The importance of ligand gated ion channels in sleep and sleep disorders.

On average, humans spend about 26 years of their life sleeping. Increased sleep duration and quality has been linked to reduced disease risk; however, the cellular and molecular underpinnings of sleep remain open questions. It has been known for some time that pharmacological modulation of neurotransmission in the brain can promote either sleep or wakefulness thereby providing some clues about the molecular mechanisms at play. However, the field of sleep research has developed an increasingly detailed understanding of the requisite neuronal circuitry and key neurotransmitter receptor subtypes, suggesting that it may be possible to identify next generation pharmacological interventions to treat sleep disorders within this same space. The aim of this work is to examine the latest physiological and pharmacological findings highlighting the contribution of ligand gated ion channels including the inhibitory GABAA and glycine receptors and excitatory nicotinic acetylcholine receptors and glutamate receptors in the sleep-wake cycle regulation. Overall, a better understanding of ligand gated ion channels in sleep will help determine if these highly druggable targets could facilitate a better night's sleep.

Ligand gated receptor interactions: A key to the power of neuronal networks.

The discovery of the chemical synapse was a seminal finding in Neurobiology but the large body of microscopic interactions involved in synaptic transmission could hardly have been foreseen at the time of these first discoveries. Characterization of the molecular players at work at synapses and the increased granularity at which we can now analyze electrical and chemical signal processing that occur in even the simplest neuronal system are shining a new light on receptor interactions. The aim of this review is to discuss the complexity of some representative interactions between excitatory and inhibitory ligand-gated ion channels and/or G protein coupled receptors, as well as other key machinery that can impact neurotransmission and to explain how such mechanisms can be an important determinant of nervous system function.

Chemogenetics a robust approach to pharmacology and gene therapy.

Modern developments in organic chemistry, molecular biology, virology, and genetics have opened new, exciting possibilities to better understand physiology and to create innovative, robust therapeutics. One such possibility is the burgeoning field of chemogenetics, a sub-field of chemical genetics that encompasses engineering macromolecules (particularly proteins) to modify how they interact with endogenous and exogenous ligands (particularly small molecules). Early efforts in chemogenetics were focused on parsing the function of a specific enzyme within a closely-related family by creating orthogonal enzyme-ligand pairs (e.g. kinases paired with antagonists). This powerful concept quickly expanded into engineered G-protein-coupled receptors (e.g. DREADDs/RASSL), and more recently into engineered ligand-gated ion channels (eLGIC). The modifications to the receptor focused on eliminating their activation by endogenous ligands, while preserving or enhancing their interaction with pharmacological agents (e.g. small molecule agonist). Creation of such an engineered receptor and delivering it selectively to specific cell types opens new possibilities of accurately and precisely controlling cellular activity. Control of this activity then increases our understanding of the cells function in normal physiology, while also creating the possibility of using it as a therapeutic to address pathophysiology. The DREADDs/RASSL and eLGIC approaches have been particularly impactful in neurosciences but have applications in multiple fields. In this work we introduce the history of the chemogenetic approach, review the seminal work with DREADDs/RASSLs and eLGIC, highlight the breadth of applications, and discuss the strengths and weaknesses associated with this technology, especially in the context of its development into a therapeutic.

GdpS contributes to Staphylococcus aureus biofilm formation by regulation of eDNA release.

In Staphylococcus aureus, the role of the GGDEF domain-containing protein GdpS remains poorly understood. Previous studies reported that gdpS mutant strains had decreased biofilm formation due to changes in icaADBC expression that were independent of cyclic-di-GMP levels. We deleted gdpS in three unrelated S. aureus isolates, and analyzed the resultant mutants for alterations in biofilm formation, metabolism and transcription. Dynamic imaging during biofilm development showed that GdpS inhibited early biofilm formation in only two out of the three strains examined, without affecting bacterial survival. However, quantification of biofilm formation using crystal violet staining revealed that inactivation of gdpS affected biofilm formation in all three studied strains. Extraction of metabolites from S. aureus cells confirmed the absence of cyclic-di-GMP, suggesting that biofilm formation in this species differs from that in other Gram-positive organisms. In addition, targeted mutagenesis demonstrated that the GGDEF domain was not required for GdpS activity. Transcriptomic analysis revealed that the vast majority of GGDEF-regulated genes were involved in virulence, metabolism, cell wall biogenesis and eDNA release. Finally, expression of lrgAB or deletion of cidABC in a strain lacking gdpS confirmed the role of GdpS on regulation of eDNA production that occurred without an increase in cell autolysis, but with a late increase in holin-mediated autolysis, in the presence of high oxacillin concentrations. In summary, S. aureus GdpS contributes to cell-to-cell interactions during early biofilm formation by influencing expression of lrgAB and cidABC mediated eDNA release. We conclude that GdpS acts as a negative regulator of eDNA release.

