Research Article
A novel background potassium channel in rat atrial cells
- Z. Shui, M. R. Boyett
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- 25 January 2001, pp. 355-361
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A K+ channel activated by intracellular ATP has been observed in inside-out patches from rat atrial cells. The channel has a slope conductance of 130 ± 5 pS in symmetrical 140 mM K+ solution, and is almost independent of voltage over the range from -80 to +80 mV. There is no detectable inactivation during application of ATP over a few minutes. In the presence of 3 mM intracellular ATP, channel openings occur as bursts with a mean open time of 1.7 ms, a mean closed time of 0.4 ms, a mean burst duration of 18 ms and a mean burst interval of 41 ms. Kinetic analysis suggests that ATP mainly affects the burst duration and the burst interval of the channel. Based on the properties above, the channel differs from other known K+ channels in cardiac cells and may contribute to background K+ current.
Epidermal growth factor regulates amino acid transport in chick embryo hepatocytes via protein kinase C
- Maria Marino*, Raffaella Mele, Silvana Spagnuolo, Fabio Maria Pulcinelli, Maria Teresa Mangiantini, Silvia Leoni
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- 25 January 2001, pp. 363-369
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System A-mediated amino acid transport, activation of different steps of signal transduction and involvement of different isoforms of protein kinase C (PKC) have been investigated in chick embryo hepatocytes after epidermal growth factor (EGF) stimulation. EGF rapidly (10 min) increased the rate of aminoisobutyric acid (AIB) uptake in chick embryo hepatocytes freshly isolated on the 19th day of embryonic life, while no change was detectable at other embryonal stages. The growth factor stimulation was abolished by PKC and tyrosine kinase inhibitors and was mimicked by 4-phorbol-12-myristate-13-acetate, dimethyl-2 (PMA). EGF treatment did not modify the phosphorylation of the γ isoform of phospholipase C (PLC-γ), and inositol trisphosphate (IP3) and intracellular calcium levels, but it induced an increase in PKC activity. Our data show that EGF regulates amino acid uptake, via PKC and without PLC-γ activation, only in the last period of chick embryo hepatocyte development. The effects of growth factor on PKC activity suggest the involvement of PKC-α and -ε isoforms in EGF modulation of amino acid transport.
Effects of resalination on intestinal glucose transport in chickens adapted to low Na+ intakes
- Carles Garriga, Miquel Moretó, Joana M. Planas
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- 25 January 2001, pp. 371-378
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In chickens, we have shown that intestinal absorption of glucose via apical SGLT1 and basolateral GLUT2 transport systems is affected by dietary Na+; low-Na+ adapted birds show a dramatic reduction of glucose transporters in both membranes in the rectum, an intermediate response in the ileum and no effects in the jejunum. We have now studied the effect of resalination of low-Na+ adapted chickens on glucose kinetics across SGLT1 (using α -methyl-D-glucoside as substrate) and GLUT2 (using D-glucose) and on the specific binding of phlorizin and cytochalasin B, respectively. Twelve-week-old male Leghorn chickens were fed wheat and barley with drinking water containing either 150 mM NaCl (high-Na+ group) or 0.015 mM (low-Na+ group) for 14 days (serum aldosterone: 242 ± 6 pg ml-1 in the low-Na+ and 46 ± 4 pg ml-1 in the high-Na+ group). On day 14, the low-Na+ group was either resalinated with an oral dose of NaCl (9 g (kg body wt)-1) or switched to the high-Na+ condition, for 1 week. Serum aldosterone measured 4 h, 1 day and 7 days after the change in NaCl intake fell to between 30 and 39 pg ml-1. The changes in apical α-methyl-D-glucoside and basolateral D-glucose transport observed in the ileum and rectum of low-Na+ adapted animals were completely reversed by resalination within 4 h of NaCl administration to the level of values observed for high-Na+ adapted birds. The good correlation between the α-methyl-D-glucoside and D-glucose Vmax and the SGLT1 and GLUT2 density, respectively, supports the view that the increase in apical and basolateral hexose transport found in the ileum and rectum of both groups of resalinated birds is due to an increase in the number of protein transporters. The rapid changes in the number of glucose transporters observed suggest that the target of the regulatory signal(s) involved are the mature enterocytes present in the villi rather than the developing enterocytes in the crypt.
