Oxygen-dependent K+ influxes in Mg2+-clamped equine red blood cells.
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1. Cl--dependent K+ (86Rb+) influxes were measured in oxygenated and deoxygenated equine red blood cells, whose free [Mg2+]i had been clamped, to examine the effect on O2 dependency of the K+-Cl- cotransporter. 2. Total [Mg2+]i was 2.55 +/- 0.07 mM (mean +/- s.e.m. , n = 6). Free [Mg2+]i was estimated at 0.45 +/- 0.04 and 0.68 +/- 0. 03 mM (mean +/- s.e.m., n = 4) in oxygenated and deoxygenated red cells, respectively. 3. K+-Cl- cotransport was minimal in deoxygenated cells but substantial in oxygenated ones. Cl--dependent K+ influx, inhibited by calyculin A, consistent with mediation via the K+-Cl- cotransporter, was revealed by depleting deoxygenated cells of Mg2+. 4. Decreasing [Mg2+]i stimulated K+ influx, and increasing [Mg2+]i inhibited it, in both oxygenated and deoxygenated red cells. When free [Mg2+]i was clamped, Cl--dependent K+ influxes were always greater in oxygenated cells than in deoxygenated ones, and changes in free [Mg2+]i of the magnitude occurring during oxygenation-deoxygenation cycles had a minimal effect. Physiological fluctuations in free [Mg2+]i are unlikely to provide the primary link coupling activity of the K+-Cl- cotransporter with O2 tension. 5. Volume and H+ ion sensitivity of K+ influx in Mg2+-clamped red cells were increased in O2 compared with those in deoxygenated cells at the same free [Mg2+]i, by about 6- and 2-fold, respectively, but again these features were not responsible for the higher fluxes in oxygenated cells. 6. Regulation of the K+-Cl- cotransporter by O2 is very similar in equine, sheep and in normal human (HbA) red cells, but altered in human sickle cells. Present results imply that, as in sheep red cells, O2 dependence of K+-Cl- cotransport in equine red cells is not mediated via changes in free [Mg2+]i and that cotransport in Mg2+-clamped red cells is still stimulated by O2. This behaviour is contrary to that reported for human sickle (HbS) cells. (+info)
Volume regulation following hypotonic shock in isolated crypts of mouse distal colon.
(10/7624)
1. A video-imaging technique of morphometry was used to measure the diameter as an index of cell volume in intact mouse distal colon crypts submitted to hypotonic shock. 2. Transition from isotonic (310 mosmol l-1) to hypotonic (240 mosmol l-1) saline caused a pronounced increase in crypt diameter immediately followed by regulatory volume decrease (RVD). 3. Exposure of crypts to Cl--free hyposmotic medium increased the rapidity of both cell swelling and RVD. Exposure of crypts to Na+-free hyposmotic medium reduced the total duration of swelling. Return to initial diameter was followed by further shrinkage of the crypt cells. 4. The chloride channel inhibitor NPPB (50 microM) delayed the swelling phase and prevented the subsequent normal decrease in diameter. 5. The K+ channel blockers barium (10 mM), charybdotoxin (10 nM) and TEA (5 mM) inhibited RVD by 51, 44 and 32 %, respectively. 6. Intracellular [Ca2+] rose from a baseline of 174 +/- 17 nM (n = 8) to 448 +/- 45 nM (n = 8) during the initial swelling phase 7. The Ca2+ channel blockers verapamil (50 microM) and nifedipine (10 microM), the chelator of intracellular Ca2+ BAPTA AM (30 microM), or the inhibitor of Ca2+ release TMB-8 (10 microM), dramatically reduced volume recovery, leading to 51 % (n = 9), 25 % (n = 7), 37 % (n = 6), 32 % (n = 8) inhibition of RVD, respectively. TFP (50 microM), an antagonist of the Ca2+-calmodulin complex, significantly slowed RVD. The Ca2+ ionophore A23187 (2 microM) provoked a dramatic reduction of the duration and amplitude of cell swelling followed by extensive shrinkage. The release of Ca2+ from intracellular stores using bradykinin (1 microM) or blockade of reabsorption with thapsigargin (1 microM) decreased the duration of RVD. 8. Prostaglandin E2 (PGE2, 5 microM) slightly delayed RVD, whereas leukotriene D4 (LTD4, 100 nM) and arachidonic acid (10 microM) reduced the duration of RVD. Blockade of phospholipase A2 by quinacrine (10 microM) inhibited RVD by 53 %. Common inhibition of PGE2 and LTD4 synthesis by ETYA (50 microM) or separate blockade of PGE2 synthesis by 1 microM indomethacin reduced the duration of RVD. Blockade of LTD4 synthesis by nordihydroguaiaretic acid (NDGA) did not produce any significant effect on cell swelling or subsequent RVD. 9. Staurosporine (1 microM), an inhibitor of protein kinases, inhibited RVD by 58 %. Taken together the experiments demonstrate that the RVD process is under the control of conductive pathways, extra- and intracellular Ca2+ ions, protein kinases, prostaglandins and leukotrienes. (+info)
Modulation of chloride, potassium and bicarbonate transport by muscarinic receptors in a human adenocarcinoma cell line.