Clinical and radiographic evaluation of disc excision for lumbar disc herniation with and without posterolateral fusion.

STUDY DESIGN: A prospective study evaluating the clinical and radiographic results in 95 patients with lumbar disc herniation. OBJECTIVES: To evaluate the results of disc excision, with and without posterolateral fusion. SUMMARY OF BACKGROUND DATA: The effect of posterolateral fusion on the outcomes and radiologic changes in patients with lumbar disc herniation has rarely been reported. METHODS: Forty-four patients underwent disc excision, and 51 patients underwent disc excision and fusion. Clinical symptoms were evaluated using the Japanese Orthopaedic Association Back scores. All medical and surgical records were examined with regard to intraoperative blood loss, operation time, and other data. Preoperative and follow-up radiographs were analyzed to determine the spinal motion and disc height. RESULTS: Clinical outcome was excellent or good in 73% of the nonfusion group and in 82% of the fusion group (P = 0.31). The reduction in lower back pain after surgery was greater in the fusion group. The rate of recurrent disc herniation at the surgical level in the nonfusion group increased, but intraoperative blood loss, operation time, length of hospital stay, and total cost of procedure were all significantly less in the patients undergoing disc excision alone than in the fusion group. The radiologic analysis provided evidence that the disc height at the level of disc excision and posterolateral fusion in the fusion group decreased with time, as in the nonfusion group. The changes in disc height and spinal motion were not related to the clinical results. CONCLUSIONS: Although there is still controversy regarding the pros and cons of fusion in association with disc excision, there is seldom an indication for primary fusion for lumbar disc herniation.

Apoptosis in microvascular endothelial cells of perfused rabbit lungs with acute hydrostatic edema.

We test the hypothesis that microvascular endothelial cells may undergo apoptosis in response to acute pulmonary venous hypertension. The isolated rabbit lungs were perfused in situ for 4 h with left atrial pressure of 0, 10, or 20 mmHg at a constant blood flow. Edema formation was monitored by lung weight gain. To assay for apoptosis, we performed agarose gel electrophoresis of DNA, in situ nick end labeling of DNA strand breaks, and electron microscopy. We also examined the levels of expression of Bcl-2, a suppressor of apoptosis, in microvascular endothelial cells using an immunohistochemical technique. In a vascular pressure-dependent fashion, we found apoptosis in endothelial cells of alveolar septal capillaries, as well as expression of Bcl-2 in arteriolar and venular endothelial cells. We conclude that acute pulmonary venous hypertension induces apoptosis in capillary endothelial cells but not in arteriolar and venular endothelial cells, suggesting that microvascular endothelial cell apoptosis is dependent on the levels of Bcl-2 expression and influences the formation or resolution of acute hydrostatic lung edema.

Effects of low-dose Beraprost sodium, a stable prostaglandin I2 analogue, on reperfusion injury to rabbit lungs.

