The effects of nitrous oxide on a glutamate-gated ion channel and their reversal by high pressure; a single channel analysis.

Nitrous oxide reversibly affects the kinetics, but not the conductance, of the qGluR channel of locust muscle. 0.5 atm N2O at 20.5 degrees C was without effect but both 1.5 and 2.7 atm significantly reduced the probability of the channel opening, the frequency of opening and the mean open time, and prolonged the mean closed time. 100 atm helium was without effect on these parameters, but when 98.5 atm He was combined with 1.5 atm N2O they, and the associated dwell time distributions, were restored to normal. 100 atm similarly combined with 2.7 atm N2O exerted a comparable trend which fell short of significance. The results are consistent with nitrous oxide binding to the channel with a significant molar volume increase, which pressure opposes. This suggests that nitrous oxide may cause conformational changes in the channel, and that the pressure reversal of nitrous oxide anaesthesia in animals could be caused by molecular antagonism.

Effects of gaseous anaesthetics and inert gases on the phase transition in smectic mesophases of dipalmitoyl phosphatidylcholine.

The phase transition in smectic mesophase of dipalmitoyl phosphatidyl-choline was studied under high pressures of helium (340 atm), nitrogen (340 atm), nitrous oxide (43 atm), cyclopropane (4.4 atm) and n-propane (8.2 atm), using a turbidimetric technique. Helium and nitrogen increased the transition temperature by 0.021 and 0.006 degree C/atm, respectively, compared with 0.024 degrees C/atm for hydrostatic pressure. Nitrous oxide reduced the transition by 0.58 degree C/atm. The hydrocarbon gases spread the transition width and lowered the transition temperature with increasing effect at higher doses. Comparisons with other membrane probes are made and the concentration of gases in the bilayer which lower the transition temperature by 1 degree C are estimated, in mol%: He, 10.2; N2, 13.2: N2O, 9.04; n-C3H8, 6.3 and cyclopropane, 12.8.

Effects of high pressure on the channel gated by the quisqualate-sensitive glutamate receptor of locust muscle and its blockade by ketamine; a single-channel analysis.

The effects of high pressure on the channel gating kinetics of the quisqualate-sensitive L-glutamate receptor (qGluR) of locust muscle have been investigated using a megaohm seal patch-clamp technique. Pressure was applied with helium gas and recordings were carried out at 20.5 degrees C with Rb+ as the main charge-carrying cation in the patch pipette. The mean open time of the qGluR channel was unaffected by 10 and 30 MPa, but it was significantly reduced at 50 MPa. A high proportion of brief openings (mean 0.808 ms) was seen at 50 MPa but not at lesser pressures. Also, in contrast to lesser pressures, 50 MPa prolonged the mean closed time and reduced both the frequency and probability of channel opening. 10(-6) M ketamine significantly reduced the mean channel open time, as previously reported. A pressure of 10 MPa which alone had no effect on the qGluR channel, restored the mean open time in the presence of 10(-6) M ketamine to the value obtained in the absence of the anaesthetic. This implies the shortening of qGluR channel open time by ketamine involves a large + delta V and, therefore, probably conformational changes in the channel. However 10 MPa did not restore the distribution of open times to normal.

Temperature, pressure and cholesterol effects on bilayer fluidity; a comparison of pyrene excimer/monomer ratios with the steady-state fluorescence polarization of diphenylhexatriene in liposomes and microsomes.

Pyrene excimer/monomer (E/M) ratios have been compared with the steady-state fluorescence polarization (P) of diphenylhexatriene (DPH) in multilamellar liposomes of dilaurylphosphatidylcholine and rat liver microsomes. The purpose was to use the well-understood properties of DPH to reveal the nature of bilayer fluidity which pyrene manifests as an E/M ratio. Reducing the temperature (from 37 degrees C to 8 degrees C), increasing the hydrostatic pressure (from 0.1 to 70 MPa), and, in liposomes, cholesterol enrichment (up to 0.30 mole fraction) separately decreased the E/M ratios and increased P. The pyrene membrane/buffer partition coefficient was affected by temperature but not by pressure, and in the case of cholesterol enrichment, it was assumed to be unaffected. Plots of P as a function of the E/M ratio showed the two to be closely correlated (r = 0.99 in liposomes and 0.96 in microsomes), independent of the treatment used to reduce fluidity. The apparent activation volume and enthalpy for excimer formation was calculated and compared with published data. Pyrene E/M ratios probably reflect the intermolecular volume (fluidity) of the outer region of the bilayer, which is reduced by a decrease in temperature and an increase in pressure and cholesterol. DPH reports the bilayer interior, which is similarly ordered by the experimental treatments. The regional distinction between the two probes, however, accounts for the divergence of E/M ratios and P, which has been reported in membranes enriched with fluidizing fatty acids.

