Masato Kanaide

Desensitization of GABAB receptor signaling by formation of protein complexes of GABAB2 subunit with GRK4 or GRK5

We investigated the role of G protein coupled‐receptor kinases (GRKs) in the desensitization of GABAB receptor‐mediated signaling using Xenopus oocytes and baby hamster kidney (BHK) cells. Baclofen elicited inward K+ currents in oocytes coexpressing heterodimeric GABAB receptor, GABAB1a subunit (GB1aR) and GABAB2 subunit (GB2R), together with G protein‐activated inwardly rectifying K+ channels (GIRKs), in a concentration‐dependent manner. Repetitive application of baclofen to oocytes coexpressing GABABR and GIRKs did not change peak K+ currents in the first and second responses, but the latter responses were significantly attenuated by coexpression of either GRK4 or GRK5 with attenuation efficacy of GRK4 > GRK5. Coexpression of other GRKs including GRK2, GRK3, and GRK6 had no effect on GABAB receptor‐mediated desensitization processes. In BHK cells coexpressing GRK4 fused to Venus (brighter variant of yellow fluorescent protein, GRK4‐Venus) with GB1aR and GB2R, GRK4‐Venus was expressed in the cytosol but was translocated to the plasma membranes by GABABR activation. In BHK cells coexpressing GRK4 fused to Cerulean (brighter variant of cyan fluorescent protein, GRK4‐Cerulean) with GB1aR and GB2R‐Venus, fluorescence resonance energy transfer (FRET) analysis demonstrated that GRK4‐Cerulean formed a protein complex with GB2R‐Venus. Immunoprecipitation and Western blot analysis confirmed GB2R‐GRK4 complex formation. GRK5 also formed a complex with GB2R on the plasma membranes as determined by FRET and Western blotting but not GRK2, GRK3, and GRK6. Our results indicate that GRK4 and GRK5 desensitize GABAB receptor‐mediated responses by forming protein complexes with GB2R subunit of GABABR at the plasma membranes. J. Cell. Physiol. 210: 237–245, 2007.

Hemodynamic and catecholamine responses during tracheal intubation using a lightwand device (Trachlight) in elderly patients with hypertension

AbstractPurpose. Tracheal intubation using a lightwand device (Trachlight) should minimize hemodynamic change by avoiding direct-vision laryngoscopy. We evaluated hemodynamic and catecholamine responses during tracheal intubation using a Trachlight in elderly patients with hypertension. Methods. Twenty-six hypertensive patients aged over 65 years undergoing orthopedic surgery were randomly divided into two groups, group L (n = 13) and group T (n = 13). Anesthesia was induced with fentanyl (2 Μg · kg−1) and propofol (1.5 mg · kg−1), and then muscle relaxation was obtained with vecuronium (0.15 mg · kg−1). The trachea was intubated with either a Macintosh laryngoscope (group L) or a Trachlight (group T). Hemodynamics, plasma catecholamine concentrations, and arterial blood gases were measured before the induction of anesthesia (T0), before tracheal intubation (T1), immediately after tracheal intubation (T2), and 3 min after tracheal intubation (T3). Results. The intubation time was shorter in group T than in group L (12.6 ± 1.7 vs 23.5 ± 2.9 s, mean ± SE; P ≪ 0.01). Compared with the preinduction (T0) value, systolic blood pressure (SBP) showed a significant decrease at T1 and T3 in group L and at T1, T2, and T3 in group T. The heart rate (HR) and plasma norepinephrine (NE) concentration showed no change in either group throughout the time course, whereas the plasma epinephrine (E) concentration showed a significant decrease at T2 and T3 in both groups. The mean values of the rate-pressure product (RPP: HR × SBP) were less than 15 000 after tracheal intubation in both groups. There was no significant difference in hemodynamic or catecholamine responses between groups at any point. No patient had ischemic ST-T changes in either group. Conclusion. A lightwand has no advantage over a laryngoscope in terms of hemodynamic and plasma catecholamine responses to tracheal intubation in elderly patients with hypertension, despite a shorter intubation time.

S(+)-ketamine suppresses desensitization of γ-aminobutyric acid type B receptor-mediated signaling by inhibition of the interaction of γ-aminobutyric acid type B receptors with G protein-coupled receptor kinase 4 or 5.

Background:Intrathecal baclofen therapy is an established treatment for severe spasticity. However, long-term management occasionally results in the development of tolerance. One of the mechanisms of tolerance is desensitization of &ggr;-aminobutyric acid type B receptor (GABABR) because of the complex formation of the GABAB2 subunit (GB2R) and G protein–coupled receptor kinase (GRK) 4 or 5. The current study focused on S(+)-ketamine, which reduces the development of morphine tolerance. This study was designed to investigate whether S(+)-ketamine affects the GABABR desensitization processes by baclofen. Methods:The G protein–activated inwardly rectifying K+ channel currents induced by baclofen were recorded using Xenopus oocytes coexpressing G protein–activated inwardly rectifying K+ channel 1/2, GABAB1a receptor subunit, GB2R, and GRK. Translocation of GRKs 4 and 5 and protein complex formation of GB2R with GRKs were analyzed by confocal microscopy and fluorescence resonance energy transfer analysis in baby hamster kidney cells coexpressing GABAB1a receptor subunit, fluorescent protein–tagged GB2R, and GRKs. The formation of protein complexes of GB2R with GRKs was also determined by coimmunoprecipitation and Western blot analysis. Results:Desensitization of GABABR-mediated signaling was suppressed by S(+)-ketamine in a concentration-dependent manner in the electrophysiologic assay. Confocal microscopy revealed that S(+)-ketamine inhibited translocation of GRKs 4 and 5 to the plasma membranes and protein complex formation of GB2R with the GRKs. Western blot analysis also showed that S(+)-ketamine inhibited the protein complex formation of GB2R with the GRKs. Conclusion:S(+)-Ketamine suppressed the desensitization of GABABR-mediated signaling at least in part through inhibition of formation of protein complexes of GB2R with GRK 4 or 5.

Preadministration of low-dose ketamine reduces tourniquet pain in healthy volunteers

We evaluated whether preadministration of low-dose ketamine could attenuate tourniquet pain and arterial pressure increase using high tourniquet pressure in ten healthy awake volunteers. Ketamine, 0.1 mg·kg−1, or normal saline was given intravenously in a double-blind fashion before tourniquet inflation with a pressure of 400 mmHg at the thigh. Visual analog scale (VAS) scores and systolic blood pressure (SBP) were measured at 5-min intervals. Ketamine significantly reduced VAS scores compared to saline just after tourniquet inflation [90 (64–100) mm, median (range), with saline versus 66 (50–81) mm with ketamine, P < 0.01] and at 30 min [92 (61–100) mm with saline versus 70 (50–100) mm with ketamine, P < 0.03), and significantly prolonged tourniquet time (28 ± 6 min with saline, mean ± SD, versus 37 ± 7 min with ketamine, P < 0.01). SBP (120 ± 9 mmHg) significantly increased before tourniquet deflation (133 ± 16 mmHg) in the saline trial, but not in the ketamine trial. The results show that preadministration of low-dose ketamine attenuates tourniquet pain and arterial pressure increase during high-pressure tourniquet application and prolongs tourniquet time in healthy volunteers.