We investigated the effects of low-dose Beraprost sodium (BPS), a stable prostaglandin I2 (PGI2) analogue, on microvascular permeability and the plasma concentrations of thromboxane and adenosine 3',5'-cyclic monophosphate (cAMP) in blood-perfused rabbit lungs subjected to ischemia-reperfusion (I/R). After an ischemic insult for 2 h, saline as a vehicle, 3 pmol/L of BPS (BPS-1), 150 to 300 pmol/L of BPS (BPS-2), 900 pmol/L of BPS (BPS-3), or 60 micromol/L of indomethacin (IND) was administered into the reservoir, then the lungs were reperfused and reventilated for 1 h. Vascular permeability was assessed by determining the microvascular filtration coefficient (Kf, ml/min/mm Hg/100 g wet lung). I/R resulted in increases in vascular resistance, Kf, and thromboxane. BPS-2, BPS-3, and IND inhibited the increase in vascular resistance, and BPS-3 and IND attenuated the increases in Kf and thromboxane. BPS-3 increased, but IND decreased, the concentrations of cAMP in the perfusate. Perfusate thromboxane released after reperfusion was significantly correlated with Kf. We conclude that cyclooxygenase products play a critical role in I/R-induced lung vascular injury and that 900 pmol/L of BPS inhibits the production of thromboxane and enhances the permeability barrier via a cAMP-elevating effect. However, vasodilatory action of BPS may exacerbate the reperfused lung injury by increasing the flow through injured capillaries via inhibition of thromboxane-induced vasoconstriction.

Effects of ventilation and pleural effusion on measurements of airway thermal volume and blood flow in dog lungs.

We studied the effects of ventilation and pleural effusion on measurements of airway thermal volume (ATV) and pulmonary blood flow (PBF) by using the airway gas thermometry method of V. B. Serikov, M. S. Rumm, K. Kambara, M. I. Bootomo, A. R. Osmack, and N. C. Staub (J. Appl. Physiol. 72: 944-953, 1992) in 39 anesthetized dogs with or without lung edema or pleural effusion. To examine the differential effects of increased-pressure and increased-permeability lung edema on accuracy and sensitivity of ATV and PBF, two models of lung edema were induced by intravenous infusion of a Dextran 70 solution and alloxan monohydrate, respectively. Dogs were hyperventilated for 3 min by using a wide range of minute ventilation (VE) to produce two steady-state conditions of airway temperature. Higher levels of VE increased an estimated amount of ATV. The ATV produced by hyperventilation at VE values of 559, 158, and 72 ml.min-1.kg-1 was consistent with the gravimetric total lung mass, the blood-free wet lung weight, and the extravascular lung water volume, respectively. The coefficient of lung thermal conductivity, a practical index of the rate of heat conduction through tissue from lung vessels, was related to the ratio of the decrease in expired air temperature to VE, and estimated PBF was consistent with the thermodilution cardiac output. Pleural effusion had little effect on measurements of ATV and PBF. However, ATV and PBF showed increased variation in dogs with dextran-induced lung edema.

Alternations in atrial natriuretic peptide release after DC cardioversion of non-valvular chronic atrial fibrillation.

The response of atrial natriuretic peptide (ANP) release to haemodynamic influences after cardioversion of atrial fibrillation has not been fully examined. We measured plasma concentrations of ANP and assessed haemodynamic changes 60-120 min after DC cardioversion in 22 patients with non-valvular chronic atrial fibrillation. Passive leg elevation to enhance volume expansion was performed 60 min after DC cardioversion. Sinus rhythm was restored in 18 of the 22 patients (successful DC cardioversion group). The control group consisted of seven patients with non-valvular chronic atrial fibrillation who did not undergo DC cardioversion (atrial fibrillation control group). In the successful DC cardioversion group, the mean pulmonary artery wedge pressure decreased significantly 15 min after cardioversion (P < 0.05) and then remained unchanged. Plasma concentrations of ANP also decreased significantly 15 min after cardioversion (P < 0.05). Furthermore, there was an additional significant decrease in ANP levels for up to 60 min after cardioversion (P < 0.05 from 15 min). Passive leg elevation for 15 min led to an increase in the mean pulmonary artery wedge pressure (P < 0.01) and right atrial pressure (P < 0.05), but did not result in increased plasma concentrations of ANP (47.1 +/- 27.6 vs 43.9 +/- 34.4 pg.ml-1, mean +/- SD, P = ns). In the atrial fibrillation control group, passive leg elevation increased the mean pulmonary artery wedge pressure (P < 0.01), the mean right atrial pressure (P < 0.05) and plasma concentrations of ANP (139.9 +/- 85.8 vs 168.1 +/- 108.2, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

[Bone union after lumbar postero-lateral fusion: comparative evaluation by x-ray and CT].

