The influence of heparin, NaCl and CaCl2 on the rate of the thrombin-antithrombin III reaction

The rate of inactivation of thrombin by antithrombin III was studied in the presence of heparin using molar concentrations of the reactants in the order: antithrombin III > thrombin > heparin. Reaction rates were calculated assuming either pseudo-first-order kinetics or second-order kinetics.

Heparin, up to 0.25 μg/ml (2.2 × 10⁻² μM), affected the reaction in such a way that for each 0.1 μg added, the second-order constant increased by 17 × 10⁵ M⁻¹ min⁻¹. Increases in the concentration of antithrombin III at a constant concentration of heparin yielded higher pseudo-first-order constants but fractional contribution of heparin to the overall increment remained approximately the same. In contrast, increases in the ratio of enzyme to heparin resulted in decreases in pseudo-first-order constants.

Sodium chloride, in the range from 0.08M to 0.22M, accelerated the inactivation reaction so that an increase by 0.1M of NaCl raised the second-order constant by 12.3 × 10⁵ M⁻¹ min⁻¹. Indirect evidence was obtained supporting the view that NaCl exerts its effect by promoting the recycling of non-stoichiometric amounts of heparin.

Calcium chloride, in the range from 1.2 mM to 5.0 mM, also accelerated the inactivation reaction by an unknown mechanism which was distinct from that of NaCl. In the presence of CaCl₂, kinetics of inactivation appear to have depended on the choice of the substrate (fibrinogen or N-benzoyl-L-Phe-L-Val-L-Arg-p-nitroanilide-HCl) for measuring residual thrombin activity. Possible reasons for this difference are discussed.     

Blood-brain barrier integrity and brain water and electrolytes during hypoxia/hypercapnia and hypotension in newborn piglets

This study examines the effects of hypoxia/hypercapnia and hypoxia/hypercapnia with hypotension (hypotensive-hypoxia/hypercapnia) on blood-to-brain transfer constants (K₁) for sodium and mannitol and brain water and electrolyte contents in newborn piglets. Hypoxia/hypercapnia was induced for 60 min with the piglets breathing a gas mixture of 15% carbon dioxide, 10–12% oxygen, and 73–75% nitrogen adjusted to achieve an arterial pH < 7.15, pO₂ < 40, and pCO₂ > 60 mmHg potension for 20 min by rapid phlebotomy to achieve a mean arterial blood pressure < 40 mmHg. Piglets were studied during 1 h of, and 24 h after resuscitation from hypoxia/hypercapnia (arterial pH 6.9 ± 0.18, pO₂ 36 ± 6 mmHg, pCO₂ 38 ± 8 mmHg, mean ± S.D.) and 10 min, and 24 h after resuscitation from hypotensive-hypoxia/hypercapnia (mean arterial blood pressure 28 ± 10 mmHg, mean ± S.D.). Values for K_t for sodium and mannitol, measured using the integral technique were 15.9 and 5.2 ml·g⁻¹·min⁻¹ × 10⁴ respectively, in 2–4-day-old controls, suggesting that the barrier is fully developed in newborn piglets. Values were not different during or after hypoxia/hypercapnia or 24 after hypotensive-hypoxia/hypercapnia. Ten to forty min after hypotensive-hypoxia/hypercapnia, there was a proportional decrease in the K₁ for sodium and mannitol of about 40%. These results suggest that the newborn piglet is similar to the adult with respect to impermeabiity of the blood-brain barrier to ions and small molecules and resistance of this barrier to systemic hypoxia/hypercapnia and hypotension. We suggest that acute decreases in K₁ for sodium and mannitol after hypotensive-hypoxia/hypercapnia reflect a reduction in capillary surface area.     

