) through the monolayers were monitored, together with the electrical resistance (Rm) of the endothelium. 2. At constant pressure, Jv/A decreased with an approximately exponential time course, towards a stable baseline value. This behaviour resembles the sealing previously described for cultured vascular endothelium. At 30‐35 cmH2O and 37 degrees C, the mean (+/‐ S.E.M.) half‐time (t1/2) of the decrease in Jv/A (the sealing t1/2) was 548 +/‐ 141 S (n = 5). The difference between the initial and baseline values of Jv/A was expressed as a fraction of the initial value. The mean (+/‐ S.E.M.) of this sealing fraction was 0.64 +/‐ 0.03 (n = 5). Mean (+/‐ S.E.M.) hydraulic permeability (Lp) was 23.9 +/‐ 6.4 x 10(‐7) cm S‐1 cmH2O‐1 (n = 9), when measured after sealing. Endothelium appeared damaged after sealing at 30‐35 cmH2O and 37 degrees C. 3. Sealing was also observed using glutaraldehyde‐fixed endothelium at 30‐33 cmH2O and 26‐28 degrees C. There was no significant difference between the mean sealing t1/2 of these fixed monolayers, and that of unfixed endothelium at 30‐35 cmH2O and 37 degrees C. However, mean sealing fraction was significantly larger for the fixed monolayers than for unfixed endothelium at 30‐35 cmH2O and 37 degrees C. There were no significant difference between the post‐sealing Lps of these fixed and unfixed monolayers, although the fixed monolayers appeared undamaged after sealing. 4. For unfixed endothelium, Rm was lower after sealing at 30‐35 cmH2O and 37 degrees C than before pressure application. There was no significant difference between endothelial Rm before and after sealing, for glutaraldehyde‐fixed monolayers. 5. Sealing was also observed at 0 degree C, using unfixed endothelium at 30 cmH2O. Mean sealing t1/2 was not significantly different from that of unfixed endothelium at 30 cmH2O and 37 degrees C. However, mean sealing fraction was significantly smaller at 0 degree C than at 37 degrees C. Unfixed endothelium appeared undamaged after sealing at 30 cmH2O and 0 degree C. Despite this, the post‐sealing Lp was not significantly different from that of unfixed endothelium sealed at 30 cmH2O and 37 degrees C, after allowance was made for the effect of temperature on Lp. Rm was not measured in these experiments. 6. It is proposed that sealing is due to pressure‐induced deformation of monolayers.(ABSTRACT TRUNCATED AT 400 WORDS)
Evidence that a long latency stretch reflex in humans is transcortical.
Authors: Palmer E , Ashby P
Published: December 2014

1. The hypothesis that the long latency reflex response to muscle stretch in humans uses a transcortical pathway was tested by looking for convergence onto cortical neurones in eleven normal subjects. 2. Postsynaptic events in single flexor pollicis longus (FPL) motoneurones were derived from changes in the firing probability of individual FPL motor units. 3. Extension of the terminal phalynx of the thumb resulted in both short latency and long latency facilitations of individual FPL motoneurones. These were not reproduced by electrical stimulation of afferents in the terminal phalynx. Magnetic stimulation over the contralateral motor cortex produced strong, short latency facilitation of FPL motoneurones. 4. When the facilitation produced by stimulation over the cortex was superimposed on the long latency facilitation produced by extension of the thumb, the facilitation produced by both stimuli was greater than the sum of the individual facilitations produced by either stimulus given alone. This was not the case when the superimposition occurred on the short latency response to stretch. 5. We conclude that afferent systems excited by the stretch of FPL converge onto cortical neurones which are known to facilitate motoneurones. Thus the cortex is likely to contribute to the long latency stretch reflex in humans.
Relationship between parasternal and external intercostal muscle length and load compensatory responses in dogs.
Authors: Romaniuk J R , Supinski G , DiMarco A F
Published: December 2014

