Mini Review Article
ACTIONS OF THE PRO-INFLAMMATORY CYTOKINE IL-1[beta] ON CENTRAL SYNAPTIC TRANSMISSION
- JOHN J. O'CONNOR, ANDREW N. COOGAN
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- 04 January 2001, pp. 601-614
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Within the last decade huge advances have been made in the understanding of the interactions between the central nervous system (CNS) and the immune system. Foremost amongst these is the realization that cytokines, classically regarded as mediators and co-ordinators of the inflammatory and immune responses, can exert a number of neuromodulatory effects within the CNS under both physiological and pathological conditions (for recent reviews see Rothwell & Hopkins, 1995; Rothwell et al. 1997; Rothwell, 1999). Consistent with this, it has been shown that many cytokines and their receptors are present in the brain (Hopkins & Rothwell, 1995). Much attention has focused on the pro-inflammatory cytokine interleukin-1 (IL-1) especially the [beta] variant. IL-1[beta] has been shown to be produced in the CNS in response to a number of stimuli including peripheral administration of lipopolysaccharide (LPS); traumatic brain injury; acute stress; anorexia and [beta]-adrenoceptor agonist administration (e.g. Maruta et al. 1997). IL-1 receptors have also been shown to be present in many brain regions, with high levels in the hippocampus and hypothalamus (Ban et al. 1991). Expression of IL-1 receptors has also been shown to be upregulated in response to insult, e.g. kainic acid administration (Nishiyori et al. 1997) and expression is higher in aged animals (Murray & Lynch, 1998). However this review will concentrate on the effects of IL-1[beta] on hippocampal synaptic transmission and long-term potentiation (LTP), a process long thought to be an important underlying mechanism of learning and memory formation (for review see Bliss & Collingridge, 1993).
The Joan Mott Prize Lecture
The integrated response to hypoxia: from circulation to cells
- JANICE M. MARSHALL
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- 30 August 2019, pp. 449-470
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I guess everyone who is asked to give one of the Physiological Society's Prize Lectures feels honoured, but I feel particularly honoured to have been asked to give the Joan Mott Prize Lecture. I first met Joan Mott in 1980 at the Oxford meeting of the Physiological Society. I had just given a communication on baro- and chemoreceptor influences on the cardiovascular system and she came to introduce herself to me. I was a young lecturer at the time, and still felt a rather junior member of the Society. I had seen Joan Mott at many Physiological Society meetings. I had heard her give communications and had heard her entering into lively discussions after papers, and I knew she was a well respected scientist. I was therefore flattered that she should have come to talk to me about my work. After that we met at many Physiological Society meetings and she always made a point of asking how our research was going and we would talk of common interests. To me this is one of the most important aspects of Physiological Society meetings, that young and older members of the Society can rub shoulders with one a nother and discuss science openly with no barriers. Joan Mott had an influence on me that I have tried to remember as I have become a more senior member of the Society.
THE SHARPEY-SCHAFER PRIZE LECTURE
NUCLEUS TRACTUS SOLITARII: INTEGRATING STRUCTURES
- JULIAN F. R. PATON
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- 30 August 2019, pp. 815-833
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Presently, many researchers have adopted a reductionistic approach to physiology. Specifically, this involves studies on acutely isolated or cultured cells. While this is essential for our understanding of important cellular and subcellular mechanisms, it should not be forgotten that physiology is about the function of an integrating body. One view point is that definitive interpretations can only be made by crossing boundaries of physiology from the molecular/ cellular to the systems/behavioural level. An aim of this lecture will stress the importance of a multidisciplinary approach to physiology, that is, 'putting the cell back into the system'. The lecture attempts to demonstrate why this is important for enhancing the functional significance and relevance of physiological data as well as for validating predictions.
Review Article
Mg-ATP binding: its modification by spermine, the relevance to cytosolic Mg2+ buffering, changes in the intracellular ionized Mg2+ concentration and the estimation of Mg2+ by 31P-NMR
- Daniel Lüthi, Dorothee Günzel, John A. S. McGuigan
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- 03 January 2001, pp. 231-252
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It is now generally accepted that the intracellular ionized magnesium concentration ([Mg2+]i) in muscle cells is around 1 mmol l-1; in heart muscle this means that from the total some 90-95 % is bound (see McGuigan et al. 1991a). Although binding will include sequestration by intracellular organelles, a large part of the binding is by ATP in the cytosol and an equilibrium exists in the cytosol between free ATP, ionized magnesium and Mg-ATP. The extend of this equilibrium depends on the equilibrium constant of the reaction, which is a function of pH, temperature and ionic strength. This equilibrium constant is also important in the estimation of [Mg2+]i using 31P-NMR. In this method the difference between the α and β peaks of ATP is measured and from this shift and the equilibrium constant between Mg2+ and ATP in the cytosol, the [Mg2+]i can be calculated (Vink, 1993).