Effect of changes in rate of vascular perfusion on release of substances into the effluent from the brain of the rabbit

The cerebral vasculature of five anaesthetised rabbits was perfused with a perfluorocarbon emulsion via the internal carotid arteries, and the effluent from the jugular veins analysed for ATP, substance P (SP), endothelin (ET) and arginine vasopressin (AVP). Viability of the preparation was monitored periodically by the electrocorticogram, oxygen uptake, carbon dioxide release and perfusion pressure. The basal rate of infusion of 7.8±1.26ml·min⁻¹ resulted in an infusion pressure of 114.0±22.1 mmHg and when increased first to 10.5±1.53ml·min⁻¹ and then to 15.0±1.87ml·min⁻¹, rose to 163.0±33.1mmHg and to 170.0±33.2mmHg, respectively. Between each 3-min period of increased flow the rate was returned to the basal rate for 6 min. of the four vasoactive substances, ET was released at the largest rate during the initial period of basal flow, 65.3±10.7pmol·min⁻¹. This increased further when the infusion rate rose to 10.5 ml·min⁻¹, but was significant only when the infusion rate was increased to 15.0 ml·min⁻¹. ATP was released at 41.5±11.5pmol·min⁻¹ during the initial period of basal flow. Its release significantly increased with flow and peaked at 15.0 ml·min ⁻¹. SP was released at a rate of 13.3±8.2pmol·min⁻¹ during the initial period of basal flow. Its rate of release was increased significantly the second time the flow was increased to 10.5 ml·min⁻¹ and increased even further when the flow was increased to 15.0 ml·min⁻¹. AVP was detected in one third of the samples collected during the initial period of basal flow, being released at a rate of 0.65±047pmol·min⁻¹. Only in the period following the 15.0 ml·min⁻¹ rate of flow was there a sustained increased release of AVP. These results are consistent with the view that ATP, SP, ET and AVP are released from vascular endothelial cells during changes in flow, and may contribute to the adjustments in local cerebrovascular tone.     

Characterization of electrogenic Na/K pump in rat neostriatal neurons

The electrogenic Na/K pump current (Ip) was studied in the dissociated neostriatal neurons of the rat by using the nystatin-perforated patch recording mode. The Ip was activated by external K⁺ in a concentration-dependent manner with an EC₅₀ of 0.7 mM at a holding potential (V_H) of −40 mV. Other monovalent cations also caused Ip and the order of potency was Tl⁺>K⁺, Rb⁺>NH₄⁺, Cs⁺>>>Li⁺. The Ip decreased with membrane hyperpolarization in an external solution containing 150 mM Na⁺, while the Ip did not show such voltage dependency without external Na⁺. Ouabain showed a steady-state inhibition of Ip in a concentration- and temperature-dependent manner at a V_H of −40 mV. The IC₅₀ values at 20 and 30°C were 7.1×10⁻⁶ and 1.3×10⁻⁶ M, respectively. The decay of Ip after adding ouabain well fitted with a single exponential function. At a V_H of −40 Mv, the association (k₊₁) and dissociation (k₋₁) rate constants estimated from the time constant of the current decay at 20°C were 4.0×10² s⁻¹ M⁻¹ and 6.3×10⁻³ s⁻¹, respectively. At 30°C, k₊₁ increased to 2.8×10³ s⁻¹ M⁻¹ while k₋₁ showed no such change with a value of 1.8×10⁻³ s⁻¹. A continuous Na⁺ influx was demonstrated by both the Na⁺-dependent leakage current and tetrodotoxin-sensitive Na⁺ current, which resulted in the continuous activation of the Na/K pump. It was thus concluded that the Na/K pump activity was well-maintained in the dissociated rat neostriatal neurons with distinct functional properties and that the activity of the pump was tightly connected with Na⁺ influxes.     

Cerebral oxygen transport and metabolism during graded isovolemic hemodilution in experimental global ischemia