1. The effects of tracheal occlusion on peak parasternal (PA) and external intercostal (EI) (3rd interspace) EMG activities were examined at different end‐expiratory lung volumes both above and below functional reserve capacity (FCR) in anaesthetized, vagotomized and spontaneously breathing dogs. 2. Parasternal (PA) and external intercostal (EI) muscle lengths were monitored in situ. The difference in peak EMG activity between free and occluded breaths (test breaths) was related to the coincident peak change in intercostal muscle length (delta L) for each muscle, respectively. 3. At FRC, tracheal occlusion resulted in compensatory augmentation of peak EI, but little change in peak PA EMG activities. At lung volumes below FRC, airway occlusion resulted in augmentation of both PA and EI activities. Responses to airway occlusion at lung volumes above FRC were variable. The magnitude and duration of these changes in EMG, however, could be linearly related to the value of delta L. With delta L = 0, there was no change in peak EI or PA EMG; for values of delta L less than 0, there was attenuation of EI and PA EMG; for delta L greater than 0, there was enhancement of EI and PA EMG activation. 4. The magnitude of the changes in EMG activity in response to tracheal occlusion was more prominent for the EI muscle compared to the PA, the latter of which are known to have much fewer muscle spindles than EI muscle. 5. Our results suggest that a difference in end‐inspiratory muscle length between the control and occluded breaths is a stimulus for the intercostal response to applied loads implicating muscle spindles as the predominant receptor moderating these responses. We hypothesize that when delta L = 0, no change in EMG occurs since the spindles sense no change in muscle length. When delta L less than 0 (i.e. peak muscle length during the occluded breath is shorter than control) muscle spindles would be disengaged, resulting in a disfacilitation of EMG activity. Where delta L greater than 0 (i.e. peak muscle length during the occluded breath is longer than control), muscle spindles are stimulated, resulting in enhancement of EMG activity. 6. Additional doses of Nembutal (20 mg), which produced significant changes in breathing pattern, did not affect the magnitude of the load compensatory responses.
Pharmacological and kinetic properties of alpha 4 beta 2 neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.
Authors: Charnet P , Labarca C , Cohen B N , Davidson N , Lester H A , Pilar G
Published: December 2014

1. Co‐injection of RNA synthesized from cloned neuronal acetylcholine receptor (nAChR) subunits (alpha 4 and beta 2) in Xenopus oocytes produced functional receptors. In macroscopic voltage‐clamp experiments, the agonist‐induced current exhibited a strong inward rectification. 2. Voltage jumps from +50 mV to more negative potentials produced relaxations of the agonist‐induced current with a single exponential time course. The relaxation rate constant was only weakly voltage dependent. 3. At the single‐channel level, three conductances were recorded of 12, 22 and 34 pS. Their burst durations were similar and varied only weakly with voltage (e‐fold for 120 to 370 mV), consistent with the poorly voltage‐dependent relaxation rate constants. However, the burst durations were less than 10 ms, or less than 1/5 the value expected from voltage‐jump relaxations. 4. Hexamethonium (Hex, 0.5 to 8 microM) inhibited the agonist‐induced current and produced voltage‐jump relaxations characterized by a rapid conductance increase and a slower conductance decrease. Analysis of these relaxations suggested that the Hex‐receptor interaction is open‐channel blockade characterized by a forward binding rate of 1 x 10(7) M‐1 s‐1 and a dissociation rate constant of about 25 s‐1. 5. For the relaxations produced by QX222, the slowest phase was a conductance increase, suggesting that the dissociation rate constant for QX222 is 10‐30‐fold greater than for Hex. 6. Hex but not QX222 produced an additional use‐dependent blockade that was manifest during repetitive hyperpolarizing pulses. 7. With mouse muscle ACh receptors expressed in oocytes, the blockade by Hex did not depend strongly on voltage. Neither Hex nor QX222 produced appreciable use‐dependent block on muscle ACh receptors. 8. Of the four conditions studied (neuronal and muscle receptors, Hex and QX222), only the blockade of the neuronal AChR by Hex is characterized by a residence time longer than the normal open time. 9. It is concluded that the modest differences in primary amino acid sequence between muscle and neuronal receptors lead to profound changes in their interactions with channels blockers.
Regulation by second messengers of the slowly activating, voltage‐dependent potassium current expressed in Xenopus oocytes.
Authors: Busch A E , Kavanaugh M P , Varnum M D , Adelman J P , North R A
Published: December 2014

