Molecular Determinants of General Anesthetic Action: Role of GABAA Receptor Structure

Abstract: Using receptors expressed from mouse brain mRNA in Xenopus oocytes, we found that enhancement of type A γ-aminobutyric acid (GABA_A) receptor-gated Cl⁻ channel response is a common action of structurally diverse anesthetics, suggesting that the GABA_A receptor plays an important role in anesthesia. To determine if GABA_A receptor subunit composition influences actions of anesthetics, we expressed subunit cRNAs in Xenopus oocytes and measured effects of enflurane on GABA-activated Cl⁻ currents. Potentiation of GABA-activated currents by enflurane was dependent on the composition of GABA_A receptor protein subunits; the order of sensitivity was α₁β₁ > α₁β₁γ_2s=α₁β₁γ_2L > total mRNA. The results suggest that anesthetics with simple structures may act on the GABA_A receptor protein complex to modulate the Cl⁻ channel activity and provide a molecular explanation for the synergistic clinical interactions between benzodiazepines and general anesthetics.     

Structure and Functional Characterization of a Novel Human Low-Voltage Activated Calcium Channel

Abstract : We have isolated and characterized overlapping cDNAs encoding a novel, voltage-gated Ca²⁺ channel α₁ subunit, α_1H, from a human medullary thyroid carcinoma cell line. The α_1H subunit is structurally similar to previously described α₁ subunits. Northern blot analysis indicates that α_1H mRNA is expressed throughout the brain, primarily in the amygdala, caudate nucleus, and putamen, as well as in several nonneuronal tissues, with relatively high levels in the liver, kidney, and heart. Ba²⁺ currents recorded from human embryonic kidney 293 cells transiently expressing α_1H activated at relatively hyperpolarized potentials (-50 mV), rapidly inactivated (τ = 17 ms), and slowly deactivated. Similar results were observed in Xenopus oocytes expressing α_1H. Singlechannel measurements in human embryonic kidney 293 cells revealed a single-channel conductance of ~9 pS. These channels are blocked by Ni²⁺ (IC₅₀ = 6.6 μ M ) and the T-type channel antagonists mibefradil (~50% block at 1 μ M ) and amiloride (IC₅₀ = 167 μ M ). Thus, α_1H-containing channels exhibit biophysical and pharmacological properties characteristic of low voltage-activated, or T-type, Ca²⁺ channels.     

The content of prostaglandins and their precursors in Mortierella and Cunninghamella species

Five strains of filamentous fungi belonging to the genera Mortierella and Cunninghamella were examined for the content of dihomo-γ-linolenic, arachidonic, eicosapentaenoic acids and prostaglandins (type E₂ and F _2α). Prostaglandins were detected using an ELISA method in mycelia of all tested strains (range 50–4800 ng g⁻¹ of PGE₂ and 6–30 ng g⁻¹ of PG F _2α). Several micro-organisms also produced prostaglandins in the culture medium (2·2–137·6 μg l⁻¹ for PGE₂ and 0·4–7·8 μg l⁻¹ for PG F _2α).     

A method for long-term measurement of respiration in intertidal fishes during simulated intertidal conditions

Aquatic and aerial respiration of the amphibious fishes Lipophrys pholis and Periophthalmus barbarus were examined using a newly designed flow-through respirometer system. The system allowed long-term measurements of oxygen consumption and carbon dioxide release during periods of aquatic and aerial respiration. The M o ₂ of L. pholis , measured at 15° C, was 2·1 μmol O₂ g^–1 h^–1 during aquatic and 1·99 μmol O₂ g^–1 h^–1 during aerial exposure. The corresponding values of the M co₂ were 1.67 and 1.59 μmol O₂ g^–1 h^–1 respectively, giving an aquatic respiratory exchange ratio (RER) of 0·80 and an aerial RER of 0·79. The M o₂ of P. barbarus , measured at 28°C, was 4·05 μmol O₂ g^–1 h^–1 during aquatic and 3·44 μmol O₂ g^–1 h^–1 during aerial exposure. The corresponding values of the Mco₂ were 3·29 μmol CO₂ g^–1 h^–1 and 2·63 μmol CO₂ g^–1 h^–1 respectively, giving an aquatic RER of 0·81 and an aerial RER of 0·77. While exposed to air for at least 10 h, both species showed no decrease in metabolic rate or carbon dioxide release. The RER of these fishes equalled their respiratory quotient. After re-immersion an increased oxygen consumption, due to the payment of an oxygen debt, could not be detected.     