Influence of the extent of haemoglobin hydrolysis on the digestive absorption of haem iron in the rat. An in vitro study
- N. Vaghefi, F. Nedjaoum, D. Guillochon, F. Bureau, P. Arhan, D. Bouglé
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- 25 January 2001, pp. 379-385
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This study was designed to assess the haem-peptide interactions which occur during progressive haemoglobin hydrolysis by digestive enzymes and their relationship with haem iron digestive absorption. The behaviour of different haemoglobin hydrolysates was studied using the Ussing chamber model. Hydrolysates were produced from enzyme digestion of bovine haemoglobin at pH 3 by pepsin and at pH 10 by subtilisin. Samples with increasing degrees of hydrolysis (0-15 %) were studied. Biochemical assays (pyridine haemochromogen method and UV absorption spectra) were used to follow haem solubility and haem-peptide interactions in samples. Increasing the hydrolysis level of haemoglobin was associated with an enhanced iron uptake; the highest uptake rate was reached between 8 and 11 % of globin hydrolysis, whichever enzyme was used. The mechanisms rendering iron soluble and available differ between the two enzymes. The comparison between biochemical and absorption data suggests that the formation of soluble peptide-haem complexes was not sufficient to enhance haem iron absorption, since globin-bound iron is poorly absorbed; an efficient absorption occurred only when haem was loosely bound to low molecular weight peptides.
Cyclic AMP-mediated inhibition of noradrenaline-induced contraction and Ca2+ influx in guinea-pig vas deferens
- Kenichi Kato, Kishio Furuya, Izuo Tsutsui, Tsuyoshi Ozaki, Shunichi Yamagishi
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- 25 January 2001, pp. 387-398
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The relaxation effects of forskolin and methylxanthines on noradrenaline (NA)-induced contractions were investigated by measuring isotonic contraction and intracellular calcium concentration ([Ca2+]i) in the epididymal side of guinea-pig vas deferens. NA (100 µM) and high K+ (55 mM) induced a biphasic contraction; fast, transient (phasic) and slow, sustained (tonic) phases. Both phases in either NA or high K+ stimulation were abolished in Ca2+-free solution. Pretreatment with 10 µM nifedipine, an L-type Ca2+ channel blocker, reduced both phasic and tonic contractions induced by high K+. In the case of NA-induced contraction, however, nifedipine reduced the phasic contraction but not the tonic contraction. The nifedipine-insensitive tonic contraction was relaxed by the application of polyvalent cations (Mn2+, Co2+, Cd2+ and La3+). These findings indicate that NA-induced biphasic contraction is mainly due to nifedipine-insensitive Ca2+ influx, especially in the tonic phase. Cyclic AMP-increasing agents such as forskolin (0.5-10 µM), IBMX (5-500 µM) and caffeine (1-20 mM) relaxed the NA-induced contraction extensively in a concentration-dependent manner. However, these agents only partially relaxed the high K+-induced contraction. Forskolin (10 µM) and IBMX (100 µM) reduced the [Ca2+]i response to NA, but had no effect on the [Ca2+]i response to high K+. These results suggest that an increase in intracellular cAMP may relax the NA-induced contraction by attenuating a nifedipine-insensitive Ca2+ influx and by a mechanism independent of a reduction in [Ca2+]i.
Nitric oxide, cGMP and cAMP modulate nitrobenzylthioinosine-sensitive adenosine transport in human umbilical artery smooth muscle cells from subjects with gestational diabetes
- Claudio Aguayo, Luis Sobrevia
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- 25 January 2001, pp. 399-409
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Adenosine transport was characterized in human umbilical artery smooth muscle cells isolated from non-diabetic and diabetic pregnant subjects. Transport of adenosine was mediated by a Na+-independent transport system inhibited by nanomolar concentrations of nitrobenzylthioinosine (NBMPR) in both cell types. Diabetes increased adenosine transport, an effect that was associated with a higher maximal velocity (Vmax) for NBMPR-sensitive (es) saturable nucleoside transport (18 ± 2 vs. 61 ± 3 pmol (µg protein)-1 min-1, P < 0.05) and the maximal number of binding sites (Bmax) for specific [3H]NBMPR binding (74 ± 4 vs. 156 ± 10 pmol (µg protein)-1, P < 0.05), with no significant changes in the Michaelis-Menten (Km) and dissociation (Kd) constants, respectively. Adenosine transport was unaltered by inhibition of nitric oxide (NO) synthase (with 100 µM NG-nitro-L-arginine methyl ester, L-NAME) or protein synthesis (with 1 µM cycloheximide), but was increased by inhibition of adenylyl cyclase activity (with 100 µM, SQ-22536) in non-diabetic cells. Diabetes-induced adenosine transport was blocked by L-NAME and associated with an increase in L-[3H]citrulline formation from L-[3H]arginine and intracellular cGMP, but with a decrease in intracellular cAMP compared with non-diabetic cells. Expression of inducible NO synthase (iNOS) was unaltered by diabetes. Dibutyryl cGMP (dbcGMP) increased, but dibutyryl cAMP (dbcAMP) decreased, adenosine transport in non-diabetic cells. dbcGMP or the NO donor S-nitrosoacetylpenicillamine (SNAP, 100 µM) did not alter the diabetes-elevated adenosine transport. However, activation of adenylyl cyclase with forskolin (1 µM), directly or after incubation of cells with dbcAMP, inhibited adenosine transport in both cell types. Our findings provide the first evidence that adenosine transport in human umbilical artery smooth muscle cells is mediated by the NBMPR-sensitive transport system es, and that its activity is upregulated by gestational diabetes.