1. Voltage‐clamp recordings of membrane current were made from Xenopus oocytes that had been injected with RNA which had been transcribed in vitro from a cloned complementary DNA. 2. Depolarization from ‐80 mV evoked outward potassium currents that developed very slowly. At ‐20 mV the time constant for activation was about 50 s, and at +40 mV about 6 s. 3. The potassium current was increased by the calcium ionophore A23187 or by intracellular injection of inositol 1,4,5‐trisphosphate (IP3), each of which should increase the intracellular calcium concentration ([Ca2+]i). The current was decreased by injection of BAPTA (1,2‐bis(2‐aminophenoxy)ethane‐N,N,N',N'‐tetraacetic acid). The current was also reduced by phorbol esters; this effect was blocked by staurosporine. 4. In oocytes that had also been injected with RNA encoding the 5‐hydroxytryptamine (5‐HT2) receptor, 5‐HT increased the potassium current. After caffeine pretreatment, to block the release of intracellular calcium, 5‐HT decreased the current; this decrease was prevented by staurosporine. 5. It is concluded that the slowly activating, voltage‐dependent potassium current expressed in Xenopus oocytes is increased by increases in [Ca2+]i and is decreased by activation of protein kinase C. Stimulation of 5‐HT2 receptors can have both these effects, but the former normally predominates.
Role of cholecystokinin in the inhibition of gastric acid secretion in dogs.
Authors: Konturek S J , Bilski J , Tasler J , Cieszkowski M
Published: December 2014

1. This study was designed to determine the involvement of cholecystokinin (CCK) in the gastric secretory responses to exogenous and endogenous secretagogues in conscious dogs with chronic gastric fistulae (GF), pancreatic fistulae (PF) and Heidenhain pouches (HP). 2. A meal of meat or intragastric application of peptone (300 mosM) increased secretion of HCl from the HP and pancreatic secretion of protein and plasma levels of gastrin, CCK and somatostatin. 3. The CCK receptor antagonist L‐364,718 caused a further increase in the postprandial HCl secretion from the HP and in the plasma levels of gastrin and CCK but pancreatic output of protein and plasma concentration of somatostatin were significantly reduced. 4. Addition to intragastric peptone of 10% oleate or its acidification to pH 3.0 profoundly inhibited the HP secretion and gastrin release but significantly increased pancreatic secretion of protein and plasma levels of CCK and somatostatin. Administration of L‐364,718 reversed the fall in the HP secretion and plasma gastrin while significantly attenuating pancreatic protein secretion and plasma somatostatin levels. 5. Intragastric administration of hyperosmolar (1200 mosM) peptone also inhibited HCl secretion from the HP but this was not affected by L‐364,718. 6. Exogenous CCK and bombesin (but not gastrin) caused a small increase in HCl secretion from the HP and marked stimulation of pancreatic protein secretion accompanied by a significant rise in plasma levels of gastrin, CCK and somatostatin. Administration of L‐364,718 resulted in a further increase in the HCl response of HP to bombesin and in plasma levels of gastrin and CCK but caused a reduction in plasma levels of somatostatin. 7. We conclude that CCK released by a meal of meat, intragastric peptone, oleate or acidified peptone and intravenous bombesin exerts tonic inhibitory influences on gastric acid secretion and that this effect is mediated, at least in part, by somatostatin.
Induction of haemodynamic oscillations in the perfused rat liver by K+ channel blockers.
Authors: Hill C E , Ajikobi D O
Published: December 2014

1. Exposure of the isolated perfused (constant flow) rat liver to the K+ channel blockers 4‐aminopyridine (4‐AP) or Cs+ causes the appearance of oscillations in portal pressure and oxygen uptake. The oscillations have a mean frequency of 0.035 Hz (2.1 cycles/min) and are fully reversible upon perfusion with blocker‐free saline. Tetraethylammonium (0.17‐24.7 mM) does not induce oscillatory behaviour. 2. Reversible block of the 4‐AP‐induced oscillations is caused by 2 mM‐EGTA, or verapamil, chlorpheniramine, phentolamine or propranolol with IC50 values of 0.42, 13.5, 15 or 11.5 microM respectively. The oscillations are transiently blocked by atropine (IC50 = 8.3 microM at peak inhibition) and are not affected by 2.7 microM‐tetrodotoxin. 3. Endothelium‐dependent vasorelaxants, Kupffer cell activity modifiers, retrograde perfusion, or removal of the portal vein from the circuit do not modify the oscillation parameters. 4. Oscillations are also caused by infusion of physiological concentrations of adrenaline or phenylephrine, but not isoprenaline. 5. The results provide new evidence for the existence of intrahepatic voltage‐sensitive Ca2+, and 4‐AP‐ and Cs(+)‐sensitive K+ channels. We propose that the K+ channel blockers reveal an intrinsic oscillator in the liver, and that phasic vasoactivity may involve a minor contribution from neurotransmitter and/or hormonal substances.
Acetylcholine activates non‐selective cation and chloride conductances in canine and guinea‐pig tracheal myocytes.
Authors: Janssen L J , Sims S M
Published: December 2014

