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 γ-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.

Presence of GABA(B) receptors forming heterodimers with GABA(B1) and GABA(B2) subunits in human lower esophageal sphincter.

Baclofen, a GABA(B)-receptor (GABA(B)R) agonist has been proposed to be useful as therapeutic agent for the management of gastro-esophageal reflux disease, but whether the compound acts directly at the lower esophageal sphincter (LES) remains to be elucidated. We performed the present study to assess the presence of GABA(B)R in human LES. Western blot analysis showed that both proteins of GABA(B1(a))/GABA(B1(b)) and GABA(B2) subunits were present in the muscle layer of LES. Immunohistochemical findings showed that both GABA(B1)- and GABA(B2)-subunit proteins were located on the neurons within the myenteric plexus, and furthermore, both proteins were observed in the same neurons. Reverse transcriptase-polymerase chain reaction analysis also revealed the presence of mRNAs for both subunits of GABA(B)R and also mRNAs for 6 isoforms of GABA(B1) subunits, from GABA(B1(a)) to GABA(B1(g)), except GABA(B1(d)), in human LES. Thus, the functional GABA(B)R-forming heterodimers with subunits of GABA(B1) and GABA(B2) are located on the myenteric neurons in human LES, suggesting that GABA(B)R agonists and antagonists act at least, at the level of the peripheral nervous system.

mu-Opioid receptor forms a functional heterodimer with cannabinoid CB1 receptor: electrophysiological and FRET assay analysis.

Interactions between mu-opioid receptor (muOR) and cannabinoid CB1 receptor (CB1R) were examined by morphological and electrophysiological methods. In baby hamster kidney (BHK) cells coexpressing muOR fused to the yellow fluorescent protein Venus and CB1R fused to the cyan fluorescent protein Cerulean, both colors were detected on the cell surface; and fluorescence resonance energy transfer (FRET) analysis revealed that muOR and CB1R formed a heterodimer. Coimmunoprecipitation and Western blotting analyses also confirmed the heterodimers of muOR and CB1R. [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) or CP55,940 elicited K+ currents in Xenopus oocytes expressing muOR or CB1R together with G protein activated-inwardly rectifying K+ channels (GIRKs), respectively. In oocytes coexpressing both receptors, either of which was fused to the chimeric Galpha protein Gqi5 that activates the phospholipase C pathway, both DAMGO and CP55,940 elicited Ca2+-activated Cl(-) currents, indicating that each agonist can induce responses through Gqi5 fused to either its own receptor or the other. Experiments with endogenous Gi/o protein inactivation by pertussis toxin (PTX) supported the functional heterodimerization of muOR/CB1R through PTX-insensitive Gqi5(m) fused to each receptor. Thus, muOR and CB1R form a heterodimer and transmit a signal through a common G protein. Our electrophysiological method could be useful for determination of signals mediated through heterodimerized G protein-coupled receptors.

Desensitization of GABA(B) receptor signaling by formation of protein complexes of GABA(B2) subunit with GRK4 or GRK5.

We investigated the role of G protein coupled-receptor kinases (GRKs) in the desensitization of GABA(B) receptor-mediated signaling using Xenopus oocytes and baby hamster kidney (BHK) cells. Baclofen elicited inward K(+) currents in oocytes coexpressing heterodimeric GABA(B) receptor, GABA(B1a) subunit (GB(1a)R) and GABA(B2) subunit (GB(2)R), together with G protein-activated inwardly rectifying K(+) channels (GIRKs), in a concentration-dependent manner. Repetitive application of baclofen to oocytes coexpressing GABA(B)R 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 GABA(B) receptor-mediated desensitization processes. In BHK cells coexpressing GRK4 fused to Venus (brighter variant of yellow fluorescent protein, GRK4-Venus) with GB(1a)R and GB(2)R, GRK4-Venus was expressed in the cytosol but was translocated to the plasma membranes by GABA(B)R activation. In BHK cells coexpressing GRK4 fused to Cerulean (brighter variant of cyan fluorescent protein, GRK4-Cerulean) with GB(1a)R and GB(2)R-Venus, fluorescence resonance energy transfer (FRET) analysis demonstrated that GRK4-Cerulean formed a protein complex with GB(2)R-Venus. Immunoprecipitation and Western blot analysis confirmed GB(2)R-GRK4 complex formation. GRK5 also formed a complex with GB(2)R 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 GABA(B) receptor-mediated responses by forming protein complexes with GB(2)R subunit of GABA(B)R at the plasma membranes.

Coupling of GABAB receptor GABAB2 subunit to G proteins: evidence from Xenopus oocyte and baby hamster kidney cell expression system.