MEMBRANE OESTROGEN RECEPTORS ON RAT PITUITARY TUMOUR CELLS: IMMUNO-IDENTIFICATION AND RESPONSES TO OESTRADIOL AND XENOESTROGENS
- CHERYL S. WATSON*, CELESTE H. CAMPBELL, BAHIRU GAMETCHU
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- 04 January 2001, pp. 1013-1022
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Our laboratory has identified plasma membrane oestrogen receptors on a GH3/B6 rat pituitary tumour cell line and several sublines which produce rapid (within minutes), non-genomic responses to oestrogens. Oestrogen receptors have been identified by their binding to nine different antibodies (Abs) which together recognize at least seven epitopes on the oestrogen receptor-α. GH3/B6/F10 cells, a membrane oestrogen receptor-enriched subline, elevate intracellular calcium levels in response to 10 nM oestradiol. Prolactin release in these cells is triggered by both 1 pM and 1 nM oestradiol and diethylstilbestrol (DES). A membrane oestrogen receptor-α immunocyto-chemical signal rapidly disappears (at 3 min) and reappears (at 12-15 min) when 1 nM oestradiol, 10 nM diethylstilbestrol, or 10 nM nonylphenol is applied to the cells. This suggests that both oestrogens and xenoestrogens can utilize this alternative pathway for oestrogenic action. Xenoestrogens, which have so far shown weak effects in genomic assay systems, should now be retested for activity in eliciting membrane-initiated oestrogenic responses.
Research Article
Spike generation from dorsal roots and cutaneous afferents by hypoxia or hypercapnia in the rat in vivo
- Gábor Pethô, Róbert Pórszász, Barna Peitl, János Szolcsányi
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- 03 January 2001, pp. 1-15
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The present study aimed at investigating the responsiveness of different parts of the primary afferent neurones to a brief hypoxia, hypercapnia or ischaemia under in vivo conditions. Action potentials were recorded in separate groups of anaesthetized rats from (i) the peripheral end of the central stump of the cut L3, L4 or L5 dorsal root (dorsal root preparation); (ii) the central end of the peripheral stump of the cut saphenous nerve (saphenous-receptor preparation); (iii) the distal end of a segment of the saphenous nerve cut at both ends (axon preparation). In paralysed animals interruption of artificial ventilation for 20-60 s elicited or increased the frequency of action potentials in both the dorsal root and saphenous-receptor preparations. Activation of these preparations was also achieved by inspiration of gas mixtures containing 10-0 % oxygen (mixed with nitrogen) or 20-50 % carbon dioxide (mixed with oxygen) which elicited in the blood a decrease in PO2 or an increase in PCO2 with a fall in pH. Occlusion of the femoral artery for 3 min also caused spike generation in the saphenous-receptor preparations with little alteration in blood pressure. All these stimuli failed to evoke action potentials in the axon preparations. Systemic (300 mg kg-1 S.C.) or perineural (2 %) capsaicin pretreatment failed to inhibit the effect of hypoxia, hypercapnia or ischaemia, indicating a significant contribution of capsaicin-insensitive neurones to the responses. It is concluded that central and peripheral terminals but not axons of primary afferent neurones are excited by a brief hypoxia or hypercapnia and the peripheral terminals by a short local ischaemia as well. Excitation of central terminals by hypoxia or hypercapnia revealed in this way an antidromic activation of dorsal roots in response to natural chemical stimuli.