1. Membrane currents activated by acetylcholine (ACh) were investigated in isolated canine and guinea‐pig tracheal myocytes using the nystatin perforated patch configuration of whole‐cell recording. ACh caused depolarization accompanied by a membrane conductance increase. 2. When cells were held under voltage clamp (holding potential, Vh = ‐60 mV), ACh elicited inward current (IACh) of up to 3900 pA, with a reversal potential (Erev) of approximately ‐20 mV. 3. Removal of extracellular Na+ (Na+o) reduced but did not eliminate IACh. IACh remaining in the absence of Na+ reversed direction close to the predicted equilibrium potential for Cl‐. Erev shifted 32 +/‐ 4 mV per 10‐fold change of [Cl‐]i. Increasing external [K+] caused Erev to shift in the positive direction. These results suggest that ACh activated chloride and non‐selective cation conductances. 4. In the absence of Na+o, the Cl‐ channel blockers SITS or niflumic acid reversibly antagonized IACh. 5. Caffeine and ryanodine elicited currents both in the presence and absence of Na+o; these currents had a reversal potential similar to that of IACh. Caffeine applied before ACh occluded the response to ACh. 6. We also observed two types of spontaneous membrane currents. Spontaneous transient outward currents (STOCs) may represent Ca(2+)‐activated K+ currents. Spontaneous inward currents were also observed which were reduced in magnitude (but not eliminated) by removal of Na+o and reversed direction at approximately the Cl‐ equilibrium potential. The spontaneous inward currents and STOCs were coincident and were reversibly suppressed by ACh. 7. ACh elicited contractions of cells under voltage clamp at ‐60 mV, an effect also observed in the absence of extracellular Ca2+ or when IACh was reduced by omission of Na+o and exposure to Cl‐ channel blockers. The number of cells which did contract in response to ACh decreased, however, when the concentration of internal Cl‐ decreased. 8. All effects of ACh on contraction and membrane currents were antagonized by atropine. 9. We conclude that activation of muscarinic receptors in mammalian tracheal myocytes causes release of Ca2+ from intracellular stores and subsequent activation of Cl‐ and non‐selective cation conductances. This is the first direct demonstration of these conductances in tracheal smooth muscle cells. Activation of these conductances does not appear to be required for contraction. However, regulation of cytosolic Cl‐ levels may be important for release and uptake of Ca2+ from internal stores.
Vagal control of mucus glycoconjugate secretion into the feline trachea.
Authors: Fung D C , Beacock D J , Richardson P S
Published: December 2014

1. We examined the effects of frequencies and patterns of electrical stimulation of the peripheral cut ends of the vagus nerves on the release of mucus glycoconjugates into feline trachea in vivo. Mucus glycoconjugates, radiolabelled biosynthetically with [35S]sulphate and [3H]glucose, were washed from a tracheal segment in situ, and dialysed before being counted and assayed chemically by the periodic acid‐Schiff (PAS) method. 2. Vagal stimulation with regular pulses (10 V, 2 ms duration) at 1, 2.25, 4.5, 9 and 18 Hz produced frequency‐dependent increases in the output of mucus glycoconjugates. 3. The muscarinic agonist pilocarpine (0.1‐10 microM), given intrasegmentally, produced dose‐dependent increases in the output of mucus glycoconjugates. 4. Pretreatment with atropine, phentolamine and propranolol reduced but did not abolish the effects of vagal stimulation. Vagus nerve stimulation still caused frequency‐dependent increases in the output of mucus glycoconjugates. 5. High frequency stimulations at 22.5 and 47.5 Hz given intermittently (1 s burst then 4 s rest), whether in the absence or presence of cholinergic and adrenergic blockade, produced similar secretory responses as the same number of pulses delivered in regular trains at 4.5 and 9.5 Hz. This suggests that neither cholinergic nor non‐adrenergic, non‐cholinergic (NANC) nerve mechanisms in this system are potentiated by high frequency, intermittent burst stimulation. 6. In the absence of atropine, regular vagal stimulation had a greater effect on heart rate than did the same number of pulses delivered in bursts. 7. High molecular weight glycoconjugates from secretions were taken from the void volume of a Sepharose CL‐2B gel filtration column and separated further by density‐gradient centrifugation. Macromolecular components were observed at two densities, a typical mucin at 1.52 g ml‐1, and a high density atypical component at 1.63 g ml‐1. In secretions collected during vagal stimulation, either in the absence or presence of cholinergic and adrenergic blockade, the ratio of low density to high density macromolecules was higher than in unstimulated secretions. This can be explained if both cholinergic and NANC nervous vagal mechanisms stimulate the output of typical (density = 1.52 g ml‐1) mucins into the feline trachea.
Sulphydryl reagents trigger Ca2+ release from the sarcoplasmic reticulum of skinned rabbit psoas fibres.
Authors: Salama G , Abramson J J , Pike G K
Published: December 2014

