Journal of Physiology
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Acetylcholine and kinin augmentation of Cl‐ secretion stimulated by prostaglandin in a canine renal epithelial cell line. 1. The actions of kinins and of acetylcholine upon transepithelial ion transport in a renal‐derived cultured epithelium (Madin‐Darby canine kidney cells, MDCK) have been investigated. 2. In voltage‐clamped epithelial layers mounted in Ussing chambers and with prior stimulation of inward short‐circuit current (SCC) by 5 or 10 microM‐prostaglandin E1 (PGE1), both bradykinin (1 microM) and acetylcholine (0.1 mM) stimulate an additional, but transient, inward SCC. In the absence of PGE1 minimal effects of both bradykinin and acetylcholine upon SCC are observed. The SCC response to bradykinin and acetylcholine are attenuated with prior stimulation by 10 microM‐adrenaline. 3. Measurements of bradykinin and acetylcholine‐stimulated inward SCC with cation and anion replacement of the bathing media and the use of the Cl channel blocker 5‐nitro‐2(3‐phenylpropylamino)‐benzoic acid (NPPB) together with bumetanide to inhibit Na(+)‐K(+)‐Cl‐ ‘co‐transport’, are consistent with the bradykinin‐ and acetylcholine‐stimulated SCC being the result of basal to apical Cl‐ secretion. 4. Bradykinin (1 microM) is capable of stimulation of inward SCC from both epithelial surfaces, whilst acetylcholine is only effective from the basolateral surface. Kallidin (lys‐bradykinin) was similar in effect to bradykinin from both epithelial surfaces whereas bradykinin (1‐8) was ineffective, suggesting that B2 bradykinin receptors mediate the effect of bradykinin upon SCC. Dose‐response relationships show that the response to kallidin and bradykinin was of higher sensitivity for additions to the apical cell aspects. 5. The data are discussed in relation to a model for epithelial Cl‐ secretion, and to the mechanism of natriuresis observed with kinins and acetylcholine in vivo.
Journal of Physiology - Tập 447 Số 1 - Trang 1-15 - 1992
Characterization of a Ca(2+)‐dependent anion channel from sheep tracheal epithelium incorporated into planar bilayers. 1. Anion‐selective channels from the apical membrane of respiratory epithelia are involved in the secretion of chloride into the airway lumen. In cystic fibrosis (CF) there is an abnormality of phosphorylation‐regulated chloride transport in this tissue, whilst a calcium‐dependent pathway appears to function normally. 2. Using incorporation of apical membrane vesicles into planar phospholipid bilayers, we have characterized the most commonly seen anion‐selective channel from sheep tracheal epithelium. 3. In symmetrical 200 mM‐NaCl solutions the channel showed rectification, with a chord conductance at negative voltages of 107 pS and at positive voltages of 67 pS. The channel characteristically demonstrated subconductance states at 1/3 and 3/4 of the fully open level. Selectivity for chloride over sodium was approximately 6:1. 4. The channel required a minimum of approximately 100 microM‐calcium on the presumed cytoplasmic surface (cis) for opening events to be observed. Open probability (Po) of the fully open state was markedly voltage dependent, but little effect of voltage was seen on the 1/3 subconductance state. 5. The relative permeabilities of monovalent anions monitored under bi‐ionic conditions gave the following sequence: NO3‐ greater than I‐ greater than Cl‐ = Br‐ much much greater than F‐. The order of conductances in symmetrical solutions was Cl‐ = NO3‐ greater than Br‐ greater than I‐ much much greater than F‐. 6. The chloride channel blocker 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate (NPPB) produced a dose‐related reduction in Po with a flickering block at 10‐50 microM and complete block at higher concentrations. 7. ATP produced a dose‐related reduction in Po with effects at 1 microM and complete closing at 1 mM. These effects were only seen with addition to the cis chamber. 8. The catalytic subunit of protein kinase A, either when incubated with vesicles prior to incorporation into bilayers, or when added directly to either chamber, produced no effect. 9. Channels with very similar properties were seen from transfected human tracheo‐bronchial cells. 10. Recent whole‐cell patch‐clamp studies have suggested a distinct calcium‐activated chloride current in secretory epithelia. The described channel has properties in common with this current and may be a candidate for its single‐channel basis.
