Hirobumi Okawa

Characterization of [Nphe1]nociceptin(1‐13)NH2, a new selective nociceptin receptor antagonist

Nociceptin (orphanin FQ) is a novel neuropeptide capable of inducing a variety of biological actions via activation of a specific G‐protein coupled receptor. However, the lack of a selective nociceptin receptor antagonist has hampered our understanding of nociceptin actions and the role of this peptide in pathophysiological states. As part of a broader programme of research, geared to the identification and characterization of nociceptin receptor ligands, we report that the novel peptide [Nphe1]nociceptin(1‐13)NH2 acts as the first truly selective and competitive nociceptin receptor antagonist and is devoid of any residual agonist activity. [Nphe1]nociceptin(1‐13)NH2 binds selectively to recombinant nociceptin receptors expressed in Chinese hamster ovary (CHO) cells (pKi 8.4) and competitively antagonizes the inhibitory effects of nociceptin (i) on cyclic AMP accumulation in CHO cells (pA2 6.0) and (ii) on electrically evoked contractions in isolated tissues of the mouse, rat and guinea‐pig with pA2 values ranging from 6.0 to 6.4. [Nphe1]nociceptin(1‐13)NH2 is also active in vivo, where it prevents the pronociceptive and antimorphine actions of intracerebroventricularly applied nociceptin, measured in the mouse tail withdrawal assay. Moreover, [Nphe1]nociceptin(1‐13)NH2 produces per se a dose dependent, naloxone resistant antinociceptive action and, at relatively low doses, potentiates morphine‐induced analgesia. Collectively our data indicate that [Nphe1]nociceptin(1‐13)NH2, acting as a nociceptin receptor antagonist, may be the prototype of a new class of analgesics.

Comparison of the effects of [Phe1Ψ(CH2‐NH)Gly2]Nociceptin (1–13)NH2 in rat brain, rat vas deferens and CHO cells expressing recombinant human nociceptin receptors

Nociceptin(NC) is the endogenous ligand for the opioid receptor like‐1 receptor (NC‐receptor). [Phe1ΨC(CH2‐NH)Gly2]Nociceptin(1–13)NH2 ([F/G]NC(1–13)NH2) has been reported to antagonize NC actions in peripheral guinea‐pig and mouse tissues. In this study, we investigated the effects of a range of NC C‐terminal truncated fragments and [F/G]NC(1–13)NH2 on NC receptor binding, glutamate release from rat cerebrocortical slices (rCX), inhibition of cyclic AMP accumulation in CHO cells expressing the NC receptor (CHONCR) and electrically evoked contractions of the rat vas deferens (rVD). In radioligand binding assays, a range of ligands inhibited [125I]‐Tyr14‐NC binding in membranes from rCX and CHONCR cells. As the peptide was truncated there was a general decline in pKi. [F/G]NC(1–13)NH2 was as potent as NC(1–13)NH2. The order of potency for NC fragments to inhibit cyclic AMP accumulation in whole CHONCR cells was NCNH2NC=NC(1–13)NH2>NC(1–12)NH2>>NC(1–11)NH2. [F/G]NC(1–13)NH2 was a full agonist with a pEC50 value of 8.65. NCNH2 and [F/G]NC(1–13)NH2 both inhibited K+ evoked glutamate release from rCX with pEC50 and maximum inhibition of 8.16, 48.5±4.9% and 7.39, 58.9±6.8% respectively. In rVD NC inhibited electrically evoked contractions with a pEC50 of 6.63. Although [F/G]NC(1–13)NH2, displayed a small (instrinsic activity α=0.19) but consistent residual agonist activity, it acted as a competitive antagonist (pA2 6.76) in the rVD. The differences between [F/G]NC(1–13)NH2 action on central and peripheral NC signalling could be explained if [F/G]NC(1–13)NH2 was a partial agonist with high strength of coupling in the CNS and low in the periphery. An alternative explanation could be the existence of central and peripheral receptor isoforms.

Partial agonist behaviour depends upon the level of nociceptin/ orphanin FQ receptor expression: studies using the ecdysone- inducible mammalian expression system