(11/7624)
1. Short-circuit current (I(SC)) responses to carbachol (CCh) were investigated in Colony 1 epithelia, a subpopulation of the HCA-7 adenocarcinoma cell line. In Krebs-Henseleit (KH) buffer, CCh responses consisted of three I(SC) components: an unusual rapid decrease (the 10 s spike) followed by an upward spike at 30 s and a slower transient increase (the 2 min peak). This response was not potentiated by forskolin; rather, CCh inhibited cyclic AMP-stimulated I(SC). 2. In HCO3- free buffer, the decrease in forskolin-elevated I(SC) after CCh was reduced, although the interactions between CCh and forskolin remained at best additive rather than synergistic. When Cl- anions were replaced by gluconate, both Ca2+- and cyclic AMP-mediated electrogenic responses were significantly inhibited. 3. Basolateral Ba2+ (1-10 mM) and 293B (10 microM) selectively inhibited forskolin stimulation of I(SC), without altering the effects of CCh. Under Ba2+- or 293B-treated conditions, CCh responses were potentiated by pretreatment with forskolin. 4. Basolateral charybdotoxin (50 nM) significantly increased the size of the 10 s spike of CCh responses in both KH and HCO3- free medium, without affecting the 2 min peak. The enhanced 10 s spike was inhibited by prior addition of 5 mM apical Ba2+. Charybdotoxin did not affect forskolin responses. 5. In epithelial layers prestimulated with forskolin, the muscarinic antagonists atropine and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, both at 100 nM) abolished subsequent 10 microM CCh responses. Following addition of p-fluoro hexahydro-sila-difenidol (pF-HHSiD, 10 microM) or pirenzepine (1 microM), qualitative changes in the CCh response time-profile also indicated a rightward shift of the agonist concentration-response curve; however, 1 microM gallamine had no effect. These results suggest that a single M3-like receptor subtype mediates the secretory response to CCh. 6. It is concluded that CCh and forskolin activate discrete populations of basolateral K+ channels gated by either Ca2+ or cyclic AMP, but that the Cl- permeability of the apical membrane may limit their combined effects on electrogenic Cl- secretion. In addition, CCh activates a Ba2+-sensitive apical K+ conductance leading to electrogenic K+ transport. Both agents may also modulate HCO3- secretion through a mechanism at least partially dependent on carbonic anhydrase. (+info)
Formal analysis of electrogenic sodium, potassium, chloride and bicarbonate transport in mouse colon epithelium.
(12/7624)
1. The mammalian colonic epithelium carries out a number of different transporting activities simultaneously, of which more than one is increased following activation with a single agonist. These separate activities can be quantified by solving a set of equations describing these activities, provided some of the dependent variables can be eliminated. Using variations in the experimental conditions, blocking drugs and comparing wild type tissues with those from transgenic animals this has been achieved for electrogenic ion transporting activity of the mouse colon. 2. Basal activity and that following activation with forskolin was measured by short circuit current in isolated mouse colonic epithelia from normal and cystic fibrosis (CF) mice. 3. Using amiloride it is shown that CF colons show increased electrogenic sodium absorption compared to wild type tissues. CF mice had elevated plasma aldosterone, which may be responsible for part or all of the increased sodium absorbtion in CF colons. 4. The derived values for electrogenic chloride secretion and for electrogenic potassium secretion were increased by 13 and 3 fold respectively by forskolin, compared to basal state values for these processes. 5. The loop diuretic, frusemide, completely inhibited electrogenic potassium secretion, but apparently only partially inhibited electrogenic chloride secretion. However, use of bicarbonate-free solutions and acetazolamide reduced the frusemide-resistant current, suggesting that electrogenic bicarbonate secretion accounts for the frusemide-resistant current. 6. It is argued that the use of tissues from transgenic animals is an important adjunct to pharmacological analysis, especially where effects in tissues result in the activation of more than one sort of response. (+info)
Binding of the transition state analog MgADP-fluoroaluminate to F1-ATPase.
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Escherichia coli F1-ATPase from mutant betaY331W was potently inhibited by fluoroaluminate plus MgADP but not by MgADP alone. beta-Trp-331 fluorescence was used to measure MgADP binding to catalytic sites. Fluoroaluminate induced a very large increase in MgADP binding affinity at catalytic site one, a smaller increase at site two, and no effect at site three. Mutation of either of the critical catalytic site residues beta-Lys-155 or beta-Glu-181 to Gln abolished the effects of fluoroaluminate on MgADP binding. The results indicate that the MgADP-fluoroaluminate complex is a transition state analog and independently demonstrate that residues beta-Lys-155 and (particularly) beta-Glu-181 are important for generation and stabilization of the catalytic transition state. Dicyclohexylcarbodiimide-inhibited enzyme, with 1% residual steady-state ATPase, showed normal transition state formation as judged by fluoroaluminate-induced MgADP binding affinity changes, consistent with a proposed mechanism by which dicyclohexylcarbodiimide prevents a conformational interaction between catalytic sites but does not affect the catalytic step per se. The fluorescence technique should prove valuable for future transition state studies of F1-ATPase. (+info)
Regulation of an amiloride-sensitive Na+-permeable channel by a beta2-adrenergic agonist, cytosolic Ca2+ and Cl- in fetal rat alveolar epithelium.
