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Masahiro Minami

Publications and source records attributed to Masahiro Minami.

4 recordsLinked to original sources
PubMed2006

Characterization of heme-regulated eIF2alpha kinase: roles of the N-terminal domain in the oligomeric state, heme binding, catalysis, and inhibition.

Heme-regulated eIF2alpha kinase [heme-regulated inhibitor (HRI)] plays a critical role in the regulation of protein synthesis by heme iron. The kinase active site is located in the C-terminal domain, whereas the N-terminal domain is suggested to regulate catalysis in response to heme binding. Here, we found that the rate of dissociation for Fe(III)-protoporphyrin IX was much higher for full-length HRI (1.5 x 10(-)(3) s(-)(1)) than for myoglobin (8.4 x 10(-)(7) s(-)(1)) or the alpha-subunit of hemoglobin (7.1 x 10(-)(6) s(-)(1)), demonstrating the heme-sensing character of HRI. Because the role of the N-terminal domain in the structure and catalysis of HRI has not been clear, we generated N-terminal truncated mutants of HRI and examined their oligomeric state, heme binding, axial ligands, substrate interactions, and inhibition by heme derivatives. Multiangle light scattering indicated that the full-length enzyme is a hexamer, whereas truncated mutants (truncations of residues 1-127 and 1-145) are mainly trimers. In addition, we found that one molecule of heme is bound to the full-length and truncated mutant proteins. Optical absorption and electron spin resonance spectra suggested that Cys and water/OH(-) are the heme axial ligands in the N-terminal domain-truncated mutant complex. We also found that HRI has a moderate affinity for heme, allowing it to sense the heme concentration in the cell. Study of the kinetics showed that the HRI kinase reaction follows classical Michaelis-Menten kinetics with respect to ATP but sigmoidal kinetics and positive cooperativity between subunits with respect to the protein substrate (eIF2alpha). Removal of the N-terminal domain decreased this cooperativity between subunits and affected the other kinetic parameters including inhibition by Fe(III)-protoporphyrin IX, Fe(II)-protoporphyrin IX, and protoporphyrin IX. Finally, we found that HRI is inhibited by bilirubin at physiological/pathological levels (IC(50) = 20 microM). The roles of the N-terminal domain and the binding of heme in the structural and functional properties of HRI are discussed.

Animals↗
PubMed2004

Effects of angiotensin II on the pericyte-containing microvasculature of the rat retina.

The aim of this study was to identify the mechanisms by which angiotensin II alters the physiology of the pericyte-containing microvasculature of the retina. Despite evidence that this vasoactive signal regulates capillary perfusion by inducing abluminal pericytes to contract and thereby microvascular lumens to constrict, little is known about the events linking angiotensin exposure with pericyte contraction. Here, using microvessels freshly isolated from the adult rat retina, we monitored pericyte currents via perforated-patch pipettes, measured pericyte calcium levels with fura-2 and visualized pericyte contractions and lumen constrictions by time-lapse photography. We found that angiotensin activates nonspecific cation (NSC) and calcium-activated chloride channels; the opening of these channels induces a depolarization that is sufficient to activate the voltage-dependent calcium channels (VDCCs) expressed in the retinal microvasculature. Associated with these changes in ion channel activity, intracellular calcium levels rise, pericytes contract and microvascular lumens narrow. Our experiments revealed that an influx of calcium through the NSC channels is an essential step linking the activation of AT(1) angiotensin receptors with pericyte contraction. Although not required in order for angiotensin to induce pericytes to contract, calcium entry via VDCCs serves to enhance the contractile response of these cells. In addition to activating nonspecific cation, calcium-activated chloride and voltage-dependent calcium channels, angiotensin II also causes the functional uncoupling of pericytes from their microvascular neighbours. This inhibition of gap junction-mediated intercellular communication suggests a previously unappreciated complexity in the spatiotemporal dynamics of the microvascular response to angiotensin II.

Alkaloids↗
PubMedDate not supplied

Electrotonic transmission within pericyte-containing retinal microvessels.

OBJECTIVE: Little is known about the electrotonic architecture of the pericyte-containing retinal microvasculature. Here, the authors focus on the cell-to-cell transmission of hyperpolarization, which can induce abluminal pericytes to relax and lumens to dilate. METHODS: With perforated-patch pipettes, the authors monitored the membrane potentials and ionic currents of pairs of pericytes located on freshly isolated rat retinal microvessels. Voltage changes were induced by administering electrical stimuli into pericytes, miniperfusing the KATP channel opener pinacidil, or using oxotremorine to activate chloride channels. RESULTS: Suggestive of extensive cell-to-cell communication, spontaneous voltage changes were strikingly similar in widely separated pericytes. In addition, injection of current into one of a pair of sampled pericytes always elicited a voltage response in the other sampled pericyte; the gap junction uncoupler, heptanol, blocked this transmission. In the dual recordings, hyperpolarization spreading from a current-injected pericyte decayed approximately 40% within 100 microm. In contrast, pinacidil-induced hyperpolarizations diminished by only approximately 2% in 100 microm. Depolarizations also appeared to spread with similar transmission efficacies. CONCLUSIONS: Based on the experiments, the authors propose that key features of the electrotonic architecture of retinal microvessels include highly efficient cell-to-cell communication within the endothelium and relatively inefficient transmission at pericyte/endothelial junctions. Thus, the endothelium is likely to provide an efficient pathway that functionally links contractile pericytes and thereby, serves to coordinate the vasomotor response of a retinal capillary.

Animals↗