Homeoviscous adaptation under pressure: the pressure dependence of membrane order in brain myelin membranes of deep-sea fish.

Steady-state and time-resolved anistropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence have been used to compare the hydrocarbon order of brain myelin membranes from a shallow water (plaice) and two deep-sea fish species (Coryphenoides rupestris and Coryphenoides armatus). At atmospheric pressure the deep sea fish displayed lower steady-state anisotropies than shallow water species although the pressure dependence of anisotropy was similar in all species. Time-resolved measurements allowed the separate determination of the rate of probe motion from the amplitude of that motion. Anisotropy decays were analysed in terms of two correlation times and a constant (r infinity). The r infinity and mean value of P2 order parameter for all species increased with pressure, the graphs for deep-sea species being translated to higher pressures relative to shallow-water species. The resulting pressure coefficients for C. armatus was distinctly less than for the two shallower species. These time-resolved studies show that the interspecific differences provide for similar order parameters in all three species when corrected to their respective habitat conditions of pressure and temperature. This indicates that myelin order is highly conserved despite the profound ordering effects of high hydrostatic pressure.

Effect of pregnancy on bone mineral density and biochemical markers of bone turnover in a patient with juvenile idiopathic osteoporosis.

Juvenile idiopathic osteoporosis (JIO) is rare, presenting with vertebral fractures in the immediate prepubertal years; however, recovery is normally observed. We report the case of a 19-year-old pregnant woman previously diagnosed with JIO. She experienced three vertebral fractures in the third trimester of pregnancy. She delivered by caesarean section at 38 weeks gestation. Spinal bone mineral density decreased by 25%, hip bone mineral density by 10%, and forearm bone mineral density by 3% during pregnancy. Bone resorption markers, free pyridinoline and deoxypyridinoline (fPYD and fDPD), were elevated at baseline and markedly increased during pregnancy (fPYD/fDPD at 0, 10, 15, 20, and 28 weeks and immediately postpartum: 36.2/11.5, 52.9/15.8, 54.3/13.3, 51.1/13.3, 90/21.8, and 95.6/22.7 nmol/mmol creatinine, respectively) The bone formation marker, bone-specific alkaline phosphatase (BSAP), was within the reference range at baseline and increased in the third trimester. (BSAP at 0, 10, 15, 20, and 28 weeks and immediately postpartum: 20.5, 18.3, 17.7, 19.8, 26.9, and 30.0 U/liter, respectively). Parathyroid hormone (PTH) was measured by two methods to assess the possible effect of PTH fragments. PTH(1-84) (Roche) showed little change during the pregnancy, whereas the Nichols assay [(1-84) and(7-84) PTH fragment], revealed increases paralleling the changes in bone resorption. This young woman's bone turnover showed an exaggerated response to pregnancy, with bone resorption predominating over formation. PTH fragments may have partially mediated this effect.

Hydrostatic pressure modifies the action of octanol and atropine on frog endplate conductance.

The effects of octanol, ethanol and atropine were examined on the time course of decay (tau D) of miniature endplate currents (m.e.p.cs) in the frog neuromuscular junction at normal and high pressure. Octanol (25-100 microM) decreased reversibly the tau D of m.e.p.cs in a dose-dependent manner, 100 microM reducing tau D to 0.39 of the control value. Higher concentrations (200-500 microM) additionally depressed the amplitude of m.e.p.cs. Hydrostatic pressure (3.19 and 5.25 MPa) reduced the tau D of octanol (25-100 microM)-shortened m.e.p.cs. Thus 3.19 MPa and 5.25 MPa reduced the tau D in the presence of 100 microM octanol to 0.75 and 0.78 of the octanol treated values. This effect was not completely reversed on decompression. The m.e.p.c. amplitude is reversibly decreased by pressure in the presence of octanol. Hydrostatic pressure (3.19-15.55 MPa) did not modify the effect of ethanol on tau D. At 10.40 and 15.55 MPa the tau D was increased equally in the absence or presence of ethanol. Atropine (60 microM) reduced the tau D and amplitude of m.e.p.cs to 0.33 and 0.63 of the control values. These effects were completely reversible. Hydrostatic pressure (3.19 and 5.25 MPa) reduced the tau D of atropine-shortened m.e.p.cs to 0.82 and 0.77 of the atropine-treated values respectively. This effect was not completely reversed on decompression. Hydrostatic pressure also reversibly depressed the amplitude of atropine-treated m.e.p.cs. The implications of these drug-hydrostatic pressure interactions are discussed.