As yet there has been no report on a uniform and precise method for evaluating bone union after lumbar postero-lateral fusion (PLF). Here we report and compare the roentgenogram (X-P) and computerized tomography (CT) methods to evaluate the union and non-union of PLF. PLF without internal fixation was undertaken in 252 patients between 1978 and 1991. Of these, 125 patients underwent both X-P and CT scanning and were followed up for more than one year (mean 4 years and 2 months, range 1 to 14 years). In the cases of complete union, X-P demonstrated that the solid fusion mass showed a continuous trabecular pattern along the grafted segment, while CT demonstrated that the solid fusion mass appeared as a long bone with the mass incorporated into the transverse process and the superior facet. In such cases, there was no micromotion on stress roentgenograms. In the cases of incomplete union, the status of the non-union was classified into three types as; Type I showing atrophy and resorption of the grafted bone, Type II showing a lack in trabecular continuity of the fusion mass, and Type III showing a gap either cranially or caudally between the fusion mass and lumbar matrix (which includes the transverse process and superior facet). X-P could easily diagnose Type I and Type II, but could not clearly diagnose Type III. CT, however, could easily diagnose Type III. Therefore, the use of only X-P might lead to a misdiagnosis in cases of Type III. The application of CT was more uniform and precise method to evaluate the state of PLF in the lumbosacral spine.

Usefulness of the pulmonary vein flow velocity-time profile as an estimate of left atrial storage fraction.

During ventricular systole, the left atrium (LA) stores a certain amount of ventricular stroke volume; this is defined as an LA storage volume. From cineangiocardiograms, an LA storage fraction is obtained as the ratio of the LA storage volume to left ventricular stroke volume. From the pulmonary vein (PV) flow velocity-time profile, the LA storage fraction may be estimated as a ratio of the PV flow velocity-time integral during systole (Sa) to a sum of that during systole and diastole (Sa+Da), namely, Sa/(Sa+Da), provided that the PV cross-sectional area remains relatively unchanged during one cardiac cycle and that the PV flow velocity-time profile is similar in any of the PVs draining to the LA. To evaluate usefulness of Doppler echocardiographic method of estimating the LA storage fraction, the authors measured the LA storage fraction from the left upper PV flow velocity-time profile by transesophageal Doppler echocardiography and compared it with the LA storage fraction from conventional cineangiocardiographic volumes. Subjects were 23 patients with a variety of cardiac diseases in normal sinus rhythm, ranging from eighteen to seventy-four years of age. The LA storage fraction was 0.58 +/- 0.12 (mean +/- SD) from cineangiocardiography and 0.64 +/- 0.08 from Doppler echocardiography larger than that from cineangiocardiography (P < 0.01), the correlation was good (r = 0.643). The authors conclude that the left upper PV flow velocity-time profile appears to provide a better correlation with that by cineangiography and may be used as a reliable quantitative estimate of the LA storage fraction.

[A patient with Legionella pneumonia diagnosed early by the PCR method].

A 72-year-old man was admitted to our hospital because of acute respiratory failure. Initially he had been diagnosed as having pneumonia in the lower left lung field and treated with cephem antibiotics by a local physician. Chest X-ray photograph revealed wide-spread infiltrates throughout both lungs, and chest CT scan revealed pleural effusions. The partial pressure of oxygen in arterial blood was 45.8 Torr. In the bronchoalveolar lavage fluid (BALF) specimen, legionella DNA was detected by the polymerase chain reaction (PCR) method. Thus, we were able to diagnose Legionella pneumonia immediately, and to treat the patient successfully.

Effects of acetylsalicylic acid on pulmonary vascular tone and membrane permeability in blood-perfused dog lung.