DEVELOPMENT OF THE FLUORESCENT MICROSPHERE TECHNIQUE FOR QUANTIFYING REGIONAL BLOOD FLOW IN SMALL MAMMALS
- D. DEVECI, S. EGGINTON
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- 04 January 2001, pp. 615-630
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1.Tissues weighing from 0·006 to 3·0 g were dissected and put directly into 15 ml screw cap polypropylene tubes with a conical base (maximum of 3 g per tube). It proved unnecessary to mince tissue, even though smaller pieces may aid quicker digestion. Five millilitres of 2 M KOH in 99 % (IMS) ethanol with 0·5 % Tween-80 was then added. Tissue digestion was usually completed in 2-4 h using a dry heating block held at 60°C, with intermittent shaking. Samples were routinely processed using fresh tissues, although storage of frozen tissue (in the dark at -20°C) introduced no detectable error in BF estimation and tended to aid tissue maceration.2.After digestion was completed the tubes were centrifuged at 3000 r.p.m. (1500 g) for 15 min.3.The supernatant was carefully aspirated until < 500 µl was left, thereby reducing the possibility of accidental loss of microspheres.4.After 1 ml dH2O was added the tubes were quickly vortexed to prevent microsphere flocculation and aid resuspension of remaining pellets, while the subsequent addition of ethanoic Tween (100 % ethanol + 0·5 % Tween-80) allowed complete sedimentation by centrifugation.5.Nine millilitres of ethanoic Tween-80 was added, and the tubes were vortexed and spun at 1500 g for 15 min.6.The supernatant was aspirated as above (Step 3).7.Five millilitres of 100 mM phosphate buffer (pH 7·00) was added to neutralize the pellet as alkaline solutions quench fluorescence. Using aqueous solutions increased the possibility of microsphere loss by adhesion to the surface of tubes or aggregation, which could readily be seen by eye when tubes were examined under bright light, and hence the buffer was followed by addition of 4 ml absolute ethanol and further vortexing, before spinning (1500 g) for 20 min.8.The supernatant was aspirated, leaving up to 300 µl depending on the amount of tissue residue, and the remaining microspheres and pellet were quickly vortexed to ensure complete resuspension.9.The tubes were left to evaporate in an oven at 60°C and then briefly vortexed during this period to disperse more of the flocculent, until around 100 µl fluid remained. This improves solvent extraction of microspheres, which may be less efficient in a completely dry pellet.10.Two or three millilitres of solvent (di(ethylene glycol) ethyl ether acetate, 98 %; Aldrich Chemical Co., Poole, Dorset, UK) was added according to the expected fluorescence intensity, and vortexed several times over 3-5 min. After 30 min the tubes were sonicated in a water bath for 5 min to complete dye extraction by the solvent. The tubes were kept as far as possible in the dark after the solvent was added to avoid photo-bleaching of the fluorescent dyes.11.After sonication, the tubes were occasionally spun (1500 g) once more to sediment any undigested/undissolved material, though this was rarely necessary in our experiments. Readings should be completed within 1 h to avoid loss of signal intensity.
Effect of extracellular cations on the inward rectifying K+ channels Kir2.1 and Kir3.1/Kir3.4
- J. M. OWEN, C. C. QUINN, R. LEACH, J. B. C. FINDLAY, M. R. BOYETT
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- 03 January 2001, pp. 471-488
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The effects of Ba2+, Mg2+, Ca2+ and Na+ as blocking ions were investigated in 90 and 10 mM extracellular K+ solutions on the cloned inward rectifying K+ channel Kir2.1 expressed in Xenopus oocytes. Some data were also obtained using another inward rectifying K+ channel Kir3.1/Kir3.4. The addition of Ba2+ caused a concentration-, voltage- and time-dependent block of both channels. Decreasing the extracellular K+ concentration augmented the block. The data suggest that Ba2+ blocks the channels by binding to a site within the channel pore and that the electrical binding distance, δ, of the site is significantly different for Kir2.1 and Kir3.1/Kir3.4 ([eqv] 0·38 and [eqv] 0·22, respectively). Mg2+ and Ca2+ caused an instantaneous concentration- and voltage-dependent block of both channels. With Kir2.1, decreasing the K+ concentration augmented the block. The voltage dependence of the block was less than that of Ba2+ (δ, [eqv] 0·1), indicating a more superficial binding site for these ions within the channel pore. The affinity of the channels for Mg2+ and Ca2+ was [eqv] 1000-fold lower than that for Ba2+. Addition of Na+ resulted in a concentration-, voltage- and time-dependent block of Kir2.1, similar to that observed with Ba2+. The competition between the blocking cations (for Kir2.1: Ba2+, Mg2+, Ca2+; for Kir3.1/Kir3.4: Ba2+) and extracellular K+ suggests that the binding sites for the blocking cations may be sites to which K+ binds as part of the normal passage of K+ through the channels. It is possible that under normal physiological conditions naturally occurring extracellular cations may partly block the two inward rectifying K+ channels.
