Toshikazu Yamashita

A region of the sulfonylurea receptor critical for a modulation of ATP-sensitive K+ channels by G-protein βγ-subunits

To determine the interaction site(s) of ATP‐sensitive K+ (KATP) channels for G‐proteins, sulfonylurea receptor (SUR2A or SUR1) and pore‐forming (Kir6.2) subunits were reconstituted in the mammalian cell line, COS‐7. Intracellular application of the G‐protein βγ2‐subunits (Gβγ2) caused a reduction of ATP‐induced inhibition of Kir6.2/SUR channel activities by lessening the ATP sensitivity of the channels. Gβγ2 bound in vitro to both intracellular (loop‐NBD) and C‐terminal segments of SUR2A, each containing a nucleotide‐binding domain (NBD). Furthermore, a single amino acid substitution in the loop‐NBD of SUR (Arg656Ala in SUR2A or Arg665Ala in SUR1) abolished the Gβγ2‐dependent alteration of the channel activities. These findings provide evidence that Gβγ modulates KATP channels through a direct interaction with the loop‐NBD of SUR.

P2Y-mediated Ca2+ response is spatiotemporally graded and synchronized in sensory neurons: a two-photon photolysis study

ATP is thought to be an initiator and modulator of noxious pain sensation. We employed two‐photon photolysis to apply ATP locally and transiently, thus mimicking ATP release upon cell damage or exocytosis. Using this technique, an increase in intracellular Ca2+ concentration ([Ca2+]i) was induced via P2Y receptors in individual sensory neurons, or in a neurite region. The ATP‐induced [Ca2+]i rise was attenuated by applications of either a phospholipase C inhibitor, or inhibitors for IP3 or ryanodine receptors. These results indicate that intracellular Ca2+ stores play a major role in contributing to the increase in [Ca2+]i. Spatiotemporal analysis revealed that local and transient applications of ATP increased [Ca2+]i by release from intracellular stores, but in a unique, graded, and synchronized manner. 1) As the duration of local ATP application was prolonged, the latency decreased and the magnitude of the [Ca2+]i rise increased; 2) The time course of the rising phase of the [Ca2+]i response to ATP was essentially the same over the cell body, once [Ca2+]i had started to rise. It is anticipated that sensory responses may be modulated variably, depending on the spatiotemporal characteristics of the ATP‐related [Ca2+]i profile.

The signalling pathway which causes contraction via P2-purinoceptors in rat urinary bladder smooth muscle

1 The signalling pathway which causes contractions to adenosine 5′‐O‐2‐thiodiphosphate (ADPβS) and α,β‐methylene adenosine 5′‐diphosphate (α,β‐Me ADP) was investigated in rat urinary bladder smooth muscle by measuring isotonic tension. 2 The responses to 10 μM α,β‐methylene adenosine 5′‐triphosphate (α,β‐Me ATP) in 0 and 3.6 mM Ca2+ were 5.9±1.3 (n=10) and 122.2±6.4 (n=8) % respectively of those obtained in 1.8 mM Ca2+, whereas those to 100 μM ADPβS were 34.6±3.3 (n=8) and 96.8±7.2 (n=8) %, in 0 and 3.6 mM Ca2+, respectively. In both experimental conditions, the responses to the two agonists expressed as % of the control responses were significantly different (P<0.01). 3 Indomethacin at high concentrations (>1 μM) decreased the responses to α,β‐Me ATP (10 μM), ADPβS (100 μM) and α,β‐Me ADP (100 μM). However, no significant difference was obtained between the responses to all the agonists at 30 μM indomethacin. 4 2‐Nitro‐4‐carboxphenyl n,n‐diphenylcarbamate (NCDC) at concentrations between 1 μM and 100 μM concentration‐dependently decreased the responses to ADPβS (100 μM) and α,β‐Me ADP (100 μM) and almost completely inhibited them at 100 μM. Although the responses to α,β‐Me ATP (10 μM) were also inhibited by the drug, at 50 and 100 μM NCDC the responses to α,β‐Me ATP were significantly larger than those to ADPβS and α,β‐Me ADP (P<0.01). 5 NCDC 100 μM significantly inhibited the KCl‐induced contraction to 65.9±4.9% (n=6) of the control (P<0.01). 6 It is suggested that the contraction via ADPβS‐sensitive receptors in the rat urinary bladder smooth muscle mainly depends on Ca2+ ions liberated from intracellular Ca2+ stores, though the contribution of Ca2+ ions from the extracellular space cannot be neglected. The release of Ca2+ ions from stores is mainly mediated by the production of inositol trisphosphate (IP3) via the activation of phospholipase C.

Effects of purinoceptor agonists on cytosolic Ca2+ concentration in swine tracheal smooth muscle cells in culture

1 The effects of various purinoceptor agonists on intracellular Ca2+ concentration ([Ca2+]i) in swine tracheal smooth muscle cells in primary culture were examined to investigate the subtype of purinoceptors in these cells. 2 ATP (1 μm to 1 mM) concentration‐dependently increased [Ca2+]i which was measured by monitoring the fluorescence signal of fura2. 3 α, β‐Me ATP at concentrations higher than 10 μm increased [Ca2+]i in the presence of extracellular Ca2+. Responses to the drug were 12 ± 5 and 61 ± 4% of responses to ATP (100 μm) at 100 μm and 1 mM, respectively (n = 7). The response to 100 μm ATP was inhibited by 62% in the presence of 1 mM α, β‐Me ATP (n = 8), though the drug at concentrations lower than that did not affect the response to ATP. 4 ATP increased [Ca2+]i in the absence of extracellular Ca2+. The response to ATP in this condition was 40% of that in the presence of extracellular Ca2+ (n = 8). 5 Neither cibacron blue 3GA (10 μm) (n = 8) nor suramin (10 and 100 μm) (n = 10) affected the response to ATP (1 μm to 100 μm). 6 The rank order of potency in the absence of extracellular Ca2+ was UTP> ATP> adenosine 5′‐o‐(3‐thiotriphosphate)>> ADP = α, β‐methylene adenosine 5′‐triphosphate > 2‐(methylthio)‐adenosine 5′‐(tetrahydrogen triphosphate). 7 UTP (1 μm to 100 μm) concentration‐dependently increased inositol 1,4,5‐triphosphate (IP3) production. 8 These results suggest that the increase in [Ca2+]i induced by purinoceptor agonists is mediated mainly via a nucleotide receptor in swine tracheal smooth muscle cells in primary culture.

Constitutive Synthesis of Physiological Networks

The relationship between the topology and collective function of a nonlinear oscillator network was investigated using nonlinear electrochemical oscillators. The constitutive experiments showed that the physiological robustness in the living system is due to their topological redundancy and asymmetry in the nonlinear network. key words: nonlinear oscillator, complex network, physiological functions, robustness, redundancy

Constitutive chemical biology: reconstruction of intercellular Ca2+ transience by nonlinear oscillators

Networks of non-linear oscillators have a potential to simulate physiological functions of living system. Expected similarity in spatiotemporal behavior was obtained in between the inter-cellular concentration pulse in Ca2+ triggered by f-sec laser irradiation and the current pulse propagation along excitable non-linear electrode pairs triggered by electric stimulus. These resemblances are owing to the same dynamical rules governing both biological and electrochemical systems.