Reflex responses from the main pulmonary artery and bifurcation in anaesthetised dogs
- N. C. McMahon, M. J. Drinkhill, D. S. Myers, R. Hainsworth
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- 25 January 2001, pp. 411-420
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This study was undertaken to determine the reflex cardiovascular and respiratory responses to discrete stimulation of pulmonary arterial baroreceptors using a preparation in which secondary modulation of responses from other reflexes was prevented. Dogs were anaesthetised with α-chloralose, artificially ventilated, the chests widely opened and a cardiopulmonary bypass established. The main pulmonary arterial trunk, bifurcation and extrapulmonary arteries as far as the first lobar arteries on each side were vascularly isolated and perfused through the left pulmonary artery and drained via the right artery through a Starling resistance which controlled pulmonary arterial pressure. Pressures distending systemic baroreceptors and reflexogenic regions in the heart were controlled. Reflex vascular responses were assessed from changes in perfusion pressures to a vascularly isolated hind limb and to the remainder of the subdiaphragmatic systemic circulation, both of which were perfused at constant flows. Respiratory responses were assessed from recordings of efferent phrenic nerve activity. Increases in pulmonary arterial pressure consistently evoked increases in both perfusion pressures and in phrenic nerve activity. Both vascular and respiratory responses were obtained when pulmonary arterial pressure was increased to above about 30 mmHg. Responses increased at higher levels of pulmonary arterial pressures. In 13 dogs increases in pulmonary arterial pressure to 45 mmHg increased systemic perfusion pressure by 24 ± 7 mmHg (mean ± S.E.M.) from 162 ± 11 mmHg. Setting carotid sinus pressure at different levels did not influence the vascular response to changes in pulmonary arterial pressure. The presence of a negative intrathoracic pressure of -20 mmHg resulted in larger vascular responses being obtained at lower levels of pulmonary arterial pressure. This indicates that the reflex may be more effective in the intact closed-chest animal. These results demonstrate that stimulation of pulmonary arterial baroreceptors evokes a pressor reflex and augments respiratory drive. This reflex is likely to be elicited in circumstances where pulmonary arterial pressure increases and the negative excursions of intrathoracic pressure become greater. They are likely, therefore, to be involved in the cardio-respiratory response to exercise as well as in pathological states such as pulmonary hypertension or restrictive or obstructive lung disease.
Absence of reflex vascular responses from the intrapulmonary circulation in anaesthetised dogs
- N. C. McMahon, M. J. Drinkhill, D. S. Myers, R. Hainsworth
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- 25 January 2001, pp. 421-430
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The aim of this investigation was to determine whether reflex cardiovascular responses were obtained to localised distension of the intrapulmonary arterial and venous circulations in a preparation in which the stimuli to other major reflexogenic areas were controlled and the lung was shown to possess reflex activity. Dogs were anaesthetised with α-chloralose, artificially ventilated, the chests widely opened and a cardiopulmonary bypass established. The intrapulmonary region of the left lung was isolated and perfused through the left pulmonary artery and drained through cannulae in the left pulmonary veins via a Starling resistance. Intrapulmonary arterial and venous pressures were controlled by the rate of inflow of blood and the pressure applied to the Starling resistance. Pressures to the carotid, aortic and coronary baroreceptors and heart chambers were controlled. Responses of vascular resistance were assessed from changes in perfusion pressures to a vascularly isolated hind limb and to the remainder of the subdiaphragmatic circulation (flows constant). The reactivity of the preparation was demonstrated by observing decreases in vascular resistance to large step changes in carotid sinus pressure (systemic vascular resistance decreased by -40 ± 5 %), chemical stimulation of lung receptors by injection into the pulmonary circulation of veratridine or capsaicin (resistance decreased by -32 ± 4 %) and, in the four dogs tested, increasing pulmonary stroke volume to 450 ml (resistance decreased by -24 ± 6 %). However, despite this evidence that the lung was innervated, increases in intrapulmonary arterial pressure from 14 ± 1 to 43 ± 3 mmHg or in intrapulmonary venous pressure from 5 ± 2 to 34 ± 2 mmHg or both did not result in any consistent changes in systemic or limb vascular resistances. In two animals tested, however, there were marked decreases in efferent phrenic nerve activity. These results indicate that increases in pressure confined to the intrapulmonary arterial and venous circulations do not cause consistent reflex vascular responses, even though the preparation was shown to be reflexly active and the lung was shown to be innervated.