We studied the effects of acetylsalicylic acid (ASA) on pressor response, microvascular filtration coefficient (Kf), extravascular lung water, and plasma concentrations of cyclooxygenase- and 5-lipoxygenase-derived products in 21 blood-perfused dog lungs with constant flow. The lungs were perfused for 1 h with an intrapulmonary injection of saline as vehicle (n = 5), a low dose of ASA [136 +/- 25 (SD) micrograms/ml perfusate; n = 5], a high dose of ASA (1,006 +/- 278 micrograms/ml perfusate; n = 6), or alloxan (1,000 mg; n = 5). Alloxan significantly increased Kf and extravascular lung water, whereas neither the low nor high dose of ASA increased Kf or extravascular lung water. The ASA-induced increase in vascular resistance did not correlate with the extent of the decrease in perfusate 6-keto-prostaglandin F1 alpha or the ratio of perfusate 6-ketoprostaglandin F1 alpha to thromboxane B2. Moreover, ASA did not enhance the generation of perfusate leukotrienes B4, D4, or E4. We conclude that pulmonary microvascular permeability is unaltered by ASA and that neither the decrease in plasma prostacyclin nor the increase in plasma sulfidopeptide leukotrienes may account for ASA-induced pulmonary vasoconstriction.

Usefulness of sodium chloride as a nondiffusible indicator in the measurement of extravascular lung thermal volume in dogs.

The authors examined the usefulness of sodium chloride as a nondiffusible indicator during the first passage through dogs' lungs, before and after increased-permeability pulmonary oedema produced by an intravenous injection of alloxan. With an injection of a mixture of ice-cold 3 per cent sodium chloride and indocyanine green dye (a nondiffusible reference indicator), the authors simultaneously recorded three dilution curves from the aortic root: dye dilution, thermal and blood electrical conductivity dilution curves in six dogs. The mean transit time of sodium chloride in the conductivity dilution curve was significantly different from, but fairly equal to, that of indocyanine green dye (6.2 +/- 1.4 s (mean +/- SD) against 6.5 +/- 1.4 s (p < 0.01) in the baseline period, and 7.6 +/- 1.9 s against 8.4 +/- 2.1 s (p < 0.01) in the oedema period, respectively). The calculated extravascular lung thermal volume with the thermal and conductivity dilution method (Y, ml kg-1) correlated well with the gravimetrically determined extravascular lung mass in a total of 12 dogs, including six other dogs without intervention (x, g kg-1) (y = 0.72 x +3.03, r = 0.96). The authors conclude that sodium chloride is useful as a nondiffusible indicator in the first passage through the lungs, and that the thermal and conductivity dilution method is also useful for measuring extravascular lung water mass.

Ultrastructural substrates for increased lung water content in experimental pulmonary edema.

We examined the relationship between the incidence of ultrastructural changes in the alveolar septum and the extravascular lung water content. Pulmonary edema was induced in 18 mongrel dogs by either dextran (n = 12) or alloxan (n = 6) administration. Six other dogs served as controls. Extravascular lung water content was measured by the thermal-dye double indicator dilution method. Specimens of lung tissue were examined with an electron microscope, and the incidence of 13 types of pathological changes in the alveolar septum was studied. For each type of pathological change, the incidence was correlated with the magnitude of lung water content. The following results were obtained. The incidence of edematous changes in the alveolar interstitium (widening of the interstitial space, and dispersion and disarray of collagen fibres in the interstitial space) was well correlated with lung water content (r = 0.78, p < 0.01, and r = 0.84, p < 0.01, respectively). The correlation was not significant in the remaining types of changes. We conclude that the incidence of the pathological changes in the alveolar septum is increased along with the increase in the content of lung water in both dextran- and alloxan-induced experimental pulmonary edema in dogs.

Correction for apparent prolongation of mean transit time resulting from response time in a thermodilution system.

We investigated a method of correcting an apparent prolongation in the measured mean transit time (MTT), resulting from the response time of the thermodilution system. We measured the mean response times (MRT) for five commercially available thermistor-tipped catheters by recording their step function response curves. MRT is the sum of the time from the point of step change to the point of the first detection of change in temperature (latency time) plus the time from the first detection to the point of 63.2% of full response (time constant). By using a flow loop model filled with saline through a mixing chamber, we recorded pairs of thermodilution curves simultaneously with pairs of catheters, and studied the influence of MRT on MTT over the constant flow rates of 1-6 L/min. The difference in MRT's (delta MRT, second) between a pair of thermodilution systems correlated with the difference in MTT's (delta MTT, second) between a corresponding pair of thermodilution curves, yielding an equation: delta MTT = 1.07 delta MRT = 0.04 (n = 72, r = 0.95), delta MTT/delta MRT = 1.02 +/- 0.18 (mean +/- SD). We conclude that an apparent prolongation of MTT due to response time is removable by subtracting MRT from measured MTT.