ION CURRENTS IN SMOOTH MUSCLE CELLS FROM HUMAN SMALL BRONCHIOLES: PRESENCE OF AN INWARD RECTIFIER K+ CURRENT AND THREE TYPES OF LARGE CONDUCTANCE K+ CHANNEL
- V. A. SNETKOV, J. P. T. WARD
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- 04 January 2001, pp. 835-846
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Bronchoconstriction of small bronchioles plays a major role in the increase in airway resistance following agonist challenge. There is evidence that the airway smooth muscle (ASM) of small bronchioles differs functionally from that in larger airways. Little is known however about the electrophysiology of small bronchioles. Ion currents were therefore studied in airway smooth muscle cells freshly dissociated from human intralobular bronchioles, with a diameter between 0·3 and 1·0 mm. As previously reported for human large airways, the major outward current in these cells was due to activity of large conductance K+ (BK) channels, with a relatively minor component due to a voltage-gated delayed rectifier current (IDR), which was only observed in [sim]30 % of cells. Three distinct types of iberiotoxin- and TEA-sensitive large conductance K+ channel contributed to large conductance K+ current (IBK). These included a highly voltage- and Ca2+-sensitive 200 pS channel previously reported in human large airways, and two smaller channels of 150 and 100 pS previously seen only in human fetal or cultured ASM. In contrast to large airways, ASM cells from bronchioles also demonstrated a voltage-gated inward rectifier current (IIR). IIR was activated by hyperpolarisation below the K+ equilibrium potential and could be blocked by submillimolar concentrations of Cs+ or Ba2+, and partially by physiological concentrations of Na+. Corresponding single channels with a conductance of [sim]>17 pS could also be recorded in the cell-attached configuration. A small voltage-independent current was also observed which was resistant to classic K+ and Cl- channel blockers but which could be abolished by replacement of Na+ with the impermeant cation N-methyl-D-glucamine (NMDG+). Corresponding non-selective single channels of [sim]20 pS could be seen in inside-out mode. These results demonstrate that ASM from small bronchioles differs in terms of ion currents and channels from ASM derived from large airways, with possible implications for function.
Review Article
Cardiac electrophysiological effects of platelet-derived substances
- N. A. Flores, A. N. S. Botchway, B. M. Stavrou, D. J. Sheridan
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- 03 January 2001, pp. 253-274
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The ability of the heart to function as an efficient pump is critically dependent on an adequate blood supply to the myocardium. When myocardial blood supply is reduced (ischaemia), contractile function of the heart is impaired and its cellular electrophysiological properties are profoundly altered. Prolonged ischaemia may result in cell necrosis which, if irreversible, leads to cell death (infarction) and predisposes the heart to ventricular arrhythmias, which in turn may be fatal. In man, myocardial ischaemia is the consequence of narrowing or occlusion of a coronary artery and is usually a manifestation of coronary artery disease which is often associated with thrombosis.
Research Article
Separate receptors mediate oxytocin and vasopressin stimulation of cAMP in rat inner medullary collecting duct cells
- E. T. Wargent, W. J. Burgess, J. F. Laycock, R. J. Balment
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- 03 January 2001, pp. 17-25
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The two neurohypophysial hormones arginine vasopressin (AVP) and oxytocin have actions in the inner medullary collecting duct (IMCD) where both peptides induce an increase in cAMP accumulation. The present study has employed a novel IMCD cell line to determine whether these two hormones induce cAMP accumulation via common or separate receptors, and to characterize the potential receptors responsible. Equal volumes of vehicle (150 mM NaCl) or hormone/antagonist solutions were added to aliquots of 104 IMCD cells in the presence of 10-3 M 3-isobutylmethylxanthine (IBMX) and incubated at 37°C for 4 min. cAMP levels were determined by radioimmunoassay and protein concentration by