1. By analogy with studies on sarcoplasmic reticulum (SR) vesicles, Ca2+ release induced by heavy metals and mercaptans (e.g. cysteine) was investigated in rabbit skinned psoas fibres through measurements of isometric tension. 2. Heavy metals (at 2‐5 microM) elicited phasic contractions by triggering Ca2+ release from the SR and had the following order of potency: Hg2+ > Cu2+ > Cd2+ > Ag+ > Ni2+. Higher concentrations produced tonic contractions due to maintained high Ca2+ permeability of SR membranes. 3. Contractions induced by heavy metals required a functional and Ca(2+)‐loaded SR, were dependent on [Ca2+]free, blocked by Ruthenium Red (RR), inhibited by free Mg2+ and reduced glutathione (GSH) but not by oxidized glutathione (GSSG). Such contractions were not elicited through direct interaction(s) of heavy metals with the myofilaments. 4. In the presence of catalytic concentrations of Hg2+ or Cu2+ (2‐5 microM), additions of cysteine (25‐100 microM) elicited rapid twitches, producing 70% of maximal force with a time to half‐peak of 2 s. Contractions induced by cysteine plus a catalyst required a functional SR network, were dependent on free [Mg2+] and were blocked by RR or GSH but not by GSSG. 5. In the presence of Hg2+ (2‐5 microM), low concentrations of cysteine (10 microM) elicited tonic contractures, but subsequent or higher additions of cysteine (50‐100 microM) caused further SR Ca2+ release and tension, followed by rapid and full relaxation. 6. High cysteine (200‐250 microM, without Cu2+ or Hg2+) blocked contractions elicited by Cl‐ induced depolarization of sealed T‐tubules. High cysteine probably acted as a sulphydryl reducing agent which promoted rapid relaxation of the fibres through the closure of Ca(2+)‐release channels and ATP‐dependent re‐uptake of Ca2+ by the SR. 7. In some batches of skinned fibres (approximately 10%), cysteine (5‐50 microM) alone (without Hg2+ or Cu2+ catalyst) produced rapid twitches. This implied that the catalyst(s) necessary to promote the sulphydryl oxidation reaction with exogenously added cysteine may be present in intact fibres but is usually lost by the skinning procedure. 8. The data demonstrate that skeletal fibres contain a highly reactive and accessible sulphydryl site on an SR protein which can be reversibly oxidized and reduced to respectively, open and close SR Ca(2+)‐release channels. A model of sulphydryl‐gated excitation‐contraction coupling is proposed where the voltage sensor on the T‐tubule membrane directly oxidizes sulphydryl sites on SR Ca(2+)‐release channels.
A potential‐dependent fast outward current in single smooth muscle cells isolated from the newborn rat ileum.
Authors: Smirnov S V , Zholos A V , Shuba M F
Published: December 2014