Journal of Physiology - Tập 443 Số 1 - Trang 137-159 - 1991
Effect of extracellular adenosine triphosphate on electrical properties of subconfluent Madin‐Darby canine kidney cells. 1. The present study has been performed to test for an influence of extracellular ATP on the potential differences across the cell membrane (PD) in subconfluent MDCK cells utilizing conventional microelectrodes. 2. In the absence of ATP, the mean measured PD was ‐47.5 +/‐ 0.3 mV (+/‐ S.E.M., n = 320). Application of 10 mumol/l ATP leads to rapid (less than 2 s) hyperpolarization of the cell membrane by ‐18.5 +/‐ 0.4 mV (n = 221), reduction of input resistance by 14 +/‐ 1 M omega (n = 106) and increase of the sensitivity of PD to alterations of extracellular potassium. 3. The concentration needed for half‐maximal effect (K1/2) of ATP is approximately 0.5 mumol/l. ATP‐gamma‐S (K1/2 approximately 0.4 mumol/l) aand ADP (K1/2 approximately 0.9 similarly effective, whereas up to 1 mmol/l AMP or adenosine does not significantly alter PD. Application of 10 mumol/l theophylline, 1 mumol/l phentolamine and 10 mumol/l indomethacin does not blunt the hyperpolarizing effect of ATP. 4. The ATP‐induced hyperpolarization is completely abolished in the presence of 1 mmol/l quinidine but only incompletely by 0.1 mmol/l quinidine or 1 mmol/l barium. In calcium‐free extracellular fluid (1 mmol/l EDTA added) PD is 18.5 +/‐ 1.7 mV (n = 18). With reduced extracellular calcium, the hyperpolarizing effect of ATP is blunted (‐12.3 +/‐ 1.6 mV, n = 18) and only transient. 5. In conclusion, ATP hyperpolarizes MDCK cells by increasing the potassium conductance. The activation of potassium channels requires calcium.
Journal of Physiology - Tập 408 Số 1 - Trang 333-343 - 1989
Effect of bradykinin, ATP and adrenaline on cell membrane resistances of Madin‐Darby canine kidney cells. 1. Previous studies have shown that bradykinin, ATP and adrenaline hyperpolarize the cell membrane of Madin‐Darby canine kidney (MDCK) cells by activation of calcium‐sensitive K+ channels. The present study has been performed to determine the effect of these hormones on the resistance of the cell membrane and the cellular coupling. To this end, cellular cable analysis has been performed. 2. As a result, all three hormones lead to the expected, marked decrease of cell membrane resistance. 3. However, the bradykinin‐induced reduction of cell membrane resistance was sustained, contrasting with only transient hyperpolarization induced by bradykinin and only transient activation of the K+ channels. Thus, the cable analysis reveals the sustained activation of an additional conductance. 4. ATP, but not the other two hormones, leads to a delayed increase of the intercellular coupling resistances. 5. Prolonged exposure of the cells to adrenaline leads to oscillations of the cell membrane potential, apparently by oscillatory activation of the K+ channels.
Journal of Physiology - Tập 443 Số 1 - Trang 45-54 - 1991
Modulation of canine antral circular smooth muscle by acetylcholine, noradrenaline and pentagastrin. 1. The effects of acetylcholine, noradrenaline and pentagastrin on the action potential of canine antral circular muscle were determined using the intracellular micro‐electrode technique. 2. Acetylcholine increased the amplitude and duration of the plateau potential of the action potential. Since these effects were blocked by atropine but not by hexamethonium, the effects of acetylcholine were on muscarinic receptors, probably located on the smooth muscle cell. 3. Pentagastrin 2 x 10(‐10) M increased the size of the plateau potential and the frequency of the action potential; pentagastrin 1 x 10(‐9) M increased the frequency of the action potential complex and produced a marked diastolic depolarization between action potentials. The effect on the size of the plateau potential was biphasic. The amplitude and half‐time duration of the plateau potential increased in the first 3 min, but thereafter, during steady‐state conditions, they were the same as or slightly greater than those obtained in Krebs solution. 4. All the effects produced by pentagastrin were due to a direct action on the smooth muscle cell. 5. Noradrenaline decreased the size of the plateau potential but increased its frequency; high concentrations (greater than 10(‐5) M) additionally produced a diastolic depolarization between action potentials. These effects were mediated primarily by alpha‐adrenoceptors presumably located on the smooth muscle cell. 6. It was concluded that the substances studied primarily alter the size of the plateau potential in antral circular muscle. Since phasic contractions are associated with the plateau potential, it is suggested that agents which increase the size of the plateau potential increase the force of the contraction whereas agents which decrease the size of the plateau potential have the opposite effect.