Swimming performance, oxygen consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-ranched coho salmon

Routine oxygen consumption ( M o ₂) was 35% higher in 1 day starved and 21% higher in 4 day starved adult transgenic coho salmon Oncorhynchus kisutch relative to end of migration ocean-ranched coho salmon. Critical swimming speed ( U _crit) and M o ₂ at U _crit ( M o _2max) were significantly lower in 4 day starved transgenic coho salmon (1·25 BL s⁻¹; 8·79 mg O₂ kg⁻¹ min⁻¹) compared to ocean-ranched coho salmon (1·60 BL s⁻¹; 9·87 mg O₂ kg⁻¹ min⁻¹). Transgenic fish swam energetically less efficiently than ocean-ranched fish, as indicated by a poorer swimming economy at U _crit ( M o _2max     ). Although M o _2max was lower in transgenic coho salmon, the excess post-exercise oxygen consumption (EPOC) measured during the first 20 min of recovery was significantly larger in transgenic coho salmon (44·1 mg O₂ kg⁻¹) compared with ocean-ranched coho salmon (34·2 mg O₂ kg⁻¹), which had a faster rate of recovery.     

Detection of Intracellular Free Na+ Concentration of Synaptosomes by a Fluorescent Indicator, Na+-Binding Benzofuran Isophthalate: The Effect of Veratridine, Ouabain, and α-Latrotoxin

Abstract: A novel fluorescent Na⁺ indicator, Na⁺-binding benzofuran isophthalate (SBFI), was used to follow changes in the intracellular free Na⁺ concentration ([Na⁺]₁) of synaptosomes. The dye, when loaded into synapto- somes in the form of its acetoxymethyl ester, was responsive to changes of [Na⁺]₁. Calibration was made using the 340/380 nm excitation ratio when the cytoplasmic Na⁺ concentration was equilibrated with different concentrations of extracellular Na⁺ in the presence of 2 μ M gramicidin D. The basal value of [Na⁺]₁ in synaptosomes in the presence of 140 m M extracellular Na⁺ was found to be 10.9 ± 1.8 m M. Veratridine, which opens potential-dependent Na⁺ channels, caused a sudden increase in [Na⁺]₁ in a concentration-dependent manner (1 -20 μ M ), whereas the effect of ouabain (20 and 50 μ M ), the inhibitor of the plasma membrane Na⁺,K⁺-ATPase, was more gradual. The rise in the fluorescence intensity upon addition of veratridine was prevented completely by 2 μ M tetrodotoxin. α-Latrotoxin, the black widow spider toxin, caused an increase in the fluorescence intensity, which became evident 1 min after the addition of the toxin. The rate of increase was proportional to the concentration of the toxin (0.19–1.5 n M ). This report confirms our earlier finding demonstrating a Na⁺-dependent component in the action of α-Iatrotoxin, and shows that changes in [Na⁺]₁ in synaptosomes can be followed by SBFI.     

Cerebrovascular Accumulation and Increased Blood-Brain Barrier Permeability to Circulating Alzheimer's Amyloid β Peptide in Aged Squirrel Monkey with Cerebral Amyloid Angiopathy

Abstract: Senescent squirrel monkey is a valuable model to study pathogenesis of cerebrovascular amyloid angiopathy (CAA). Cerebrovascular sequestration and blood-brain barrier (BBB) permeability to ¹²⁵I-amyloid β(1-40) synthetic peptide (sAβ_1-40) were studied in adult versus aged squirrel monkey 1 h after a single intravenous injection. In aged monkey, the half-time of elimination of sAβ_1-40, t ^e_1/2, was prolonged by 0.6 h, the systemic clearance, Cl _SS, was reduced from 1.8 to 1.1 ml/min/kg, and the mean residence time of intact peptide in the circulation was increased by 1 h (45%). In adult monkey, cerebrovascular sequestration of intact sAβ_1-40 was significant, and the BBB permeability was 18.6-fold higher than for inulin. In aged monkey, the sequestration of intact sAβ_1-40 by cortical and leptomeningeal microvessels and the BBB permeability were increased by 5.9, 1.8-, and 2.1-fold, respectively, in the presence of an unchanged barrier to inulin. In brain parenchyma of aged animals, 76.1% of circulating sAβ_1-40 remained intact versus 45.7% in adult. We conclude that multiple age-related systemic effects, i.e., reduced body elimination and systemic clearance of sAβ_1-40, and reduced peripheral metabolism, may act in concert with BBB mechanisms, i.e., increased transendothelial transport and microvascular accumulation of blood-borne sAβ_1-40, and reduced brain metabolism to enhance the development of CAA.     