Bradford assay. Both AVP and oxytocin elicited dose-dependent increases in cAMP generation, though oxytocin was less potent than AVP (EC50 = 1Σ6 × 10-8 M vs. 7Σ4 × 10-10 M). AVP at 10-8 M and oxytocin at 10-6 M, concentrations sufficient to elicit near-maximal cAMP accumulation, resulted in cAMP levels of 73Σ4 ± 1Σ7 and 69Σ0 ± 3Σ3 pmol (mg protein)-1 (4 min)-1, respectively (n = 10), compared with the vehicle-treated basal value of 37Σ7 ± 2Σ2 pmol (mg protein)-1 (4 min)-1 (P < 0Σ001, n = 10). Combined AVP (10-8 M) and oxytocin (10-6 M) resulted in cAMP accumulation of 63Σ8 ± 3Σ1 pmol (mg protein)-1 (4 min)-1 (n = 10), which was not significantly different from the effect of oxytocin alone, but slightly less than that for AVP alone (P < 0Σ05). A submaximal concentration of AVP (10-10 M) induced cAMP accumulation of 48Σ6 ± 2Σ5 pmol (mg protein)-1 (4 min)-1 (P < 0Σ01 compared with basal level of 34Σ9 ± 2Σ4 pmol (mg protein)-1 (4 min)-1, n = 10), which was blocked in the presence of a vasopressin V2 receptor antagonist (10-7 M OPC-31260) but not by the oxytocin receptor antagonist (10-6 M [Pen1,pMePhe2,Thr4,Orn8]oxytocin) (36Σ3 ± 6Σ1 and 45Σ1 ± 1Σ3 pmol (mg protein)-1 (4 min)-1 respectively, P < 0Σ05, n = 10). A submaximal concentration of oxytocin (10-7 M) induced a cAMP accumulation of 45Σ8 ± 1Σ8 pmol (mg protein)-1 (4 min)-1 (n = 10), which was reduced by addition of 10-6 M oxytocin antagonist (36Σ3 ± 2Σ1 pmol (mg protein)-1 (4 min)-1, P < 0Σ05, n = 10), whereas co-incubation with 10-6 M of the V2 receptor antagonist had no effect (43Σ2 ± 1Σ3 pmol (mg protein)-1 (4 min)-1, n = 10). These results indicate that AVP and oxytocin induce cAMP accumulation from a common ATP pool in IMCD cells, and that separate vasopressin V2 and oxytocin receptor systems are involved, perhaps coupled to a common adenylate cyclase system.
Review Article
ICln, AN ION CHANNEL-FORMING PROTEIN ASSOCIATED WITH CELL VOLUME REGULATION
- M. GSCHWENTNER, J. FÜRST, M. RITTER, C. BAZZINI, E. WÖLL, A. DIENSTL, M. JAKAB, M. KÖNIG, E. SCANDELLA, J. RUDZKI, G. BOTTA, G. MEYER, F. LANG, P. DEETJEN, M. PAULMICHL
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- 04 January 2001, pp. 1023-1031
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It is not resolved whether the anionic channel involved in volume regulation after cell swelling comprises one or more subunits. Moreover, it remains to be determined which of the different proteins cloned so far, for which an involvement in cell volume regulation has been postulated, is the ideal candidate. In this review, we consider the role of the ICln protein, cloned from MDCK cells, in cell volume regulation.
Research Article
H+-zwitterionic amino acid symport at the brush-border membrane of human intestinal epithelial (CACO-2) cells
- David T. Thwaites, Beverley C. Stevens
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- 03 January 2001, pp. 275-284
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Transport of a number of dipolar amino acids (and the orally active antibiotic D-cycloserine) across the apical membrane of human intestinal epithelial (Caco-2) cell monolayers is mediated by a Na+-independent, pH-dependent transport mechanism. Relatively little is known about the mode of action of this transport system so to differentiate between pH dependence and proton coupling three experimental protocols were designed and tested. The results demonstrate, firstly, that it is the transapical pH gradient and its maintenance (rather than apical acidity alone) that is important in amino acid uptake. Secondly, Na+-independent uptake of seven dipolar amino acids (with pKa (-log of acid dissociation constant) values between 1.50 and 4.23) showed a similar dependence on apical pH (half-maximal uptake being observed at pH 5.99-6.20). Thirdly, the pattern of pH-dependent amino acid (β-alanine) uptake is similar irrespective of whether the cationic substrate concentration is varied or constant, demonstrating no relationship between uptake and concentration of the cationic form of the amino acid. These observations demonstrate that the transport mechanism is a H+-zwitterionic amino acid symporter and suggest that the presence of a H+ gradient at the apical surface of the human small intestine (in the form of the acid microclimate) may be important in driving nutrient absorption.