Partial agonism is primarily dependent upon receptor density and coupling efficiency. As these parameters are tissue/model dependent, intrinsic activity in different tissues can vary. We have utilised the ecdysone‐inducible expression system containing the human nociceptin/orphanin FQ (N/OFQ) peptide receptor (hNOP) expressed in Chinese hamster ovary cells (CHOINDhNOP) to examine the activity of a range of partial agonists in receptor binding, GTPγ35S binding and inhibition of adenylyl cyclase studies. Incubation of CHOINDhNOP cells with ponasterone A (PON) induced hNOP expression ([leucyl‐3H]N/OFQ binding) of 24, 68, 191 and 1101 fmol mg−1 protein at 1, 2, 5 and 10 μM PON, respectively. At 191 fmol mg−1, protein hNOP pharmacology was identical to that reported for other traditional expression systems. pEC50 values for GTPγ35S binding ranged from 7.23 to 7.72 (2–10 μM PON) for the partial agonist [Phe1ψ(CH2–NH)Gly2]N/OFQ(1–13)–NH2 ([F/G]N/OFQ(1–13)–NH2) and 8.12–8.60 (1–10 μM PON) for N/OFQ(1–13)–NH2 and Emax values (stimulation factor relative to basal) ranged from 1.51 to 3.21 (2–10 μM PON) for [F/G]N/OFQ(1–13)–NH2 and 1.28–6.95 (1–10 μM) for N/OFQ(1–13)–NH2. Intrinsic activity of [F/G]N/OFQ(1–13)–NH2 relative to N/OFQ(1–13)–NH2 was 0.3–0.5. [F/G]N/OFQ(1–13)–NH2 did not stimulate GTPγ35S binding at 1 μM PON, but competitively antagonised the effects of N/OFQ(1–13)–NH2 with a pKB=7.62. pEC50 values for cAMP inhibition ranged from 8.26 to 8.32 (2–10 μM PON) for [F/G]N/OFQ(1–13)–NH2 and 9.42–10.35 for N/OFQ(1–13)–NH2 and Emax values (% inhibition) ranged from 19.6 to 83.2 for [F/G]N/OFQ(1–13)–NH2 and 40.9–86.0 for N/OFQ(1–13)–NH2. The intrinsic activity of [F/G]N/OFQ(1–13)–NH2 relative to N/OFQ(1–13)–NH2 was 0.48–0.97. In the same cellular environment with receptor density as the only variable, we show that the profile of [F/G]N/OFQ(1–13)–NH2 can be manipulated to encompass full and partial agonism along with antagonism.

Stereoselective Interaction of Ketamine with Recombinant [micro sign], [small kappa, Greek], and [small delta, Greek] Opioid Receptors Expressed in Chinese Hamster Ovary Cells

BackgroundThe authors examined the interaction of ketamine with recombinant [micro sign], [small kappa, Greek], and [small delta, Greek] opioid receptors and recombinant orphan opioid receptors expressed in Chinese hamster ovary cells (CHO-[micro sign], CHO-[Greek small letter kappa, CHO-[small delt

A new non-invasive continuous cardiac output trend solely utilizing routine cardiovascular monitors.

Objective. Three of the us developed a new non-invasive continuous cardiac output (CCO) measurement method utilizing routine clinical monitors based on the pulse-contour analysis combined with pulse wave transit time (PWTT). Using pulmonary artery catheter (CCOpa), we compared this estimated CCO (esCO) with the thermodilution CCO early after cardiac surgery, and tested whether the esCO method has potential of being an alternative measure of CCO. Methods. Thirty-six patients without continued arrhythmias were studied. esCO was computed using electrocardiogram (ECG) monitor, arterial pressure monitor and pulse-oximetry system. Both sets of data (esCO and CCOpa), by averaging the results of the preceding 10 min, were compared at 30-min intervals throughout the 15.8± 3.3 h (S.D.) of study. Bland–Altman plots and correlation analysis were used for statistical comparison. Results. A total of 981 paired sets of data (89.9%) among 1093 measurements were compared in the absence of displacement of either pulse-oximetry or ECG probes and/or inaccurate detection of R wave. The difference between esCO and CCOpa results was −0.06 ± 0.82 L/min (S.D.), and there was a linear correlation between them (r = 0.80, p < 0.0001). The difference between them was 0.00± 0.48 L/min at the first 1 h, which remained unchanged throughout 20 h after the start of measurement. Conclusions. The results demonstrate that esCO has a close correlation with the CCOpa, even though the two methods are not interchangeable. The results suggest that esCO method has potential of being an alternative non-invasive cardiac output trend, unless there are apparent arrhythmias.

Density Detection in Dependent Left Lung Region Using Transesophageal Echocardiography

Background Densities in dependent lung regions worsen oxygenation in patients with acute respiratory distress syndrome. Identification of these densities requires examination using computed tomography (CT). In this study, the authors evaluated the use of transesophageal echocardiography (TEE) to estimate densities in the dependent lung. Methods Forty consecutive patients with acute lung injury or acute respiratory distress syndrome who underwent CT and TEE examination were included in this study. Densities in the lower left lung area were detected through the descending aorta by TEE. Density areas observed by TEE were compared with those obtained by CT. The effect of positive end-expiratory pressure (PEEP) application on density area was also evaluated. Results Density areas in the dependent lung region measured by TEE were 12.0 ± 6.1 cm2 (mean ± SD) at mid esophageal position. Density areas evaluated using TEE in the left lung correlated significantly with those estimated with CT in the left and right lungs (P < 0.01 in both lungs). In addition, the authors observed a significant correlation between Pao2/Fio2 and density areas estimated using TEE (P < 0.05). During positive end-expiratory pressure application, the area of density estimated with TEE decreased and Pao2 improved. Conclusions The authors clearly demonstrated that it is possible to estimate the density area of the dependent left lung regions in patients with acute lung injury or acute respiratory distress syndrome using TEE. It is also possible to observe the changes of density areas during application of positive end-expiratory pressure.