Inhibition, by 2-Oxo Acids That Accumulate in Maple-Syrup-Urine Disease, of Lactate, Pyruvate, and 3-Hydroxybutyrate Transport Across the Blood-Brain Barrier

Abstract: Data are presented in support of the transport of (-)- d -3-hydroxybutyrate across the blood-brain barrier (BBB) being a carrier-mediated process. The kinetic parameters in 21-day-old pentobarbital-anaesthetized rats were V_max 2.0 μmol.g⁻¹.min⁻¹, K _m 29 m M , and K _D 0.024 ml.g⁻¹.min⁻¹. The value for V_max was the same as that for l -lactate and pyruvate transport in animals of the same age. The transport of all three substrates was sensitive to inhibition by low concentrations of either 2-oxo-3-methylbutanoate or 2-0x0-4-methylpentanoate, the 2-oxo acids that can accumulate in patients with maple-syrup-urine disease. The K _m values for the 2-oxo acids were severalfold lower than the respective K _m values. 2-oxo-3-phenylpropionate was a poor inhibitor. The relative affinities of the various monocarboxylic acids for the transport system of the BBB distinguished it from similar systems described in brain, heart, and liver mitochondria; human erythrocytes; and Ehrlich ascites-tumour cells.     

Does calcium ameliorate the negative effect of NaCl on melon root water transport by regulating aquaporin activity?

The hydraulic conductance ( L ₀) of detached, exuding root systems from melon ( Cucumis melo cv. Amarillo oro) was measured. All plants received a half-strength Hoagland nutrient solution, and plants stressed either solely with NaCl (50 mM) or with NaCl (50 mM) following treatment (2 d) with CaCl₂ (10 mM) were compared with controls and CaCl₂-treated (10 mM) plants. The L ₀ of NaCl-treated plants was markedly decreased when compared to control and CaCl₂-treated plants, but the decrease was smaller when NaCl was added to plants previously treated with CaCl₂. A similar effect was observed when the flux of Ca²⁺ into the xylem and the Ca²⁺ concentration in the plasma membrane of the root cells were determined. In control, CaCl₂- and NaCl + CaCl₂-treated plants, HgCl₂ treatment (50 μM) caused a sharp decline in L ₀ to values similar to those of NaCl-stressed roots, but L ₀ was restored by treatment with 5 mM DTT. However, in NaCl roots only a slight effect of Hg²⁺ and DTT were observed. The effect of all treatments on L ₀ was similar to that on osmotic water permeability ( P _f) of individual protoplasts isolated from roots. The results suggest that NaCl decreased the passage of water through the membrane and roots by reducing the activity of Hg-sensitive water channels. The ameliorative effect of Ca²⁺ on NaCl stress could be related to water-channel function.     

Involvement of Intracellular Stores in the Ca2+ Responses to N-Methyl-D-Aspartate and Depolarization in Cerebellar Granule Cells

Abstract: The [Ca²⁺]₁ of cerebellar granule cells can be increased in a biphasic manner by addition of NMDA or by depolarization (induced by elevating the extracellular K⁺ level), which both activate Ca²⁺ influx. The possibility that these stimuli activate Ca²⁺-induced Ca²⁺ release was investigated using granule cells loaded with fura 2-AM. Dantrolene, perfused onto groups of cells during the sustained plateau phase of the [Ca²⁺]₁ response to K⁺ or NMDA, was found to reduce the response to both agents in a concentration-dependent manner. Preincubation with thapsigargm (10 μ M ) substantially reduced the plateau phase of the [Ca²⁺], response to K⁺ and both the peak and plateau phases of the NMDA response. Preincubation with ryanodine (10 μ M ) also reduced both the K⁺-evoked plateau response and both phases of the NMDA response. Neither had a consistent effect on the peak response to K⁺. The effects of thapsigargin and ryanodine on the NMDA response were partially additive. These results demonstrate that in cerebellar granule cells a major component of both K⁺- and NMDA-induced elevation of [Ca²⁺]₁ appears to be due to release from intracellular stores. The partial additivity of the effects of thapsigargin and ryanodine suggests that these agents affect two overlapping but nonidentical Ca²⁺ pools.     