Patch-clamp experiments with porins extracted from a marine bacterium (Photobacterium profundum strain SS9) and reconstituted in liposomes.

The reconstitution of bacterial porins in liposome bilayers for patch-clamp recording is well established. However, the solutions used in the dehydration, rehydration, and osmotic swelling of the liposomes have been developed for porins from enteric bacteria. Porins from marine bacteria normally function in contact with seawater whose ionic composition and osmotic pressure would appear to be incompatible with the established methods. Here, we show that, contrary to expectation, an established reconstitution and patch-clamp method works well with porins, mainly OmpH and OmpL, extracted from the deep-sea marine bacterium Photobacterium profundum strain SS9 and that seawater can be introduced at a supplementary stage.

Effects of hydrostatic pressure and inert gases on platelet aggregation in vitro.

A novel cuvette was used to subject citrated platelet-rich plasma (PRP) to high hydrostatic pressure with negligible contamination by He (used for compression of the apparatus). Aggregation was induced at pressure by ADP and quantified turbidimetrically. The maximum degree of aggregation (MDA) was reduced from a control level of 82.2 to 53.6% by exposure to 101 ATA. Because decompression bubbles did not form, aggregation was also measured immediately after a compression cycle. After exposure to 101 ATA hydrostatic pressure, platelets responded normally to ADP at 1 ATA. In a matching apparatus, PRP was equilibrated with high partial pressures of inert gases. Normal physiological plasma Po2 and pH were maintained during equilibration. N2O (5 ATA) reduced the MDA from 86.5 (control) to 58.1%. N2 (51 ATA) reduced the MDA from 74.7 (control) to 51.6%, and 101 ATA Pn2 reduced the MDA from 78.0 (control) to 32.3%. He (100 ATA) reduced the MDA from 83.6 to 38.6%. It was concluded that platelet aggregation was relatively sensitive to hydrostatic pressure and less sensitive to inert gases than predicted from their anesthetic potency ratios.

Effects of gas bubbling and other forms of convection on platelets in vitro.

Citrated platelet-rich human plasma was subjected to one of three experimental treatments at 37 degrees C for 15 min: stirring, bubbling (with stirring), and gentle agitation achieved by a rocking motion. The last two were "equiconvective" as judged by equilibration rates with CO2 and O2 but presumably differed in the shear stress they imposed on the cells. Stirring platelets in normal air or 5% CO2-air caused no significant aggregation. Bubbling air through platelet-rich plasma increased its pH and marked aggregation occurred. Bubbling CO2-air caused the platelet-rich plasma pH to attain its physiological level of 7.4 with less aggregation. In both cases, subsequent ADP-induced aggregation was diminished. Rocking (without stirring) in the presence of CO2-air caused negligible aggregation in platelets and an enhanced response to ADP. Because of the marked difference between the two equiconvective treatments, bubbling and rocking, the main factor in activating the human platelets is suggested to be shear stress (potentiated by high pH), with perhaps a lesser contribution from the air-plasma interface.

Effects of subanesthetic doses of inert gases on behavioral thermoregulation in mice.

Mice exposed to subanesthetic partial pressures of N2O (0.25 to 0.75 atm) or N2 (5.7 or 11.33 atm) and allowed to choose between a warm and a cool environment showed a marked preference for the cooler environment. This behavior was associated with the onset of hypothermia, with deep body temperature falling by up to about 3 degrees C, usually to a new, steady level. Both the length of time spent in the cooler environment and the degree of the hypothermia produced increased with the partial pressure of N2O or N2 used. The effects of N2O on behavioral thermoregulation and body temperature were reversible. There was a correlation between anesthetic potency and the ability of both gases to alter thermoregulation, suggesting that the effect of these agents on thermoregulation was caused by the same molecular interactions as those which underlie anesthesia. Since both gases elicited changes in behavioral thermoregulation promoting rather than opposing the onset of hypothermia, it is concluded that they may have acted to lower the level at which deep body temperature was being regulated.

Behavioral thermoregulation in mice subjected to high pressure.