Evaluation of density area in dorsal lung region during prone position using transesophageal echocardiography

ObjectiveTo evaluate the changes of density area in the dorsal lung regions of acute respiratory distress syndrome patients during prone position using transesophageal echocardiography. DesignRetrospective clinical study. SettingGeneral intensive care unit in a university hospital. PatientsTen patients with acute respiratory distress syndrome who underwent prone position therapy. InterventionsDensity areas in the left dorsal lung region were observed using transesophageal echocardiography before and after patients were in the prone position for 2 hrs. In five patients, a pediatric transesophageal echocardiography probe was left in the esophagus and used for observation during the prone procedure. Measurements and Main ResultsChanges of density area and Pao2/Fio2 were observed. The density areas decreased after prone position compared with those of preprone position (preprone 11.4 ± 5.1 cm2, after prone 5.6 ± 3.5 cm2, mean ± sd, p < .01, respectively). There was also a significant correlation between the percentage change of density area and Pao2/Fio2 (r = .47, p < .05) after prone position. During prone position, the density area decreased; however, there was no correlation between the percent changes of density area and Pao2/Fio2. ConclusionIt was possible to observe the change in density area during prone position using transesophageal echocardiography. The change of density area estimated with transesophageal echocardiography during prone position was useful to estimate the effectiveness of the procedure.

Does Indocyanine Green Accurately Measure Plasma Volume Early After Cardiac Surgery

Potential overestimation of plasma volume (PV) determination by the conventional indocyanine green (ICG) dilution method (PV-ICG) can occur when generalized capillary protein leakage is present, because ICG binds to proteins. We recently reported that this overestimation can be recognized by simultaneous measurement of the initial distribution volume of glucose (IDVG). We examined whether overestimation of PV-ICG and further ICG-pulse dye densitometry-derived plasma volume (PV-PDD) can occur early after cardiac surgery by using the PV-ICG/IDVG ratio as an indicator. Possible overestimation was defined as a ratio higher than 0.45. Twenty-four consecutive postcardiac surgical patients were enrolled. PV-ICG, PV-PDD, and IDVG were calculated simultaneously after admission to the intensive care unit and on the first postoperative day. The mean ± sd PV-ICG/IDVG ratio for 47 recordings was 0.38 ± 0.05. Four had a PV-ICG/IDVG ratio higher than 0.45, and the highest was 0.48. The mean PV-PDD/IDVG ratio for a total of 47 recordings was 0.39 ± 0.10. There were extremely high or low ratios observed in PV-PDD determinations, but they were not observed in PV-ICG determinations. Results suggest that most of the PV-ICG measurements are accurate, but inaccuracy of PV-PDD can occur early after cardiac surgery.

Effects of nociceptinNH2 and [Nphe1]nociceptin(1-13)NH2 on rat brain noradrenaline release in vivo and in vitro.

We have investigated the effects of nociceptin/orphanin FQ (NC) receptor agonist NCNH2 and a competitive NC receptor antagonist, [Nphe1]NC(1-13)NH2 on noradrenaline (NA) release in vivo using microdialysis in freely moving animals and in vitro from cerebrocortical slices. One nmol of NCNH2 injected into rat locus coeruleus inhibited NA release from the prefrontal cortex (E(max) 27.4+/-5.7% 30 min after injection) which was partially (33%) reversed by 100 nmol of [Nphe1]NC(1-13)NH2. In cerebrocortical slices NCNH2 inhibited NA release in a concentration-dependent manner (EC50 12 nM, E(max) 29.4%) that was reversed by [Nphe1]NC(1-13)NH2. In both preparations, [Nphe1]NC(1-13)NH2 per se was inactive. These data demonstrate an inhibition of NA release by NCNH2 in a [Nphe1]NC(1-13)NH2 sensitive manner in both in vivo brain microdialysis and in vitro cerebrocortical slices studies in rats.

Can Initial Distribution Volume of Glucose Predict Hypovolemic Hypotension After Radical Surgery for Esophageal Cancer

We recently reported that the initial distribution volume of glucose (IDVG) reliably measures the central extracellular fluid volume in the presence or absence of fluid gain or loss. We examined which variables, including IDVG, can predict subsequent hypovolemic hypotension produced by the continuous shift of the extracellular fluid from the central to the peripheral compartment early after radical surgery for esophageal cancer. IDVG and plasma volume were calculated after measuring cardiac index (CI), central venous pressure, and pulmonary artery wedge pressure immediately after admission to the intensive care unit. Intraoperative fluid balance and urine volume were also recorded. Postoperative hypovolemic hypotension was clinically defined as systolic blood pressure < 80 mm Hg responsive to IV fluid administration. Either IDVG < 105 mL/kg or CI < 3.4 L · min−1 · m−2 was associated with subsequent hypovolemic hypotension (P = 0.002 for the former and P = 0.00 03 for the latter), while remaining variables were not. IDVG and CI were well correlated (r = 0.8 7, n = 25, P = 0.0001). Our results suggest that IDVG can help predict the subsequent hypovolemic hypotension early after radical surgery for esophageal cancer.