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  • 1
    ISSN: 1432-2013
    Keywords: α1-Adrenoceptor ; Inositol 1,4,5-trisphosphate ; GTP-binding protein ; Ca2+ release ; Ca2+-dependent K+ conductance ; Mouse peritoneal macrophage
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In mouse peritoneal macrophages, α1-adrenoceptor stimulation evokes a Ca2+-dependent K+ current [I 0(Adr)] [Hara et al. (1991) Pflügers Arch 419:371–379]. The roles of D-myo-inositol 1,4,5-trisphosphate (InsP 3) and a GTP-binding protein (G protein) in I 0(Adr) were investigated with tight-seal whole-cell recordings and fura-2 fluorescence measurements. Intracellular injection of lnsP 3 (5–50 μM) evoked transient outward currents [I 0(InsP 3)] with or without damped oscillations in membrane currents at -40 mV. Dialysis with 0.2 mM guanosine 5′-[3-thio]triphosphate (GTP[γS], a poorly hydrolysable GTP analogue) at -40 mV activated oscillatory outward currents or a slowly developing steady current on which such oscillations were superimposed after a delay of 10–90 s. I 0(InsP 3) and the GTP[γS]-induced current {I 0(GTP[γS])} were accompanied by an increase in conductance. Reversal potentials of both responses closely depended on the extracellular K+ concentration. Fura-2 measurements revealed that I 0(InsP 3) and I 0(GTP[γS]) result from a rise in intracellular free Ca2+ concentration ([Ca2+]i). Removal of extracellular Ca2+ did not abolish I 0(InsP 3) and I 0(GTP[γS]). Both were blocked by bath-applied charybdotoxin. Intracellular D- myo-inositol 1,3,4,5-tetrakisphosphate (InsP 4, 50 μM) did not evoke any responses, whereas D-myo-inositol 2,4,5-trisphosphate [InsP 3(2,4,5), 20 μM] elicited an outward current at -40 mV. I0(InsP 3) was completely blocked by prior dialysis with the InsP 3 receptor antagonist heparin (5 mg/ml). Inclusion of guanosine 5′-[2-thio] diphosphate (GDP[βS], 2 mM) or heparin (5 mg/ml) together with GTP[γS] in the patch pipette solution completely blocked I 0(GTP[γS]). These results indicate that intracellular injection of InsP 3 or GTP[γS] mimic I 0(Adr). Furthermore, intracellular dialysis with heparin (3 mg/ ml) or GDP[βS] (2 mM) greatly accelerated a run-down of I 0(Adr). On the other hand, I 0(Adr) was markedly prolonged in a cell dialysed with GTP[γS] (0.2 mM). Therefore, it is concluded that I 0(Adr) results from stimulation of α1-adrenoceptor and InsP 3 formation via a G protein.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Pflügers Archiv 419 (1991), S. 371-379 
    ISSN: 1432-2013
    Keywords: Adrenaline ; α 1-Adrenoceptor ; Ca2+ release ; Ca2+-dependent K+ conductance ; Patch clamp ; Mouse peritoneal macrophage
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Responses to adrenaline in mouse peritoneal macrophages were investigated with perforated and cell-attached patch-clamp recording, and with a combination of the perforated-patch recording and fura-2 fluorescence measurements. Extracellularly applied adrenaline induced a transient outward current (4–10s in duration, 100–500 pA in amplitude) at −40 mV associated with a marked increase in conductance. The adrenaline-induced current [I o (Adr)] reversed polarity near −80 mV. The reversal potential depended distinctly on the external K+ concentration but not on external Cl− concentration. Removal of external Ca2+ did not affect I o(Adr) within 2–4 min but subsequent responses to adrenaline were progressively depressed. In contrast, treatment with an intracellular Ca2+ chelator, the acetoxymethyl ester of 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid completely abolished I o(Adr). Furthermore, I o(Adr) was blocked by bath-applied quinidine and charybdotoxin, but not by tetraethylammonium or apamin. Extracellular application of an α 1-adrenoceptor agonist phenylephrine and of noradrenaline mimicked I o(Adr). On the other hand, I o(Adr) was antagonized by a non-selective α-adrenoceptor antagonist phentolamine (0.2 μM) and an α 1-adrenoceptor antagonist prazosin (0.2 μM), but was not affected by an α2-adrenoceptor antagonist yohimbine (1 μM) or a β-adrenoceptor antagonist propranolol (1 μM). Cell-attached single-channel recordings with the pipette solution containing 145 mM KCl revealed the activation of single-channel currents with a conductance of 40 pS during application of adrenaline outside the patch. Parallel measurements of membrane current and fura-2 fluorescence in the same cell demonstrated a correlation between the rise in [Ca2+]i and an increase in K+ conductance. Therefore, it is concluded that adrenaline activates a Ca2+-dependent K+ conductance by release of Ca2+ from internal stores through an activation of an α 1-adrenoceptor.
    Type of Medium: Electronic Resource
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