Mice exposed to normoxic He and Ne at increased pressure and allowed to choose between a neutral and a cool environment showed a preference for the cooler environment. This behavior was apparent at 5.7 but not at 2.5 atm He. At 11.3 atm He and Ne, the behavior was associated with a similar reduction in the deep body temperature to a new steady level. The reduction in body temperature increased with pressure, up to 35 atm He, the maximum studied. Since the heat transfer of the He and Ne gas mixtures is different and both gases exert negligible anesthetic effects, the hydrostatic pressure most likely affects behavioral thermoregulation by affecting neuronal function.

Effect of high hydrostatic pressure on the porin OmpC from Escherichia coli.

Porin OmpC from Escherichia coli was reconstituted in liposomes and its gating kinetics were recorded at high hydrostatic pressure, up to 90 MPa, using a development of the patch clamp technique. The composition of the recording solution influenced the results but generally high hydrostatic pressure favoured channel opening.

Single channel analysis of ketamine interaction with a quisqualate receptor.

The interactions of the general anaesthetic ketamine with the quisqualate-sensitive L-glutamate receptor (QUIS-R) of locust muscle have been investigated at the single channel level using a M omega seal patch clamp technique. Low concentrations (10(-10) to 10(-9) M) of ketamine did not significantly alter the kinetics of the QUIS-R channel. Higher concentrations of ketamine decreased the probability of the channel being open, the frequency of channel opening and the channel mean open time, and increased the channel mean closed time. Probability density functions of channel dwell times indicate that during application of greater than 10(-8) M ketamine the distribution of channel openings becomes restricted mainly to brief events. These results are consistent with the view that ketamine blocks the open, and possibly also the closed, channel of locust muscle QUIS-R and that this anaesthetic dissociates only slowly from its blocking site(s).

Avascular necrosis of the femoral head in patients with prostate cancer treated with cyproterone acetate and radiotherapy.

We report the case histories of two patients with histologically confirmed adenocarcinoma of the prostate, both of whom had been treated with steroidal anti-androgen therapy in the form of cyproterone acetate prior to radical or palliative pelvic irradiation, and who subsequently developed femoral head avascular necrosis. This is a diagnosis that should be considered in patients with prostate cancer who present with hip pain in the absence of biochemical evidence of disease progression.

Microwave thermography as a noninvasive assessment of disease activity in inflammatory arthritis.

Microwave thermography is the technique of measuring natural thermal radiation from body tissues of clinical interest. It is safe, requires no control of environmental conditions and exposes the patient to no ionising radiation. In this study, we found that microwave thermography of the knee joints showed weak correlation with some global parameters of disease activity. Bed rest alone for three days had no significant effect on microwave thermographic index (MTI). Intra-articular steroid injection, however, was associated with a marked fall in MTI both in the injected knee and the contra-lateral knee. We conclude that microwave thermography can measure inflammatory activity in the knee joints of patients with inflammatory arthritis and can respond to clinical change brought about by major treatment intervention.

Triploidy induction in recently fertilized Atlantic salmon ova using anaesthetics.

The comparative effectiveness of five general anaesthetics as agents for inducing triploidy in recently fertilized Atlantic salmon ova is reported. Triploid rates and triploid yields following the exposures of eggs from a single female fish to nitrous oxide, and separately to Freon 22, were shown to be related to the partial pressure and duration of exposure. The maximum triploid yield (79.7%) was observed following exposure to nitrous oxide at 11 atmospheres (atm) for the first 0 min after fertilization. Triploid yields following exposure to Freon (at 1 and 3 atm) were generally lower, the highest value (43.7%) occurring after exposure at ambient pressure for the first hour after fertilization. Triploid yields in these same eggs were low ( < 10%) when halothane and ethrane were used at partial pressures in the range 0.05-0.2 atm and cyclopropane at atmospheric pressure was ineffective. In a subsequent season the eggs from ten different individual female Atlantic salmon were shown to vary little in susceptibility following exposure to nitrous oxide treatment (0-30 min after fertilization, 11 atm) and experienced high yields of triploids (mean=80.4 +/- 6.6% SD).

A method of patch clamp recording in hyperbaric oxygen.

A method of patch clamp recording in hyperbaric oxygen is described which is derived from a method for patch clamping at high hydrostatic pressure. Excised patches equilibrate with hyperbaric oxygen within 10 minutes. Experiments with BK channels expressed in HEK 293 cells showed that 1 MPa O2 caused an increase in the ionic permeability of the patch, an effect accelerated in the presence of ferrous ions. Seal resistance was unaffected. Small negative currents, (VH - 40 to - 65 mV, symmetrical KCl solutions), apparent in skewed all-points amplitude histograms, reduced the signal to noise ratio. In some patches the BK channel activity was inhibited and in others it persisted with the currents becoming irresolvable in the increased noise.