1. Whole‐cell outward currents have been studied in single smooth muscle cells isolated from newborn and adult rat ileum, using fire‐polished glass micropipettes. 2. Two major outward currents, delayed (I(do)) and fast inactivating potential‐dependent (I(fo)), have been observed in the newborn rat ileal cells. I(fo) is activated between ‐50 and ‐40 mV from the holding potential of ‐80 mV, whereas I(do) usually starts to activate at membrane potentials positive to ‐20 mV. Activation of I(do) was fast, its time‐to‐peak decreased from 10.8 +/‐ 0.9 ms (n = 5) at ‐30 mV to 4.5 +/‐ 0.7 ms (n = 4) at 20 mV. 3. I(fo) decay was monoexponential and its time constant did not depend on the membrane potential. Dependence of I(fo) inactivation on membrane voltage in normal physiological salt solutions (PSS) could be described by the Boltzmann equation with the following parameters: a half‐inactivation potential, V0.5 = ‐70.8 mV and slope factor, k = 7.7 mV. 4. Recovery of I(fo) from inactivation was fitted by a single exponential and was potential dependent. The average time constant was 28.4 +/‐ 2.4 ms (n = 11) at ‐120 mV, 47.7 +/‐ 3.0 ms (n = 6) at ‐100 mV and 89.6 +/‐ 5.3 ms (n = 13) at ‐80 mV. 5. Removal of Ca2+ ions from the PSS (in the presence of 5 mM‐Mg2+) increased I(fo) amplitude by about two times, and shifted its voltage dependence of inactivation towards negative membrane potentials by about 16 mV (V0.5 = ‐87.2 mV). Removal of Mg2+ from the PSS (in the presence of 2.5 mM‐Ca2+) exerted no effects upon either inactivation dependence (V0.5 = ‐74.2 mV) or I(fo) amplitude. 6. I(do) and I(fo) had different sensitivities to K+ channel blockers. With 10 mM‐external TEA+ I(do), was preferentially suppressed, while 5 mM‐4‐aminopyridine (4‐AP) completely blocked I(fo). I(fo) was also partially blocked by a higher TEA+ concentration (30 mM), which suppressed I(fo) to 0.55 +/‐ 0.02 (n = 9). The blocking effect of 4‐AP on I(fo) was potential, use and time dependent. 7. Ileal cells isolated from the adult rat demonstrated the presence of two populations of smooth muscle cells. One has an outward current which seems to be similar to that described in the newborn rat. However, in other cells spontaneous transient outward currents, well described in other single smooth muscle cells, but not found in newborn rat ileal cells, have been observed.
Phosphorylation restores activity of L‐type calcium channels after rundown in inside‐out patches from rabbit cardiac cells.
Authors: Ono K , Fozzard H A
Published: December 2014

1. Rundown of L‐type calcium channels was studied in inside‐out patches made from single isolated rabbit ventricular myocytes, using barium as the charge carrier. 2. In the cell‐attached patches single‐channel activity was stable for more than 15 min after the patch pipette sealed. beta‐Receptor stimulation by isoprenaline caused a characteristic increase in opening probability and the appearance of prolonged openings. When the patch was excised to the inside‐out configuration and exposed to a simple ionic solution, channel activity disappeared within 1‐2 min and never reappeared spontaneously. 3. After rundown of L‐type channel activity in the excised patch, exposure of the inside face of the patch to MgATP and the catalytic subunit of the cyclic AMP‐dependent protein kinase (PKAc) resulted in recovery of Ca2+ channel activity. Under these conditions channel activity could be even greater than under control cell‐attached conditions, resembling channel activity after exposure to isoprenaline. This recovery of activity persisted many minutes, usually until the patch was lost. Addition of MgATP alone caused a small transient increase in channel activity in some patches. 4. Recovery of activity by MgATP and PKAc could be prevented by prior exposure of the excised patch to protein kinase inhibitor (PKI), or it could be abruptly terminated by exposure to PKI after recovery of activity. Addition to the pipette solution of okadaic acid, a protein phosphatase inhibitor, greatly slowed rundown. These findings support the proposal that dephosphorylation is an important component of rundown, and that phosphorylation is needed for channel opening activity. 5. Single‐channel conductance was not altered by patch excision, but it was reduced after exposure of the excised patch to MgATP and PKAc. Mg2+ was responsible for this effect, probably by direct channel block from the inside, and Mg2+ also caused a negative shift in the channel activation, as expected from shielding of inside fixed negative charges.
A change from HCO3(‐)‐CO2‐ to hepes‐buffered medium modifies membrane properties of rat CA1 pyramidal neurones in vitro.
Authors: Church J
Published: December 2014