Doppler sonography to monitor flow in different cerebral arteries in the rabbit
- Silvestro Roatta*, Silvia Roncari, Giuseppe Micieli, Daniele Bosone, Magda Passatore
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- 25 January 2001, pp. 431-438
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Most of the transcranial Doppler (TCD) experimental studies on cerebral haemodynamics have been performed in the rabbit because of the similarity between its Willis circle and that of the human, but these studies have mainly been limited to the basilar artery. The present study was aimed at extending the use of TCD sonography to all other large cerebral arteries. In anaesthetised rabbits, these arteries were insonated from three different recording sites, i.e. top-cranial, suboccipital and orbital, using a two-channel pulsed Doppler device equipped with 4 and 8 MHz probes. First, discrimination between intra- and extracranial arteries was achieved through a standard 'rebreathing' test (hypercapnic-hypoxic stimulation). The distinctive blood velocity response patterns, reflecting the different extents of metabolic reactivity in intra- and extracranial territories, are described and discussed. Intracranial arteries were then identified on the basis of their response to ipsi- and contralateral common carotid artery occlusion. This procedure allowed recording from the following arteries: anterior common trunk, anterior cerebral, internal carotid, middle cerebral and basilar; the latter could be simultaneously monitored with any of the others. This study provides an experimental model allowing investigation of regional differences in the haemodynamic response to neurogenic and pharmacological stimuli.
Effect of feeding level and thyroxine on adipose tissue development and growth in postnatal lambs
- J. J. Gate, L. Clarke, J. A. Bird, M. A. Lomax, M. E. Symonds
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- 25 January 2001, pp. 439-444
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We investigated the influence of exogenous thyroxine (T4) administration in conjunction with level of feeding on adipose tissue and liver growth in postnatal lambs. Pairs of lambs were fed either 100 g (i.e. low fed) or 200 g (i.e. high fed) of milk powder per litre of reconstituted milk replacer over the first month of life. Half of the pairs of lambs were fed a bolus dose of T4 (15 mg (kg body weight)-1) daily until 8 days of age. Perirenal adipose tissue and hepatic tissue were sampled at either 8 or 35 days of age. High fed lambs grew faster, possessed more adipose tissue and had larger livers than low fed lambs at 8 and 35 days of age. T4 administration resulted in a lower thermogenic activity (i.e. GDP binding) in adipose tissue at 8 days of age in low, but not high fed lambs. There was no difference between groups in colonic temperature or oxygen consumption. Between 17 and 35 days of age high fed lambs previously treated with T4 had lower daily milk consumption than untreated siblings, but still attained the same growth rate. Plasma insulin-like growth factor-I concentrations were greater in high than low fed lambs, a relationship that was not influenced by T4 treatment. In adipose tissue, iodothyronine 5′ deiodinase activity was not influenced by T4 administration and was greater in high than low fed lambs. Hepatic iodothyronine 5′ deiodinase activity was not influenced by T4 administration in low fed lambs, but was reduced by T4 in high fed lambs. In conclusion, T4 administration over the first 8 days of life can accelerate the rate of decline in thermogenic activity of uncoupling protein-1. This effect is not observed when the level of feeding is increased. Following withdrawal of T4 treatment, high fed T4-treated lambs were able to maintain the same growth rate as untreated lambs despite having a lower food intake.
Stretch reflexes in the rectus abdominis muscle in man
- S. E. Myriknas, I. D. Beith, P. J. Harrison
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- 25 January 2001, pp. 445-450
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The spinal reflex circuitry of the rectus abdominis (RA) muscle in man was investigated by the application of a mechanical tap to the muscle. Electromyographic recordings were made in ten healthy subjects, performing a series of manoeuvres, using pairs of surface electrodes placed bilaterally. The reflex responses elicited largely depended on the amount of tonic (postural) activity of the trunk. When standing in the upright position, no reflex activity was recorded in response to the tap. Reflex activity due to mechanical tap was readily recorded when the muscle became tonically active. Moderate, backward trunk extension introduced short-latency reflexes at 18.8 ± 1.9 ms (mean ± S.D.) ipsilaterally and 20.8 ± 1.8 ms contralaterally. Excitatory reflex activity of longer latency was also recorded contralaterally in all subjects (latency 45.1 ± 4.3 ms) and ipsilaterally in five of the ten subjects (latency 47.2 ± 2.6 ms). Vibration of the tapped muscle produced a reduction in the amplitude of the early reflex responses, whilst increasing the amplitude of the late responses. Moreover, the early reflexes were facilitated by the Jendrassik manoeuvre. Such observations are consistent with the early responses being mediated, at least partly, monosynaptically, and the late responses being of polysynaptic nature. This implies that muscle spindle afferents from rectus abdominis monosynaptically activate motoneurones contralaterally.