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1. In cell-attached patches formed on the apical membrane of fetal alveolar epithelium, terbutaline (a specific beta2-adrenergic agonist) increased the open probability (Po) of an amiloride-sensitive Na+-permeable non-selective cation (NSC) channel (control, 0.03 +/- 0.04; terbutaline, 0.62 +/- 0.18; n = 8, P < 0. 00001) by increasing the mean open time 100-fold without any significant change in the mean closed time and without any change in the single channel conductance (control, 27.8 +/- 2.3 pS; terbutaline, 28.2 +/- 2.1 pS; n = 8). 2. The Po of the unstimulated channel increased when the apical membrane was depolarized due to a decrease in the closing rate and an increase in the opening rate, while the Po of the terbutaline-stimulated channel did not depend on the membrane potential. 3. Increased cytosolic [Ca2+] also increased the Po of the channel in a manner consistent with one Ca2+-binding site on the cytosolic surface of the channel. Terbutaline increased the sensitivity of the channel to cytosolic Ca2+ by shifting the concentration of cytosolic Ca2+ ([Ca2+]c) required for half-maximal activation to a lower [Ca2+]c value, leading to an increase in Po. 4. An increase in the cytosolic Cl- concentration ([Cl-]c) decreased the Po of the channel consistent with two Cl--binding sites by increasing the closing rate without any significant change in the opening rate. Terbutaline increased Po by reducing the effect of cytosolic Cl- to promote channel closing. 5. Taken together, these observations indicate that terbutaline activates a Ca2+-activated, Cl--inhibitable, amiloride-sensitive, Na+-permeable NSC channel in fetal rat alveolar epithelium in two ways: first, through an increase in Ca2+ sensitivity, and second, through a reduction in the effect of cytosolic Cl- to promote channel closing. (+info)
Properties and functional roles of hyperpolarization-gated currents in guinea-pig retinal rods.
(15/7624)
1. The inward rectification induced by membrane hyperpolarization was studied in adult guinea-pig rods by the perforated-patch-clamp technique. 2. CsCl blocked the rectification observed in both voltage- and current-clamp recordings at voltages negative to -60 mV, while BaCl2 blocked the inward relaxation observed at voltages positive to -60 mV. The current activated at -90 mV had a low selectivity between sodium and potassium and reversed at -31.0 mV. 3. These observations suggest that two inward rectifiers are present in guinea-pig rods: a hyperpolarization-activated (Ih) and a hyperpolarization-deactivated (Ikx) current. The functional roles of Ih and Ikx were evaluated by stimulating rods with currents sinusoidally modulated in time. 4. Rods behave like bandpass amplifiers, with a peak amplification of 1.5 at about 2 Hz. For hyperpolarizations that mainly gate Ikx, amplification and phase shifts are fully accounted for by a rod membrane analogue model that includes an inductance. For hyperpolarizations that also gate Ih, a harmonic distortion became apparent. 5. Bandpass filtering and amplification of rod signals, associated with Ih and Ikx gating by membrane hyperpolarization, are strategically located to extend, beyond the limits imposed by the slow phototransductive cascade, the temporal resolution of signals spreading to the rod synapse. (+info)
Sympathetic neuroeffector transmission in the rat anococcygeus muscle.
(16/7624)
1. When intracellular recordings were made from preparations of rat anococcygeus muscle, transmural nerve stimulation evoked noradrenergic excitatory junction potentials (EJPs) made up of two distinct components. Both components were abolished by either guanethidine or alpha-adrenoceptor antagonists, indicating that they resulted from the release of transmitter from sympathetic nerves and the subsequent activation of alpha-adrenoceptors. 2. The first component was associated with a transient increase in the intracellular concentration of calcium ions ([Ca2+]i) and a contraction. Although the second component was often associated with a long lasting increase in [Ca2+]i it was not associated with a contraction unless the second component initiated an action potential. 3. The increase in [Ca2+]i associated with the first component resulted from Ca2+ release from an intracellular store and from entry of Ca2+ through voltage-dependent Ca2+ channels. The increase in [Ca2+]i associated with the second component resulted only from the entry of Ca2+ through L-type Ca2+ channels (CaL channels). The depolarization associated with the initial increase in [Ca2+]i was abolished by reducing the external concentration of chloride ions ([Cl-]o), suggesting that it involved the activation of a Cl- conductance. 4. When the relationships between changes in [Ca2+]i, membrane depolarization and contraction produced by an increasing number of sympathetic nerve stimuli were determined in control, and caffeine- and nifedipine-containing solutions, it was found that an increase in [Ca2+]i recorded in nifedipine produced a larger contraction and larger membrane depolarization than did a similar increase in [Ca2+]i recorded in either control or caffeine-containing solutions. These observations indicate that Ca2+ released from stores more readily triggers contraction and membrane depolarization than does Ca2+ entry via CaL channels. (+info)