1. Intracellular recordings were obtained from CA1 pyramidal neurones in rat hippocampal slices. Perfusion with a HCO3(‐)‐CO2‐free, HEPES‐buffered medium at pH 7.4 produced a wide variety of reversible effects on neuronal excitability, compared to responses obtained under standard (21 mM‐HCO3‐, 5% CO2, pH 7.4) conditions. 2. Introduction of HCO3(‐)‐CO2‐free medium most commonly elicited, within 5‐20 min, a fall in resting membrane potential (Vm), a rise in threshold for Na(+)‐dependent action potential generation, and a reduction in input resistance. Anomalous inward rectification in the hyperpolarizing direction and subthreshold inward rectification were commonly reduced in HEPES‐buffered medium. More prolonged exposure (> or = 25 min) to HCO3(‐)‐CO2‐free medium produced, on occasion, Na+ spike inactivation. 3. The amplitudes of the fast and medium after‐hyperpolarizations (AHPs) following a single depolarizing current‐evoked action potential were attenuated during perfusion with HEPES‐buffered medium at pH 7.4, as was the composite AHP following a train of action potentials. 4. Perfusion with HEPES‐buffered medium at pH 7.4 reduced the degree of spike frequency adaptation and abolished depolarizing current‐evoked burst‐firing behaviour when this was present under standard conditions. 5. In tetrodotoxin (TTX)‐ and tetraethylammonium (TEA)‐poisoned neurones, perfusion with HCO3(‐)‐CO2‐free medium at pH 7.4 slightly raised the threshold for activation of Ca(2+)‐dependent potentials and slightly reduced their duration, compared to responses obtained in HCO3(‐)‐CO2‐buffered medium at the same pH. The AHP following the Ca2+ spike was, however, markedly attenuated. 6. Perfusion with a low‐pH HCO3(‐)‐CO2‐buffered medium (7 mM‐HCO3‐, 5% CO2, pH 6.9) produced changes qualitatively similar to those observed during perfusion with HEPES‐buffered medium at pH 7.4. Raising the pH of the HEPES‐buffered medium to 7.8 or 7.9 reversed inconsistently and then only in part the changes noted on the transition from a HCO3(‐)‐CO2‐ to a HEPES‐buffered medium at the same pH (7.4). 7. The effects noted are unlikely to be due to a direct action of HEPES itself on neuronal membrane conductances. Rather, I suggest that they are likely to be caused by intracellular acidosis consequent upon the omission of HCO3‐ and CO2 from the extracellular medium.
Neurosteroid regulation of GABAA receptor single‐channel kinetic properties of mouse spinal cord neurons in culture.
Authors: Twyman R E , Macdonald R L
Published: December 2014

1. Single‐channel kinetics of steroid enhancement of single gamma‐aminobutyric acidA (GABA) receptor currents obtained from somata of mouse spinal cord neurones in culture were investigated using the excised outside‐out patch‐clamp recording technique. GABA (2 microM) and GABA (2 microM) plus androsterone (5 alpha‐androstan‐3 alpha‐ol‐17‐one, AND, 10 nM‐10 microM) or pregnanolone (5 beta‐pregnan‐3 alpha‐ol‐20‐one, PRE, 100 nM‐10 microM) applied by pressure ejection from micropipettes evoked inward currents when patches were voltage clamped at ‐75 mV in symmetrical chloride solutions. Averaged GABA receptor currents were increased in the presence of the steroids. 2. GABA receptor currents were recorded with at least two conductance levels, a predominant or main‐conductance level of about 28 pS (which contributed 96% of the current evoked) and a minor or sub‐conductance level of about 20 pS. The current amplitudes of the two conductance levels were unchanged by the steroids. The gating (opening and closing) kinetics of both of the conductance levels were analysed. Findings for the main‐conductance level are summarized below. 3. Both steroids increased the average GABA receptor channel open duration. Consistent with the increased GABA receptor channel average open duration, the steroids shifted frequency histograms of GABA receptor channel open durations to longer durations. Three exponential functions were required to fit best the frequency histograms of GABA open durations, consistent with at least three kinetic open states of the main‐conductance level. Time constants obtained from the GABA receptor channel open‐duration frequency histograms were unchanged in the presence of the steroids. The basis for the increased average GABA receptor channel open durations by the steroids was due to an increased relative proportion of the two longer open‐duration time constants. The GABA receptor channel average open durations were increased by AND and PRE in a concentration‐dependent manner by shifting the proportion of openings to the longer open time constants. At a concentration of 10 microM, the prolongation of the average open duration was decreased, suggesting that the GABA receptor channel was blocked by these steroids. 4. GABA receptor channel opening frequency was increased and average channel‐closed duration was decreased by AND or PRE. Consistent with this, areas of the frequency histograms of channel closed durations were shifted to shorter durations. Closed frequency distributions were fitted best with five to six exponential functions, suggesting that the channel had multiple kinetic closed states. The three briefest time constants were not greatly altered by the steroids.(ABSTRACT TRUNCATED AT 400 WORDS)