Pretreatment with catalase or dimethyl sulfoxide protects alloxan-induced acute lung edema in dogs.

We tested the preventive effects of catalase, an enzymatic scavenger of hydrogen peroxide, or dimethyl sulfoxide (DMSO), a hydroxyl radical scavenger, on intravenous alloxan-induced lung edema in four groups of pentobarbital sodium-anesthetized, ventilated dogs for 3 h: saline (20 ml.kg-1.h-1) infusion alone (n = 5), alloxan (75 mg/kg) + saline infusion (n = 5), catalase (150,000 U/kg) + alloxan + saline infusion (n = 5), or DMSO (4 mg/kg) + alloxan + saline infusion (n = 5). Catalase or DMSO significantly prevented the increase in plasma thromboxane B2 and 6-keto-prostaglandin F1 alpha over 3 h after alloxan and the accumulation of extravascular lung water after 3 h [3.95 +/- 0.52 (SE) g/g with catalase, 3.06 +/- 0.42 g/g with DMSO] but not early pulmonary arterial pressor response. An electron microscopic study indicated that catalase or DMSO significantly reduced the endothelial cellular damages after alloxan. These findings strongly suggest that hydrogen peroxide and hydroxyl radical are major mediators responsible for intravenous alloxan-induced edematous lung injury in anesthetized ventilated dogs.

Reliability of extravascular lung thermal volume measurements by thermal conductivity technique in sheep.

We tested the accuracy, sensitivity, and reproducibility of a new lung water computer, based on the thermal conductivity technique, in 22 anesthetized closed-chest ventilated sheep with different treatments: 1) controls (n = 8), 2) 0.05 ml/kg of oleic acid + 100 ml/kg of lactated Ringer solution (n = 6), and 3) airway instillation of saline [3.1 +/- 1.3 (SD) g/kg, n = 8]. After 4 h, we determined the extravascular lung water gravimetrically. We found a significant overall correlation between the final extravascular lung thermal volume and the gravimetric extravascular lung mass (P < 0.001). Although the average ratio of extravascular lung thermal volume to extravascular lung mass was 0.97 +/- 0.25 ml/g for all groups, the computer overestimated extravascular lung mass in controls by 10% (17 g) and underestimated it in sheep with oleic acid by 15% (95 g) and in sheep with airway instillation by 8% (37 g). The computer also underestimated the small quantities of saline placed via the airway in the alveolar space by 75% (61 g). Reproducibility of three consecutive measurements was 4.3% (SE). We conclude that the thermal conductivity technique has an ability to detect the baseline extravascular lung mass but has a poor ability to detect an accurate increment of the extravascular lung water under poor tissue perfusion in anesthetized ventilated sheep.

Increases in urinary enzyme excretion in rats depleted of glutathione inhibited by scavenger of oxygen free radicals.

Urinary excretion of enzymes by rats was assessed after glutathione (GSH) was depleted by treatment with a mixture of the GSH depletors D,L-buthionine-S,R-sulfoximine (BSO) and diethylmaleate (DEM). Renal GSH was low 2 h after treatment and later returned to the control level. The urinary excretion of gamma-glutamyltranspeptidase (gamma-GTP) and N-acetyl-beta-D-glucosaminidase (NAG) remained high for at least 3 d after the injection of BSO (100 mg/kg) and DEM (0.5 ml/kg), with no effect on the blood urea nitrogen level. N,N'-Dimethylthiourea (DMTU), a scavenger of oxygen free radicals, inhibited this increase in the urinary excretion of gamma-GTP. DMTU also inhibited the increase in cisplatin-induced NAG excretion caused by the GSH depletors. These results suggested that the urinary excretion of these enzymes is an index of renal tubular injury caused by short-term depletion of renal GSH, and that the generation of free radicals may be involved in renal tubular injury during GSH depletion or caused by cisplatin together with GSH depletors.