Activation of ionic channels by deoxycholate in frog and human cell lines
- A. C. MAURÍCIO, K. T. G. FERREIRA
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- 03 January 2001, pp. 489-499
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Humans, after extensive ileal resection, frequently suffer from diarrhoea, which may be due to an increased delivery of deoxycholate (DOC) to the large intestine. In the frog skin the addition of DOC (0·5 mM) to the apical side induced the activation of amiloride-sensitive Na+ channels and an increase in the unidirectional Cl- fluxes. Here we used two established cell lines (A6 and Caco2) to study the effect of DOC on ion channels at cell and membrane level using the patch-clamp technique. In A6 cells subcultured directly on Petri dishes and studied in the whole-cell configuration, DOC induced an increase in cell conductance of 110·3 ± 4 pS pF-1 (N = 8) which was reduced to 89 ± 14 pS pF-1 (N = 8) by the addition of DIDS (0·5 mM). The absolute values of these two effects were not statistically different (P < 0·2). In Caco2 cells, the addition of DOC (0·5 mM) induced, after 1 min, an increase in cell conductance of 583 ± 16 pS pF-1 (N = 8) which was reduced to 560·4 ± 16 pS pF-1 (N = 8) by DIDS (0·5 mM) and N-phenylanthranilic acid (DPC; 0·5 mM). The two values were not statistically different (P < 0·4). In Caco2 cells subcultured under the same conditions, DOC induced an increase in cell conductance of 1710 ± 64 pS pF-1 (N = 6). Subsequent addition of amiloride (0·1 mM) reduced the cell conductance to 1558 ± 33 pS pF-1 (N = 6). These two mean values were statistically different allowing for an error of the second kind < 0·05. In cells in which DOC produced a conductance increase of 1010 ± 10 pS pF-1, gadolinium (0·5 mM) induced a fall in cell conductance of 1800 ± 10 pS pF-1. In Caco2 cells, addition of DOC (0·5 mM) to the bath reversibly induced the appearance of or an increase in channel activity in patches studied in cell-attached and excised inside-out configuration. In inside-out experiments (N = 13) DOC (0·5 mM) induced the appearance of channel activity with conductances and reversal potentials (Er) of 27·7 ± 1·9 pS and 0·8 ± 5·7 mV, respectively. In cell-attached patches (N = 13) these values were 24·9 ± 4·4 pS and -18·1 ± 6·4 mV. In excised inside-out patches from Caco2 cells, subjected to electrochemical gradients for Na+, K+ and Cl-, (+85, -85 and 0 mV, respectively), addition of DOC also induced an increase in the baseline conductance and a shift in the reversal potential from values around +25 mV to values around 0 mV. Bile salts activated both anionic and cationic channels and did not require the presence of intracellular factors for these effects. We suggest that they act at the membrane level.
Anion efflux from cytotrophoblast cells derived from normal term human placenta is stimulated by hyposmotic challenge and extracellular a23187 but not by membrane-soluble cAMP
- M. A. Turner, M. K. Sides, C. P. Sibley, S. L. Greenwood
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- 03 January 2001, pp. 27-40
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The regulation of placental anion transport influences fetal accretion and placental homeostasis. We investigated whether efflux of 125I- or 36Cl- from multinucleated cytotrophoblast cells derived from human term placenta is regulated by one of three stimuli: (a) the calcium ionophore A23187, (b) a 'cocktail' of agents designed to raise intracellular levels of cAMP, (c) a hyposmotic solution. After loading with the appropriate isotope for 2 h and thorough washing, cells were exposed to sequential aliquots of buffer applied and removed each minute. Following an equilibration period of 5 min one of the stimuli was applied at room temperature At the end of the experiment the cells were lysed to give a lysate count which was used to express the count obtained from each aliquot as percentage efflux of that possible for that minute. The cAMP 'cocktail' and A23187 were applied for 5 min; the hyposmotic solution was applied for 10 min. The results for 125I- at 7 min showed that the mean efflux in the presence of hyposmotic shock was greater than control (5Σ7 ± 1Σ0 % min-1versus 2Σ2 ± 0Σ1 % min-1, respectively; mean ± S.E.M., n = 4 placentas). Similarly mean efflux at 6 min in the presence of A23187 was also significantly greater than control (6Σ5 ± 1Σ9 % min-1versus 2Σ6 ± 1Σ0 % min-1, respectively, n = 3 placentas). The mean efflux in the presence of the cAMP cocktail was not different from control at any time point. The results were qualitatively the same if 36Cl- was used in the place of 125I- and when the experiment was performed with 36Cl- in a HCO3- buffer gassed with CO2. Mean 125I- efflux at 6 min in response to hyposmotic challenge was 33 % less (P < 0Σ01) in the presence of 1 mM 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) and 37 % less (P < 0Σ005) in the presence of 10 µM tamoxifen but no different if the hyposmotic solution was nominally calcium free. We conclude that there are differential effects of second messengers on anion efflux from the differentiated cytotrophoblast cells.