To verify the optimal hematocrit (Hct) level in the treatment of cerebral ischemia, cerebral oxygen transport (CTO₂) and cerebral oxygen metabolism (CMRO₂) in graded isovolemic hemodilution were evaluated during cerebral ischemia. Isovolemic hemodilution with low molecular weight dextran to stepwise lower Hct from 43% to 36%, 31%, and 26% was carried out in 13 splenectomized dogs, 6 h after global cerebral ischemia. Global ischemia of the animals was produced by multiple intra- and extracranial ligations of cerebral arteries. Cerebral blood flow (CBF) was measured with radioisotope labeled microspheres. CTO₂, CMRO₂, and oxygen extraction fraction (OEF) were calculated from CBF, arterial oxygen content (CaO₂), and venous oxygen content (CvO₂). In dogs with global cerebral ischemia, CBF increased with graded isovolemic hemodilution (r=−0.73, P<0.05). CTO₂ reached its highest value at a Hct level of 31.3%. CTO₂ at Hct of 36.1% and 31.3% was statistically different from the value measured at a Hct of 43.3%, and there was a decrease when Hct was lowered to 25.9%. CMRO₂ was the highest when Hct was at 31.3% and differed significantly from the value measured at a Hct of 43.3%. There was a 10% increase of OEF when Hct was at 25.9%; however this change was not statistically significant compared with the OEF at Hct of 36.1% and 31.3%, respectively. These findings indicate that CTO₂ and CMRO₂ were the highest when Hct was reduced to 31% in hemodilution. Hct at 31% is the optimum for cerebral metabolism in ischemic status. Uncoupling of CTO₂, CMRO₂ with CaO₂ was also observed in this study. This phenomenon suggests that hemodilution to augment cerebral circulation may be at least partially attributed to the beneficial effects of hemorheologic improvement in the microcirculation of the ischemic brain.     

Binding of [125I]insulin to specific receptors and stimulation of nucleotide incorporation in cells cultured from rat brain

The occurrence of insuling receptors and biological responses to insulin has been investigated in trypsin-dissociated fetal rat brain cells maintained in culture for 8 days. Binding of [¹²⁵]insulin to brain cells in culture was time- and pH-dependent and 85–90% specific. Porcine insulin competed for [¹²⁵]insulin binding in a dose-dependent manner. Unrelated polypeptides, including angiotensin II, glucagon, bovine growth hormone, and bovine prolactin did not compete for [¹²⁵]insulin binding. The half-life of [¹²⁵]insulin dissociation from receptors at 24°C was 15 min and a plot of ln[B/Bo] vs time suggested two dissociation rate constants of2.7 × 10⁻⁴ sec⁻¹ and5.0 × 10⁻⁵ sec⁻¹. Scatchard analysis of the binding data gave a curvelinear plot which may indicate negative cooperativity or the occurrence of both high affinity(K_a = 2 × 10¹¹M⁻¹) and low affinity(K_a = 4 × 10¹⁰M⁻¹) sites. Of the estimated total of 4.9 × 10⁴ binding sites per cell, 28–30% appear to be high affinity sites.

Incubation of cultures with insuling caused a time- and dose-dependent stimulation of [³H]thymidine and [³H]uridine incorporation into TCA-precipitable material. Maximum stimulation of thymidine incorporation (2–5-fold) occured 11 h after incubation with 167 nM insulin. The same concentration of insulin caused a 2.2-fold increase in [³H]uridine incorporation in 2 h. These results indicate that cells cultured from rat brain contain specific insulin receptors capable of mediating effects of insulin on macromolecular synthesis in the central nervous system.     

Differences in uptake kinetics of cis-3-aminocyclohexane carboxylic acid into neurons and astrocytes in primary cultures

The uptake of [³H]ACHC and [³H]GABA into cultured neurons and astrocytes was studied. [³H]ACHC uptake was less efficient than that of GABA in both cell types and K_m values for ACHC uptake into neurons and astrocytes were 40.3 μM and 210.8 μM, respectively. The corresponding V_max values were 0.321 and 0.405 nmol·min⁻¹·mg⁻¹ cell protein, respectively. Kinetic studies of the effects of GABA on ACHC uptake and vice versa showed that GABA is a linear competitive inhibitor of ACHC uptake in both cell types with a K_i value of 15 μM. On the other hand, ACHC turned out to be a complex inhibitor of astrocytic GABA uptake being competitive at lower concentrations and non-competitive at higher concentrations. ACHC inhibited GABA uptake into neurons competitively with a K_i of 69 μM. It is concluded that ACHC acts primarily on neuronal GABA uptake sites but its uptake is much more complicated than hitherto anticipated.     