Activation of Xenopus Eggs by an Extract of Cynops Sperm

An extract obtained from Cynops sperm induced the activation of both Cynops and Xenopus eggs with accompanying changes in the potential of the egg membrane that were quite similar to those caused by the Cynops sperm. The activation-inducing properties of the extract were abolished by treatment with proteinase K or by heating (60°C, 15 min) and were associated with a protease activity against peptidyl Arg-MCA substrates. The activation of Xenopus eggs by the extract was inhibited by those substrates, or by protease inhibitors, aprotinin or leupeptin. The protease activity was localized in the acrosomal region of Cynops sperm. The activation of Xenopus eggs by the extract was prevented when the exterior concentration of Ca²⁺ions, [Ca²⁺]₀, was reduced to 1.5 μM, but it was enhanced when [Ca²⁺]₀ was increased to 340 μM. The activation of Xenopus eggs by the extract was not affected by positive clamping when [Ca²⁺]₀ was 340 μM. These results suggest that the sperm extract contains a protease that causes an increase in the influx of Ca²⁺ions that results in voltage-insensitive activation of the egg.     

Stimulation of Cyclic GMP Accumulation by Sodium Nitroprusside Is Potentiated via a Gs Mechanism in Intact Pinealocytes

Abstract: Cyclic GMP accumulation in pinealocytes is elevated>100-fold by norepinephrine (NE) through a mechanism involving conjoint activation of α₁- and β₁-adrenergic receptors. Little or no stimulation occurs if either α₁- or β₁-adrenergic receptors alone are activated. It appears that α₁-adrenergic effects are mediated by Ca²⁺ acting in part through nitric oxide (NO), and β₁-adrenergic effects are mediated by G_s. In the study presented here we investigated effects of adrenergic agonists or related postreceptor-active agents on stimulation of pineal cyclic GMP accumulation by the NO generator sodium nitroprusside (NP). The cyclic GMP response to NP (1 m M ) was potentiated by NE and isoproterenol (ISO) but not by phenylephrine, indicating that activation of β₁-adrenergic receptors potentiates the effects of NP. Similarly, vasoactive intestinal peptide (VIP), cholera toxin (CTX), and forskolin, all of which are known to mimic the effects of ISO in this system, also potentiated the effects of NP. In contrast, neither dibutyryl cyclic AMP nor agents that elevate intracellular Ca²⁺ levels caused marked potentiation of the effects of NP on pineal cyclic GMP. Depletion (90%) of G_sα by 21-h treatment with CTX reduced β-adrenergic potentiation of NP. These findings indicate that β-adrenergic agonists and VIP potentiate the effects of NP through a mechanism involving G_s. The molecular basis of this action may be an increase in guanylyl cyclase responsiveness to NO.     

Histamine Evokes Greater Increases in Phosphatidylinositol Metabolism and Catecholamine Secretion in Epinephrine-Containing than in Norepinephrine-Containing Chromaffin Cells

Abstract: Chromaffin cells have H₁ histamine receptors. Histamine, acting at these receptors, increases the metabolism of inositol-containing phospholipids and stimulates catecholamine secretion from Chromaffin cells. We have investigated the effects of histamine and other agents on the accumulation of inositol monophosphate (InsP₁) and catecholamine secretion in purified cultures of norepinephrine-containing and epinephrine-containing bovine Chromaffin cells. Histamine-stimulated InsP, accumulation in epinephrine cells was three times greater than that in norepinephrine cells. In contrast, bradykinin caused roughly equivalent increases in InsP¹ accumulation in the two Chromaffin cell subtypes. Histamine-stimulated catecholamine secretion was also greater in epinephrine cells than in norepinephrine cells, whereas high K⁺, bradykinin, phorbol 12, 13-dibutyrate, and angiotensin II all caused greater secretion from norepinephrine cells than from epinephrine cells. The density of H₁ receptors in epinephrine cells was approximately three times greater than that in norepinephrine cells. The greater density of H₁ receptors on epinephrine cells may account for the greater effects of histamine on InsP₁ accumulation and catecholamine secretion in these cells.     