Contractile properties, fatigue and recovery are not influenced by short-term creatine supplementation in human muscle
- J. M. Jakobi, C. L. Rice, S. V. Curtin, G. D. Marsh
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- 25 January 2001, pp. 451-460
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There have been several studies on the effect of short-term creatine (Cr) supplementation on exercise performance, but none have investigated both voluntary and stimulated muscle contractions in the same experiment. Fourteen moderately active young men (19-28 years) were randomly assigned, in a double blind manner, to either a creatine (Cr) or placebo (P) group. The subjects supplemented their regular diet 4 times a day for 5 days with either 5 g Cr + 5 g maltodextrin (Cr group), or 5 g maltodextrin (P group). Isometric maximal voluntary contraction (MVC), muscle activation, as assessed using the modified twitch interpolation technique, electrically stimulated contractile properties, electromyography (EMG), endurance time and recovery from fatigue were measured in the elbow flexors. The fatigue protocol involved both voluntary and stimulated contractions. Following supplementation there was a significant weight gain in the Cr group (1.0 kg), whereas the P group did not change. For each group, pre-supplementation measures were not significantly different from post-supplementation for MVC, twitch and tetanic tensions at rest, time to peak tension, half-relaxation time and contraction duration. Prior to Cr supplementation time to fatigue was 10 ± 4 min (mean ± S.E.M.) for both groups, and following supplementation there was a non-significant increase of 1 min in each group. MVC force, muscle activation, EMG, stimulated tensions and durations were similar for the Cr and P groups over the course of the fatigue protocol and did not change after supplementation. Furthermore, recovery of MVC, stimulated tensions and contractile speeds did not differ as a result of Cr supplementation. These results indicate that short-term Cr supplementation does not influence isometric elbow flexion force, muscle activation, stimulated contractile properties, or delay time to fatigue or improve recovery.
Rapid Communication
A putative alternatively spliced variant of the P2X1 purinoreceptor in human bladder
- L. A. Hardy, I. J. Harvey, P. Chambers, J. I. Gillespie
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- 25 January 2001, pp. 461-463
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Activation of purinergic P2X receptors, putatively P2X1, may be important in the initiation of contraction in human detrusor. Purinergic transmission may be more important in muscle taken from patients with bladder instability. In this study the presence of the P2X1 receptor subtype was confirmed using RT-PCR. In addition, the results indicate, at the mRNA level, the presence of a splice variant of P2X1 that is lacking part of the second transmembrane domain. It is therefore possible that human bladder expresses multiple isoforms of the P2X1 receptor which may be potential sites for modifying or regulating putative purinergic activation of the human bladder.
Human ejaculatory duct: parameters of smooth muscle motor activity and modulatory role of autonomic drugs
- R. Mancinelli, P. Usai, R. Vargiu, A. De Lisa, R. M. Scarpa, E. Usai
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- 25 January 2001, pp. 465-467
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The contractile behaviour and effects of several autonomic drugs on the motor activity of human isolated ejaculatory ducts were investigated. Ejaculatory ducts exhibited spontaneous contractions characterised by an amplitude of 2.35 ± 0.28 mN, a duration of 62.9 ± 3.72 s and a frequency of 0.64 ± 0.014 waves min-1. Acetylcholine (10-5-10-4 m) induced a slight increase in basal tone and in the frequency of the contraction waves. These effects were suppressed by atropine (10-4 m). Noradrenaline (norepinephrine) increased the basal tone and frequency of spontaneous contractions in a dose-dependent manner. These responses were competitively inhibited by HEAT, a selective a1-adrenoceptor antagonist. These preliminary functional findings, indicating the presence of spontaneous motor activity of human ejaculatory ducts and its possible control by adrenergic agonists, suggests a physiological role for human ejaculatory duct in the propulsion of semen from the seminal vesicle towards the urethra. Experimental Physiology (2000) 85.4, 465-467.