BLOCKAGE OF MOUSE MUSCLE NICOTINIC RECEPTORS BY SEROTONERGIC COMPOUNDS
- JESÚS GARCÍA-COLUNGA, RICARDO MILEDI
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- 04 January 2001, pp. 847-864
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Xenopus laevis oocytes were used to analyse the effects of serotonin (5-hydroxytryptamine, 5-HT) and serotonergic agents on ionic currents elicited by the activation of mammalian muscle nicotinic acetylcholine receptors (AChRs). 5-HT as well as other serotonergic agents, such as ketanserin, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), methysergide, spiperone, or fluoxetine alone (up to 1 mM), did not elicit membrane currents in Xenopus oocytes expressing AChRs, but they reversibly reduced the current elicited by acetylcholine (ACh-current). Serotonin was applied before, together with or after ACh application, and its effects were examined on desensitizing and non-desensitizing ACh-currents. 5-HT reduced the amplitude and accelerated the desensitization of the desensitizing currents. In contrast, non-desensitizing currents were reduced in amplitude but their time course was not significantly affected. With the same concentration of 5-HT the inhibition was stronger on desensitizing than on non-desensitizing ACh-currents. For example, 100 µM 5-HT reduced the peak of a desensitizing ACh-current to 0·48 ± 0·06 (peak current ratio) and after 40 s the current was reduced to a ratio of 0·25 ± 0·04, whereas a non-desensitizing ACh-current was reduced to a ratio of 0·66 ± 0·01. All the serotonergic agents tested inhibited the ACh-currents rapidly and reversibly, suggesting that they are acting directly on the AChRs. The half-inhibitory concentration, IC50, of 5-HT acting on non-desensitizing currents elicited by 250 nM ACh was 247 ± 26 µM and the Hill coefficient was [sim]0.88, suggesting a single site for the interaction of 5-HT with the receptor. It appears that 5-HT inhibits AChRs non-competitively because neither the half-effective concentration of ACh, EC50, for ACh-current nor the Hill coefficient were affected by 5-HT. Furthermore, the extent of inhibition of 5-HT on AChRs did not depend on the nicotinic agonist (suberyldicholine, ACh or nicotine). The inhibition of AChRs by serotonergic agents was voltage-dependent. The electrical distance of the binding site for 5-HT was [sim]0.75, whereas for the other serotonergic agents tested it was [sim]0.22, suggesting that ketanserin, 8-OH-DPAT, methysergide, spiperone and fluoxetine act within the ion channel, but at a site more external than that for 5-HT. These substances inhibited the ACh-current more potently than 5-HT. We conclude that 5-HT and serotonergic agents inhibit, in a non-competitive manner, the ACh-current in muscle AChRs by blocking the open receptor-channel complex. Moreover, 5-HT appears to promote the desensitized state of the receptor when the current is elicited by high ACh concentrations.
NON-MUSCARINIC AND NON-NICOTINIC INHIBITION BY THE ACETYLCHOLINE ANALOGUE CARBACHOL OF THE DELAYED RECTIFIER POTASSIUM CURRENT, iK, IN RABBIT ISOLATED SINO-ATRIAL NODE CELLS
- MING LEI, PETER KOHL, HILARY BROWN, DENIS NOBLE
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- 04 January 2001, pp. 631-638
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The effect of carbachol, an analogue of acetylcholine, on the delayed rectifier potassium current, iK, was investigated in rabbit isolated sino-atrial node cells using the whole cell patch clamp technique with amphotericin-permeabilized patches. In the presence of 500 nM atropine and 500 nM hexamethonium to block muscarinic and nicotinic receptors, respectively, 500 nM carbachol decreased the amplitude and rate of deactivation of iK without, however, affecting the slope of the iK activation curve. The same concentration of carbachol decreased the pacemaking rate of spontaneously active sino-atrial node cells by more than 13 %. Thus, there is a non-muscarinic and non-nicotinic pathway for cholinergically induced reduction in the amplitude and rate of deactivation of iK that would appear to contribute to negative chronotropy in rabbit sino-atrial node pacemaker cells.