Effects of prostaglandin E2, forskolin and cholera toxin on cAMP production and in vitro LH-RH release from the rat hypothalamus

The present study attempts to elucidate the possible role of adenosine 3′,5′-monophosphate (cAMP) and prostaglandin E₂ (PGE₂) in the function of the neural luteinizing hormone-releasing hormone (LH-RH) apparatus. To this end, in vitro LH-RH release from superfused hypothalamic fragments and cAMP production by hypothalamic P₂ membrane fractions were measured. Immature female rats (day 28) were ovariectomized and implanted with Silastic capsules containing estradiol (235 μg/ml). Two days later, animals were sacrificed and the mediobasal hypothalamic preoptic area (hypothalamic units or fragments) were removed. To examine in vitro LH-RH release from superfused hypothalamic fragments, effluents were collected into tubes on ice at 10-min intervals and LH-RH concentration was determined by radioimmunoassay (RIA). Following a 50-min control period, a step-wise increment in several doses of PGE₂ (each dose for a 50-min interval) evoked a dose-related increase in LH-RH release. PGE₂ induced significant (P<0.01) increments in LH-RH release at doses of 5.68 × 10⁻⁷, 5.68 × 10⁻⁶, and 5.68 × 10⁻⁵ M, respectively. When adenylate cyclase activators, such as forskolin and cholera toxin were infused in a step-wise manner (each dose for a 50-min interval) following a 50-min control period, a dose-related increase in LH-RH release was also obtained; forskolin and cholera toxin significantly (P<0.01) stimulated LH-RH release at doses of 1 × 10⁻⁴ and 5.4 × 10⁻¹⁰ M, respectively. These two substances were ineffective in stimulating LH-RH release when hypothalamic fragments were superfused in calcium-free plus EGTA (10 mM) containing medium. An intermittent infusion of dibutyryl cAMP (dbcAMP: 1 × 10⁻⁷ M, 10-min on, 20-min off) resulted in rhythmic LH-RH release from median eminences superfused in vitro. In separate experiments, to examine adenylate cyclase activity, P₂ membrane fractions from the mediobasal hypothalamus were preincubated with appropriate test agents. Adenylate cyclase reaction was initiated by adding adenosine triphosphate. After a 15-min incubation, the reaction was terminated by boiling, the supernatant recovered and subjected to cAMP determination by RIA. The following results were obtained: (1) in vivo E priming of ovariectomized animals significantly increased basal adenylate cyclase activity of P₂ membrane preparations as compared to those from unprimed rats; (2) known adenylate cyclase activators, such as forskolin and cholera toxin clearly produced a dose-related increase in cAMP production; and (3) PGE₂ at the concentration of 5.68 × 10⁻⁶ M stimulated cAMP production. It appears that cAMP and PGE₂ may be involved in the activation of the LH-RH neural apparatus. It is tempting to postulate that PGE₂ may stimulate adenylate cyclase to increase intracellular cAMP levels which, in turn, trigger the release of LH-RH from the median eminence nerve terminals.     

Adenosine A1 receptor-mediated inhibition of evoked glutamate release is coupled to calcium influx decrease in goldfish brain synaptosomes

Binding of [³H]cyclohexyladenosine (CHA) to the cellular fractions and P₂ subfractions of the goldfish brain was studied. The A₁ receptor density was predominantly in synaptosomal membranes. In goldfish brain synaptosomes (P₂), 30 mM K⁺ stimulated glutamate, taurine and GABA release in a Ca²⁺-dependent fashion, whereas the aspartate release was Ca²⁺-independent. Adenosine, R-phenylisopropyladenosine (R-PIA) and CHA (100 μM) inhibited K⁺-stimulated glutamate release (31%, 34% and 45%, respectively). All of these effects were reversed by the selective adenosine A₁ receptor antagonist, 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, K⁺ (30 mM) stimulated Ca²⁺ influx (46.8±6.8%) and this increase was completely abolished by pretreatment with 100 nM ω-conotoxin. Pretreatment with 100 μM R-PIA or 100 μM CHA, reduced the evoked increase of intra-synaptosomal Ca²⁺ concentration, respectively by 37.7±4.3% and 39.7±9.0%. A possible correlation between presynaptic A₁ receptor inhibition of glutamate release and inhibition of calcium influx is discussed.     