Coupling of functional GABAB receptors (GABABR) to G proteins was investigated with an expression system of baby hamster kidney (BHK) cells and Xenopus oocytes. Fluorescence resonance energy transfer (FRET) analysis of BHK cells coexpressing GABAB1a receptor (GB1aR) fused to Cerulean, a brighter variant of cyan fluorescent protein, and GABAB2 receptor (GB2R) fused to Venus, a brighter variant of yellow fluorescent protein, revealed that GB1aR-Cerulean and GB2R-Venus form a heterodimer. The GABABR agonists baclofen and 3-aminopropylphosphonic acid (3-APPA) elicited inward-rectifying K+ currents in a concentration-dependent manner in oocytes expressing GB1aR and GB2R, or GB1aR-Cerulean and GB2R-Venus, together with G protein-activated inward-rectifying K+ channels (GIRKs), but not in oocytes expressing GB1aR alone or GB2R alone together with GIRKs. Oocytes coexpressing GB1aR + Galphai2-fused GB2R (GB2R-Galphai2) caused faster K+ currents in response to baclofen. Furthermore, oocytes coexpressing GB1aR + GB2R fused to Galphaqi5 (a chimeric Galphaq protein that activates PLC pathways) caused PLC-mediated Ca2+-activated Cl- currents in response to baclofen. In contrast, these responses to baclofen were not observed in oocytes coexpressing GB1aR-Galphai2 or GB1aR-Galphaqi5 together with GB2R. BHK cells and Xenopus oocytes coexpressing GB1aR-Cerulean + a triplet tandem of GB2R-Venus-Galphaqi5 caused FRET and Ca2+-activated Cl- currents, respectively, with a similar potency in BHK cells coexpressing GB1aR-Cerulean + GB2R-Venus and in oocytes coexpressing GB1aR + GB2R-Galphaqi5. Our results indicate that functional GABABR forms a heterodimer composed of GB1R and GB2R and that the signal transducing G proteins are directly coupled to GB2R but not to GB1R.

Human middle cerebral artery flow velocity during controlled hypotension combined with hemodilution-transcranial Doppler study.

STUDY OBJECTIVE: To evaluate the effects of controlled hypotension combined with hemodilution on human middle cerebral artery flow velocity (Vmca) by transcranial Doppler ultrasonography. DESIGN: Randomized prospective study. SETTING: Inpatient surgery at Nagasaki Rosai Hospital. PATIENTS: Thirty American Society of Anesthesiologists physical status I and II patients scheduled for total hip arthroplasty. INTERVENTIONS: Anesthesia was maintained with nitrous oxide-oxygen (N(2)O-O(2)) and sevoflurane during normocapnia. Hemodilution was carried out after induction of anesthesia, in which blood was withdrawn then replaced with the same amount of hydroxyethyl starch to achieve a final hematocrit level of 32% (group A = mild hemodilution group, N = 15) or 24% (group B = moderate hemodilution group, N = 15). In both groups, controlled hypotension was induced with prostaglandin E(1) to maintain mean arterial pressure at approximately 55 mm Hg for 80 minutes. MEASUREMENTS AND MAIN RESULTS: Vmca and blood gas were measured before hemodilution, after hemodilution, 80 minutes after starting hypotension, and 60 minutes after recovery from hypotension. Vmca significantly increased in group A (+122%) and group B (+156%) after each hemodilution. In group B, Vmca was significantly greater than baseline values at 80 minutes after starting hypotension (+135%) and 60 minutes after recovery from hypotension (+140%). CONCLUSION: The combination of moderate hemodilution, such as hematocrit value of 24%, and prostaglandin E(1)-induced hypotension would not impair middle cerebral artery flow during sevoflurane-N(2)O-O(2) anesthesia during normocapnia.

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 x 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.

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

PURPOSE: 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 microg x kg(-1)) and propofol (1.5 mg x kg(-1)), and then muscle relaxation was obtained with vecuronium (0.15 mg x 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 x SBP) were less than 15000 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.

[The effects of colloid preload on hemodynamics and plasma concentration of atrial natriuretic peptide during spinal anesthesia in elderly patients].

We evaluated the effects of moderate colloid preloading on hemodynamics and plasma concentration of atrial natriuretic peptide (ANP) during spinal anesthesia in elderly patients undergoing low extremity surgery. Twenty patients (aged 66-90 yr) were randomly divided into two groups. Control group (n = 10) received no prehydration, and hydration group (n = 10) received colloid (6% hydroxyethyl starch; HES) preloading of 8 ml.kg-1 before spinal anesthesia. Systolic blood pressure decreased significantly 10 and 30 min after spinal anesthesia in either group, and there was no difference between the groups in the incidence of hypotension. The concentration of ANP decreased significantly by 23% in control group, whereas it increased significantly by 86% in hydration group, suggesting that cardiac preload might increase with volume expansion effect of prehydration with HES. In conclusion, colloid preloading with moderate volume might prevent the decrease in cardiac preload with increasing ANP, whereas it did not prevent spinal-induced hypotension in elderly patients.