A MONOCARBOXYLATE TRANSPORTER MCT1 IS LOCATED AT THE BASOLATERAL POLE OF RAT JEJUNUM
- M. N. ORSENIGO, M. TOSCO, C. BAZZINI, U. LAFORENZA, A. FAELLI
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- Published online by Cambridge University Press:
- 04 January 2001, pp. 1033-1042
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We have functionally expressed and identified a monocarboxylate transporter (MCT1) from rat jejunal enterocyte and we provide evidence for its basolateral localization. Poly(A)+ RNA isolated from rat jejunum was injected into Xenopus laevis oocytes and expression of a proton-lactate symporter was investigated by means of L-[14C]lactate uptake. The existence of an endogenous capacity for L-lactate transport was demonstrated; when, however, oocytes were injected with jejunal mRNA, an expressed L-lactate uptake was seen which differed from the endogenous transporter since it was significantly pH dependent. After sucrose density gradient fractionation, the highest expression of the pH-dependent lactate uptake was detected with the mRNA size fraction of about 2-3 kb in length. The substrate specificity, stereoselectivity and sensitivity to pCMBS (an organomercurial thiol reagent that modifies cysteine residues) of the expressed transport were in good agreement with results previously obtained using isolated jejunal basolateral membranes. Using the reverse transcriptase-polymerase chain reaction, the presence of mRNA coding for the MCT1 isoform was demonstrated in jejunal enterocytes. These data, together with previous results, suggest that MCT1 is a major route for lactate efflux across the basolateral membrane of rat jejunum; this is in contrast to current opinion which restricts the presence of MCT1 to the apical membrane of the whole small intestine.
Drug extrusion, 125I- efflux and the control of intracellular [Ca2+] in drug-resistant ovarian epithelial cells
- H. L. McAlroy, D. L. Bovell, J. A. Plumb, P. Thompson, S. M. Wilson
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- Published online by Cambridge University Press:
- 03 January 2001, pp. 285-297
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Experiments were undertaken using an ovarian adenocarcinoma cell line (A2780) and a drug-resistant strain (A2780.ad) derived from this line. P-glycoprotein could not be detected in A2780 cells but was essentially ubiquitous in A2780.ad cells, although removing the selective pressure for drug resistance led to reduced expression. However, the amount of P-glycoprotein present was used to predict the capacity of these cells to extrude rhodamine-123 (R-123) and their resistance to adriamycin, a cytotoxic drug. This accords with the role of P-glycoprotein as a drug pump. Although hypotonic solutions increased anion efflux from A2780 and A2780.ad cells, larger responses occurred in the parental line. Moreover, R-123 extrusion and anion efflux appeared to be mutually independent processes and so these data do not support the view that P-glycoprotein is involved in the control of volume-sensitive anion channels. Hypotonic solutions increased intracellular free calcium ([Ca2+]i) in drug-resistant cells but not in the parental line, and so establishing a drug-resistant strain may affect the control of [Ca2+]i during osmotic swelling. This could account for effects that were previously attributed to P-glycoprotein.
COMPARISON OF STEP AND RAMP VOLTAGE CLAMP ON BACKGROUND CURRENTS IN GUINEA-PIG VENTRICULAR CELLS
- A. J. SPINDLER, S. J. NOBLE, D. NOBLE
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- Published online by Cambridge University Press:
- 04 January 2001, pp. 865-879
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Isolated cardiac ventricular myocytes from guinea-pig were used to investigate the effect of voltage clamp protocols on background Na+ current (ib.Na) and inward rectifier current (iK1). Using long (4 s) clamp pulses and very long step clamps, the i-V relations showed that removal of Na+ reduces the amplitude and shifts the voltage dependence of iK1 (Spindler et al. 1998). Ramp clamps, however, gave more complicated results, with slower ramps more often giving the same results as steps and pulses. Both iK1 itself and, during faster ramps, other currents show hysteresis, so masking the steady-state changes. Using pulses, TTX had no effect on steady-state current. Small differences occurred in the ramps, but even at fast ramp speeds the effects are very much smaller than in Purkinje tissue. Only part of ib,Na is TTX sensitive and the effect does not occur in all cells.