The kinetic and structural characterization of the reaction of nafamostat with bovine pancreatic trypsin

Nafamostat mesilate (FUT-175), a synthetic serine protease inhibitor, is active against a number of the serine proteases involved in coagulation. This has been proposed as the basis of its anticoagulant activity. We investigated the reaction of Nafamostat with bovine pancreatic trypsin as a model system. It was shown to act as a time-dependent competitive inhibitor, and the inhibition constants for the binding of Nafamostat to trypsin (i.e., K_i) and the overall inhibition constants (i.e., K_i^*) were calculated to be 11.5 μM and 0.4±0.14 nM, respectively. The second-order rate constant for the reaction was 4.5±0.19×10⁵ M⁻¹s⁻¹, and the product released following the acylation step, 6-amidino-2-naphthol, showed mixed-type inhibition. The competitive (K_ic) and uncompetitive (K_iu) inhibition constants were 14.7 μM and 19.5 μM, respectively. Formation of the acyl-enzyme intermediate was dissected into at least two steps, with rates of 0.9 s⁻¹ and 195 s⁻¹. The deacylation step was relatively much slower (3.2±0.19×10⁻⁵ s⁻¹), enabling the mass spectroscopic analysis of the acyl-enzyme intermediate, which confirmed the covalent attachment of 4-guanidinobenzoic acid to trypsin. The product of the deacylation step, 4-guanidinobenzoic acid, showed no inhibition up to a concentration of 200 μM. These data strongly suggest that while Nafamostat is a potent inhibitor of trypsin, it is actually an extremely poor substrate, and that apparent inhibition is due to the competitive formation of a very stable acyl-enzyme intermediate, analogous to some other active site titrants.     

High metabolic rates of 4-hydroxynonenal in brain capillary endothelial cells during hypoxia/reoxygenation

We measured the accumulation of 4-hydroxynonenal (HNE), a major lipid peroxidation product during hypoxia/reoxygenation of brain capillary endothelial cells (BCEC). The concentration of HNE after 2 h of hypoxia was 0.23 nmol/mg protein and rose up to 0.28 nmol/mg protein after 30 min of reoxygenation. That reflects a 1.5-fold increase, whereas aortic endothelial cells (AEC) increased the HNE level 5-fold, compared to the control. Therefore, the ability of BCEC to degrade exogenously added HNE was tested. The HNE consumption in BCEC achieved a rate of about 600 nmol · min⁻¹ mg protein⁻¹, about two times higher than in AEC. The higher ability of BCEC to degrade FINE is probably the reason of the 2-fold higher IC₅₀ value against the aldehyde. Therefore, we concluded that the high ability of BCEC to degrade HNE is a substantial part of the secondary antioxidative defense of the brain.     

pO2-dependent distribution of potassium in hippocampal slices of the guinea pig

The distribution of extracellular K⁺-concentration (cK_s⁺) in 200–1000-μm thick hippocampal slices was studied with ion-selective microelectrodes. In ca. 500-μm thick slices cK_s⁺ increased from the surface to the innermost layers by ca. 2 mmol/liter if the pO₂ of the bath (p_BO₂) ranged from 300–600 mm Hg and if the temperature was 28 °C. In thicker slices and lowered pO₂-values further elevations of cK_s⁺ were observed. In vital slices thinner than 500 μm cK_s⁺-values exceeded the potassium-concentration of the bath (CK_B⁺) only when p_BO₂ was markedly lowered. When p_BO₂ was reincreased in such thin slices, cK_s⁺ rapidly declined and often decreased transiently below ck_B⁺. Similar undershoots of cK_s⁺ were observed when cK_B⁺ was lowered from high to normal levels. The rapid decline was blocked by hypoxia, ouabain, antimycine and a temperature of 18 °C. A stepwise rise of cK_B⁺ also caused rapid changes of cK_s⁺ in vital thin slices. The rates of changes, however, were hardly affected e.g. by a transient hypoxia. Diffusion did not contribute significantly to these steep changes of cK_s⁺. These rapid distribution modes were widely missing in slices thicker than 500 μm. Therefore in such preparations, the extracellular microenvironment of neurons may markedly differ from the ionic concentrations in the bath.     