The nitrite oxidizing system of Nitrobacter winogradskyi

Abstract Cytochrome components which participate in the oxidation of nitrite in Nitrobacter winogradskyi have been highly purified and their properties studied in detail. Cytochrome a ₁ c ₁ is an iron-sulphur molybdoenzyme which has haems a and c and acts as a nitrite-cytochrome c oxidoreductase. Cytochrome c -550 is homologous to eukaryotic cytochrome c and acts as the electron mediator between cytochrome a ₁ c ₁ and aa ₃-type cytochrome c oxidase. The oxidase is composed of two kinds of subunits, has two molecules of haem a and two atoms of copper in the molecule, and oxidizes actively eukaryotic ferrocytochrome c as well as its own ferrocytochrome c -550. Further, a flavoenzyme has been obtained which has transhydrogenase activity and catalyses reduction of NADP⁺ with benzylviologen radical. This enzyme may be responsible for production of NADPH in N. winogradskyi . The electron transfer against redox potential from NO₂⁻ to cythochrome c could be pushed through prompt removal by cytochrome aa ₃ of H⁺ formed by the dehydrogenation of NO₂⁻+ H₂O. As cytochrome c in anaerobically kept cell-free extracts is rapidly reduced on addition of NO₂⁻, a membrane potential does not seem necessary for the reduction of cytochrome c by cytochrome a ₁ c ₁ with NO₂⁻ in vivo.     

Isoform-Specific Effects of Apolipoproteins E2, E3, and E4 on Cerebral Capillary Sequestration and Blood-Brain Barrier Transport of Circulating Alzheimer's Amyloid β

Abstract: Cerebral capillary sequestration and blood-brain barrier (BBB) permeability to apolipoproteins E2 (apoE2), E3 (apoE3), and E4 (apoE4) and to their complexes with sAβ_1–40, a peptide homologous to the major form of soluble Alzheimer's amyloid β, were studied in perfused guinea pig brain. Cerebrovascular uptake of three apoE isoforms was low, their blood-to-brain transport undetectable, but uptake by the choroid plexus significant. Binding of all three isoforms to sAβ_1–40 in vitro was similar with a K _D between 11.8 and 12.9 n M . Transport into brain parenchyma and sequestration by BBB and choroid plexus were negligible for sAβ_1–40-apoE2 and sAβ_1–40-apoE3, but significant for sAβ_1–40-apoE4. After 10 min, 85% of sAβ_1–40-apoE4 taken up at the BBB remained as intact complex, whereas free sAβ_1–40 was 51% degraded. Circulating apoE isoforms have contrasting effects on cerebral capillary uptake of and BBB permeability of sAβ. ApoE2 and apoE3 completely prevent cerebral capillary sequestration and blood-to-brain transport of sAβ_1–40. Conversely, apoE4, by entering brain microvessels and parenchyma as a stable complex with sAβ, reduces peptide degradation and may predispose to cerebrovascular and possibly enhance parenchymal amyloid formation under pathological conditions.     

A new type of metabolism chamber for the determination of active and postprandial metabolism offish, and consideration of results for coregonid and salmon juveniles