Journal of Physiology - Tập 279 Số 1 - Trang 309-320 - 1978
Preferential motor unit loss in the SOD1<sup>G93A</sup> transgenic mouse model of amyotrophic lateral sclerosis The present study investigated motor unit (MU) loss in a murine model of familial amyotrophic lateral sclerosis (ALS). The fast‐twitch tibialis anterior (TA) and medial gastrocnemius (MG) muscles of transgenic SOD1G93A and SOD1WT mice were studied during the presymptomatic phase of disease progression at 60 days of age. Whole muscle maximum isometric twitch and tetanic forces were 80% lower (P < 0.01) in the TA muscles of SOD1G93A compared to SOD1WT mice. Enumeration of total MU numbers within TA muscles showed a 60% reduction (P < 0.01) within SOD1G93A mice (38 ± 7) compared with SOD1WT controls (95 ± 12); this was attributed to a lower proportion of the most forceful fast‐fatigable (FF) MU in SOD1G93A mice, as seen by a significant (P < 0.01) leftward shift in the cumulative frequency histogram of single MU forces. Similar patterns of MU loss and corresponding decreases in isometric twitch force were observed in the MG. Immunocytochemical analyses of the entire cross‐sectional area (CSA) of serial sections of TA muscles stained with anti‐neural cell adhesion molecule (NCAM) and various monoclonal antibodies for myosin heavy chain (MHC) isoforms showed respective 65% (P < 0.01) and 28% (P < 0.05) decreases in the number of innervated IIB and IID/X muscle fibres in SOD1G93A , which paralleled the 60% decrease (P < 0.01) in the force generating capacity of individual fibres. The loss of fast MUs was partially compensated by activity‐dependent fast‐to‐slower fibre type transitions, as determined by increases (P < 0.04) in the CSA and proportion of IIA fibres (from 4% to 14%) and IID/X fibres (from 31% to 39%), and decreases (P < 0.001) in the CSA and proportion of type IIB fibres (from 65% to 44%). We conclude that preferential loss of IIB fibres is incomplete at 60 days of age, and is consistent with a selective albeit gradual loss of FF MUs that is not fully compensated by sprouting of the remaining motoneurons that innervate type IIA or IID/X muscle fibres. Our findings indicate that disease progression in fast‐twitch muscles of SOD1G93A mice involves parallel processes: (1) gradual selective motor axon die‐back of the FF motor units that contain large type IIB muscle fibres, and of fatigue‐intermediate motor units that innervate type IID/X muscle fibres, and (2) activity‐dependent conversion of motor units to those innervated by smaller motor axons innervating type IIA fatigue‐resistant muscle fibres.
Journal of Physiology - Tập 586 Số 14 - Trang 3337-3351 - 2008
Okadaic acid‐sensitive activation of Maxi Cl<sup>−</sup> channels by triphenylethylene antioestrogens in C1300 mouse neuroblastoma cells
Journal of Physiology - Tập 536 Số 1 - Trang 79-88 - 2001
Regulation of arterial diameter and wall [Ca<sup>2+</sup>] in cerebral arteries of rat by membrane potential and intravascular pressure
The regulation of intracellular [Ca2+ ] in the smooth muscle cells in the wall of small pressurized cerebral arteries (100‐200 μm) of rat was studied using simultaneous digital fluorescence video imaging of arterial diameter and wall [Ca2+ ], combined with microelectrode measurements of arterial membrane potential. Elevation of intravascular pressure (from 10 to 100 mmHg) caused a membrane depolarization from ‐63 ± 1 to ‐36 ± 2 mV, increased arterial wall [Ca2+ ] from 119 ± 10 to 245 ± 9 nM, and constricted the arteries from 208 ± 10 μm (fully dilated, Ca2+ free) to 116 ± 7 μm or by 45 % (‘myogenic tone’). Pressure‐induced increases in arterial wall [Ca2+ ] and vasoconstriction were blocked by inhibitors of voltage‐dependent Ca2+ channels (diltiazem and nisoldipine) or to the same extent by removal of external Ca2+ . At a steady pressure (i.e. under isobaric conditions at 60 mmHg), the membrane potential was stable at ‐45 ± 1 mV, intracellular [Ca2+ ] was 190 ± 10 nM, and arteries were constricted by 41 % (to 115 ± 7 μm from 196 ± 8 μm fully dilated). Under this condition of ‐45 ± 5 mV at 60 mmHg, the voltage sensitivity of wall [Ca2+ ] and diameter were 7.5 nM mV−1 and 7.5 μm mV−1 , respectively, resulting in a Ca2+ sensitivity of diameter of 1 μm nM−1 . Membrane potential depolarization from ‐58 to ‐23 mV caused pressurized arteries (to 60 mmHg) to constrict over their entire working range, i.e. from maximally dilated to constricted. This depolarization was associated with an elevation of arterial wall [Ca2+ ] from 124 ± 7 to 347 ± 12 nM. These increases in arterial wall [Ca2+ ] and vasoconstriction were blocked by L‐type voltage‐dependent Ca2+ channel inhibitors. The relationship between arterial wall [Ca2+ ] and membrane potential was not significantly different under isobaric (60 mmHg) and non‐isobaric conditions (10‐100 mmHg), suggesting that intravascular pressure regulates arterial wall [Ca2+ ] through changes in membrane potential. The results are consistent with the idea that intravascular pressure causes membrane potential depolarization, which opens voltage‐dependent Ca2+ channels, acting as ‘voltage sensors’, thus increasing Ca2+ entry and arterial wall [Ca2+ ], which leads to vasoconstriction.