Impact of exercise training on plasma adrenocorticotropin response to a well-learned vigilance task

Plasma adrenocorticotropin hormone (ACTH), cortisol, heart rate, self-reported state anxiety and total reaction time responses in physically active (n=8) and sedentary (n=11) middle-aged men were compared during performance of a well-learned, psychologically challenging task. The groups were similar in age (active: 39.9±8.4 yr vs. sedentary: 44.4±7.2 yr) and trait anxiety (29.4±5.2 vs. 34.5±12.6), but they had different maximal oxygen consumption relative to lean body weight (68.6±7.1 vs. 44.7±4.6 ml · kg⁻¹ · min⁻¹). During performance of the psychological protocol, the total group had significant increases in heart rate and state anxiety, whereas both ACTH and cortisol remained near baseline levels. ACTH and cortisol were not different between the groups. The sedentary group exercise trained for 4 mo, which resulted in an 18% improvement in maximal aerobic capacity ( O₂ max). Exercise intervention did not significantly alter the neuroendocrine response compared to pre-training values or compared to a corresponding control group. These findings suggest that enhanced cardiovascular fitness resulting from exercise training does not affect anterior pituitary-adrenocortical activity during performance of a practiced vigilance task.     

The primary wave of epinephrine-induced platelet aggregation represents alpha 2-adrenoceptor status

We examined relationships between epinephrine-induced slope of primary wave of aggregation and the alpha₂-adrenoceptor status on platelets. A concentration (10^-9 to 10^-6 )-dependent increase in slope of primary wave with EC50 of epinephrine at 4.5 ± 0.4 × 10^-7 was observed. In studies on epinephrine binding to alpha₂adrenoceptors in competition with ³H-yohimbine to platelets, (IC₅₀) of epinephrine was 4.8 ± 3.4 x 10^-7 M. There was a significant (P<0.02) correlation between EC₅₀ of epinephrine to evoke biological response and IC₅₀ of epinephrine to bind to alpha₂-adrenoceptors (r-0.75). There was no relationship between number of receptor sites or dissociation constant of ³H-yohimbine binding and primary wave of platelet aggregation. These data show that the slope of primary wave in response to epinephrine reflects alpha₂-adrenoceptor binding of the agonist.     

Opiate peptide receptors on intact NIE-115 neuroblastoma: radioligand binding properties, intracellular response, and effects of increasing membrane cholesterol

1. Binding of [³H]methionine-enkephalin to intact NIE-115 neuroblastoma cells (competing ligand: naloxone) revealed a homogenous population of receptors with a density (B_max) of 79.0 ± 6.5 mol/mg protein (mean SEM, N=3) and an apparent K, of 5.33 ± 1.63 mM.

2. The order of displacement of [³H]met-enkephalin was met-/leu-enkephalin > naloxone > morphine, suggesting that it is of the delta receptor class.

3. Specific binding was heat-labile, stereospecific and sensitive to Na⁺.

4. Adding met-enkephalin to intact neuroblastoma caused reductions of both basal and prostaglandin E₁-stimulated levels of cyclic AMP (41.4 ± 4.0% (N=6) and 45.1 ± 2.4% (N=3) of control levels, respectively). Maximum inhibition (naloxone-reversible) was observed as low as 10⁻⁷ M met-enkephalin.

5. Preliminary results suggest that cells grown in cholesterol-supplemented medium show reduced binding of [³H]met-enkephalin.     

Vasopressin induced cyclooxygenase dependent superoxide generation contributes to K(+) channel function impairment after brain injury