The optomotor reaction of juvenile Coregonus schinzipalea Val. et Cuv. and Salmo salar L. was utilized to develop a circular tube metabolism chamber to measure oxygen consumption and ammonia excretion as a function of swimming speed. The metabolism chamber with a constant water flow assured the maintenance of stable conditions. The unidirectional movement of fish was measured in a circular tube with a single narrowing. The relationships between the swimming speed and oxygen consumption or ammonia excretion described by exponential equations allowed the extrapolation towards the standard metabolism, i.e., zero swimming speed. For a juvenile coregonid (0.1–0.15 g individual weight, 2.6–2.8 cm total length) standard metabolism at 14° C was estimated as 0.65 mg0₂ g⁻¹ h⁻¹ and 17.3 μg N(NH₃)g⁻¹ h⁻¹, whereas for juvenile salmon (136mg individual weight) respective values at 22° C were 0.047mg0₂g⁻¹h⁻¹ and 0.61 μg N(NH₃)g⁻¹ h⁻¹. The feeding test with juvenile salmon was also performed in this circular chamber, and in both energy and nitrogen budgets after a meal the partitioning could be precisely attributed to standard metabolism, active metabolism and specific dynamic action (in the case of oxygen consumption) or postprandial nitrogen increase.
The new metabolism chamber allowed the relationship between metabolism and swimming velocity of juvenile fish with developed rheotactic response. It could be used with adult fish for similar purposes.     

Scaling of oxygen consumption of Lake Magadi tilapia, a fish living at 37°C

Rates of oxygen consumption were measured in the geothermal, hot spring fish, Oreochromis alcalicus grahami by stopped flow respirometry. At 37° C, routine oxygen consumption followed the allometric relationship: V o₂=0.738 M ^0.75, where V o₂ is ml O₂ h ⁻¹ and M is body mass (g). This represents a routine metabolic rate for a 10 g fish at 37° C of 0.415 ml O₂ g⁻¹ h ⁻¹ (16.4 μmol O₂ g ⁻¹ h ⁻¹). Acutely increasing the temperature from 37 to 42° C significantly elevated the rate of O₂ consumption from 0.739 to 0.970 ml O₂ g ⁻¹ h ⁻¹ ( Q ₁₀=l.72). In the field, O. a. grahami was observed to be 'gulping' air from the surface of the water especially in hot springs that exceeded 40° C. O. a. grahami may utilize aerial respiration when O₂ requirements are high.     

Epinephrine, norepinephrine, and cortisol concentrations in cannulated seawater-acclimated rainbow trout (Oncorhynchus mykiss) following black-box confinement and epinephrine injection

The present study investigates the effect of cannulation and chronic'black-box' confinement, as well as epinephrine administration (4–0 μg kg⁻¹), on the degree and time-course of alterations in trout ( Oncorhynchus mykiss ) catecholamine and cortisol concentrations. Plasma cortisol concentrations in seawater trout acclimated to 3–6° C reached 104 ng ml⁻¹ 1 day after cannulation/confinement and remained elevated above resting levels (8 ng ml⁻¹) until 6 days post-confinement. Although plasma epinephrine and norepinephrine generally declined over the period of confinement (day 1 approx. 12 nM; day 7 approx. 6 nM), norepinephrine titres were usually higher and more variable. Epinephrine injection caused elevations in plasma epinephrine levels but not in norepinephrine levels; epinephrine titres reaching 107 ± 26 nM (range 65–238 nM) at 2 min post-injection and returning to pre-injection levels by 30 min post-injection. Plasma cortisol increased by 20 ng ml⁻¹ following epinephrine administration. Based on the time-course for post-confinement alterations in plasma cortisol, it appears that up to a week may be required before cannulated fish are completely acclimated to 'black-box' confinement. The findings suggest that meaningful results from experiments utilizing epinephrine injection and 'black-box confinement are contingent upon: (1) knowledge of circulating epinephrine levels shortly after injection (i.e. within 2 min post-injection); and (2) an experimental design that takes into account the elevated cortisol titres that are inherent with cannulation/confinement and epinephrine injection.     

In vitro evidence for the ionoregulatory role of rainbow trout mucus in acid, acid/aluminium and zinc toxicity

Rainbow trout body mucus dialysed with acidified distilled water at pH 7,5 and 3 experienced ion depletion which was greatest at pH 3 and minimal between pH 7 and 5. Mucus Na⁺ loss is exacerbated in the presence of 1 mg I⁻¹ aluminium as A1₂(SO₄), at pH 5 and 7. Al₂(SO₄), causes greater depletion of Na⁺ from mucus than A1C1₃. A lethal level of zinc (2 mg 1⁻¹) does not deplete mucus Na⁻ or K⁺, unlike a lethal level of aluminium (1 mg 1⁻¹) at pH 7. The results are discussed in terms of the ionoregulatory role of mucus in heavy metal and acid toxicity.