Journal of Physiology - Tập 508 Số 1 - Trang 199-209 - 1998
Prenatal cocaine exposure increases heart susceptibility to ischaemia–reperfusion injury in adult male but not female rats The present study tested the hypothesis that prenatal cocaine exposure differentially regulates heart susceptibility to ischaemia–reperfusion (I/R) injury in adult offspring male and female rats. Pregnant rats were administered intraperitoneally either saline or cocaine (15 mg kg−1 ) twice daily from day 15 to day 21 of gestational age. There were no differences in maternal weight gain and birth weight between the two groups. Hearts were isolated from 2‐month‐old male and female offspring and were subjected to I/R (25 min/60 min) in a Langendorff preparation. Preischaemic values of left ventricular (LV) function were the same between the saline control and cocaine‐treated hearts for both male and female rats. Prenatal cocaine exposure significantly increased I/R‐induced myocardial apoptosis and infarct size, and significantly attenuated the postischaemic recovery of LV function in adult male offspring. In contrast, cocaine did not affect I/R‐induced injury and postischaemic recovery of LV function in the female hearts. There was a significant decrease in PKCɛ and phospho‐PKCɛ levels in LV in the male, but not female, offspring exposed to cocaine before birth. These results suggest that prenatal cocaine exposure causes a sex‐specific increase in heart susceptibility to I/R injury in adult male offspring, and the decreased PKCɛ gene expression in the male heart may play an important role.
Journal of Physiology - Tập 565 Số 1 - Trang 149-158 - 2005
Quantitative investigations of the adhesiveness of circulating polymorphonuclear leucocytes to blood vessel walls 1. A new simple method is described for quantitating the adhesiveness of circulating polymorphonuclear leucocytes, or granulocytes, to the walls of blood vessels. The cheek pouch of anaesthetized hamsters or a small part of the mesentery of anaesthetized mice were prepared for continuous microscopic observation of selected venules. Those granulocytes which moved sufficiently slowly to be individually visible were counted for 1 or 2 min periods as they rolled past a selected point on one side of a vessel. The velocity distribution of these cells was determined by analysing films. Films were used also to measure mean blood flow velocity in the venules by observing embolizing platelet thrombi induced by the iontophoretic application of adenosine diphosphate. Emigration of granulocytes into the tissues was quantitated by enumerating them in standard areas of stained histological sections. 2. In control experiments with hamster cheek pouch venules, the rolling granulocyte count usually passed through a maximum shortly after the preparation was set up and then fell to a low constant value. In mouse mesentery venules the count remained at a low approximately constant value from the beginning for at least 3 hr. 3. The mean velocity of blood flow in the venules was between 900 and 200 μ/sec. All rolling granulocytes moved much more slowly; in hamster cheek pouch venules the mean velocity was about 20 μ/sec and in mouse mesentery venules about 10 μ/sec. Around these means the velocity distribution of individual cells was narrow. 4. Rolling of granulocytes was abolished by superfusing ethylenediamine tetra‐acetate (EDTA, 0·1
M ) suggesting that the phenomenon depends on calcium or magnesium ions. 5. Agents were applied locally to the observed venules. Human serum albumin, trypsin or histamine in high concentrations did not affect the rolling granulocyte count. 6. The rolling granulocyte count was increased during the application of Hammarsten casein or Escherichia coli culture filtrate which are chemotactic to granulocytes in vitro . These agents did not cause alterations in mean blood flow velocity in the observed venules which might have accounted for the effect on the rolling granulocyte counts. When E. coli culture filtrate was perfused through mouse intestine the increase in rolling granulocyte count in the draining venous blood was proportional to the logarithm of the concentration of filtrate. 7. The rolling granulocyte count was also increased by the local application of plasma globulin permeability factor or lymph node permeability factor. 8. Granulocyte counts in standard histological sections showed no significant increases in control preparations but considerable increases following the application of Hammarsten casein.
Journal of Physiology - Tập 222 Số 2 - Trang 447-474 - 1972
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