This study determined if vasopressin generates superoxide anion (O⁻₂) in a cyclooxygenase dependent manner and if such production contributes to impairment of dilation to activators of ATP sensitive K⁺ (K_ATP) and calcium sensitive K⁺ (K_ca) channels following fluid percussion brain injury (FPI) in newborn pigs equipped with closed cranial windows. Superoxide dismutase (SOD) inhibitable nitroblue tetrazolium (NBT) reduction was determined as an index of O⁻₂ generation. Under non-brain injury conditions, topical vasopressin (40 pg/ml, the concentration present in CSF following FPI) increased SOD inhibitable NBT reduction from 1±1 to 25±4 pmol/mm². Indomethacin, a cyclooxygenase inhibitor, blunted such NBT reduction (1±1 to 5±1 pmol/mm²), while the vasopressin antagonist, l-(β-mercapto-β β-cyclopentamethylene propionic acid) 2-(o-methyl)-Tyr-AVP (MEAVP) blocked NBT reduction. MEAVP and indomethacin also blunted the NBT reduction observed after FPI. Under non-brain injury conditions, vasopressin (40 pg/ml) coadministered with the K_ATP and K_ca channel agonists, cromakalim and NS1619 (10⁻⁸, 10⁻⁶ M) diminished dilation to these K⁺ channel agonists while indomethacin partially prevented such impairment (13±1 and 23±1 vs. 4±1 and 10±2 vs. 8±1 and 19±1% for cromakalim in untreated, vasopressin, and vasopressin plus indomethacin treated piglets, respectively). Cromakalim and NS1619 induced pial artery dilation was attenuated following FPI, while indomethacin or MEAVP preadministration partially prevented such impairment (13±1 and 23±1, sham control; 1±1 and 4±1, FPI; 8±1 and 16±3%, FPI–indomethacin pretreated for responses to cromakalim 10⁻⁸, 10⁻⁶ M, respectively). These data show that vasopressin increased O⁻₂ production in a cyclooxygenase dependent manner and contributed to this production after FPI. These data also show that vasopressin blunted K_ATP and K_ca channel mediated cerebrovasodilation in a cyclooxygenase dependent manner. These data suggest that vasopressin induced cyclooxygenase dependent O⁻₂ generation contributes to K_ATP and K_ca channel function impairment after FPI.     

The resolution of dopamine and beta 1- and beta 2-adrenergic-sensitive adenylate cyclase activities in homogenates of cat cerebellum, hippocampus and cerebral cortex.

The stimulation of adenylate cyclase by dopamine and various β-adrenergic agonists has been investigated in homogenates from 3 areas of cat brain: the cerebral cortex, cerebellum and hippocampus. The purpose of the study was to determine whether the β-adrenergic receptors coupled to adenylate cyclase could be classified as either β₁ and β₂ subtypes in the different regions studied.

The stimulation of adenylate cyclase by the β-adrenergic agonist, (−)isoproterenol (5 × 10⁻⁶M), was completely blocked by the specific β-adrenergic antagonist, (−)alprenolol (10⁻⁵ M), but not by the dopaminergic antagonist, fluphenazine (10⁻⁵ M), whereas the stimulation of adenylate cyclase by (−)epinephrine (10⁻⁴ M) was blocked to varying extents by these two drugs in each of the 3 regions studied. The (−)epinephrine effect was always blocked in the combined presence of (−)alprenolol and fluphenazine. The adenylate cyclase stimulation by (−)epinephrine which is not blocked by (−)alprenolol was due to interaction of (−)epinephrine with a dopaminergic-sensitive adenylate cyclase which has been characterized in cerebral cortex, hippocampus and cerebellum.

Regional differences in the affinity of β-adrenergic-sensitive adenylate cyclase for various agonists were investigated in the presence of fluphenazine (10⁻⁵ M). In the cerebellum the potency order was (±)protokylol> (±)hydroxybenzylisoproterenol> (±)isoproterenol> (−)epinephrine> (±)salbutamol> (−)norepinephrine, indicating the presence of a β₂-adrenergic receptor. In the cerebral cortex the potency order was (−)isoproterenol> (±)protokylol> (±)hydroxybenzylisoproterenol> (−)epinephrine= (−)norepinephrine((±)salbutamol being inactive). A similar pattern was found in the hippocampus indicating the presence of a β₁-adrenergic receptor in these two regions. (±)Salbutamol was a partial agonist in the cerebellum and a competitive antagonist in the cerebral cortex.

The ratio of the antagonist potencies of (±)practolol and (±)butoxamine preferential β₁- and β₂-adrenergic antagonists respectively, to block the stimulation of adenylate cyclase was 25 in the cerebellum, compared to 0.5 in the cerebral cortex and 1.6 in the hippocampus. These results confirm the presence of a β₂ subtype of receptor coupled to adenylate cyclase in the former and β₁ subtypes in the latter two regions. The comparison between the affinities of a series of β-adrenergic agonists and antagonists for the β-adrenergic receptors coupled with an adenylate cyclase in cerebral cortex and cerebellum with their affinities for well characterized β₂-adrenergic receptors in lung and β₁-adrenergic receptor in heart substantiated this conclusion.     

Physiological roles of glycine and γ-aminobutyric acid in dissociated neurons of rat visual cortex

The effects of glycine (Gly) and γ-aminobutyric acid (GABA) on the neurons acutely dissociated from rat visual cortex (VC) were investigated in the whole-cell mode using a conventional patch-clamp technique. GABA and Gly evoked Cl⁻ currents (I_Cl) in a concentration-dependent manner at a holding potential (V_H) of −50 mV. The half maximum effective concentrations (EC₅₀) were 4.64 × 10⁻⁶ M for GABA and 6.67 × 10⁻⁵ M for Gly. Strychnine and bicuculline reversively inhibited both 10⁻⁵ M GABA -and 10⁻⁴ M Gly-induced I_Cl in a concentration-dependent manner. The half maximum inhibitory concentrations (IC₅₀) of strychnine on GABA- and Gly-induced currents were 4.00 × 10⁻⁶ M and 8.26 × 10⁻⁸ M, respectively. The IC₅₀ values of bicuculline on GABA and Gly responses were 1.18 × 10⁻⁶ M and 2.97 × 10⁻⁴, respectively. GABA at 10⁻⁵ M, which is near the EC₅₀ of the GABA response, induced I_Cl in all neurons tested n = 83). However, Gly of 10⁻⁴ M, which is near the the Gly response, induced I_Cl in 34 out of 83 neurons tested (41%). Moreover, the maximum amplitude of the Gly response was about 60% of that of the GABA response. On the other hand, the enhancement of N-methyl-d-aspartate (NMDA, 3 × 10⁻⁴M) response by Gly (10⁻⁶ M) was observed in all neurons (n=36) whether they had the Gly-induced I_Cl or not. In conclusion, it was proved that the VC neurons of immature rat could be classified into two classes, i.e. Gly sensitive and Gly insensitive in respect to activation of Cl⁻ channels. There were no significant correlations between the Gly response and the facilitatory action of Gly on the NMDA response, nor between the Gly and GABA responses. The GABA response was closely correlated to the NMDA response, having a correlation coefficient of 0.86.     

Differences in cation transport properties of primary astrocyte cultures from mouse and rat brain

⁴²K and²²Na contents and unidirectional fluxes, as well as net accumulation of⁴²K in response to elevated extracellular K⁺, were investigated in primary cultures of astrocytes prepared from neonatal rat and mouse brain. The major difference between both species affected the unidirectional K⁺ influx which was up to 75 times higher in mouse as compared to rat cultures. The flux rates in mouse astrocytes were doubled by measuring uptake in salt solution instead of growth medium, while⁴²K influx in rat astrocytes was unaffected by such treatment.²²Na transport was very similar in astrocytes from both species. The length of culture period and treatment with DBcAMP (2′,3′-dibutyryl cyclic adenyl monophosphate) modified K⁺ transport but not Na⁺ transport. Both types of cultures showed the same accumulation of⁴²K in response to raised medium K⁺. Amiloride inhibited⁴²K influx by 41% and 13% in mouse and rat cultures, respectively. In contrast, furosemide inhibited⁴²K uptake in rat astrocytes cultures by 50% but had no effect on mouse astrocyte cultures. 50 μM barium chloride markedly inhibited⁴²K uptake in mouse cultures by 96% (or 1491 nmol·mg⁻¹·min⁻¹), but inhibited⁴²K uptake in rat cultures by only 23% (or 9 nmol·mg⁻¹·min⁻¹). Ouabain was similarly effective in both types of astrocyte cultures. We conclude that Na⁺ transport as well as net K⁺ accumulation and Cl⁻ transport (based on previous studies) properties are reasonably stable and reproduced in primary cultures from both mouse and rat brain. However, unidirectional K⁺ influx showed a large species difference and also a larger variation due to differences in culturing conditions. There were also differences in the sensitivity to some inhibitors. Possible causes could be differences in the number or characteristics of K⁺ channels as well as other specific K⁺ transport systems.