Acidosis, Acetazolamide, and Amiloride: Effects on 22Na Transfer Across the Blood-Brain and Blood-CSF Barriers

Sprague-Dawley rats were given treatments, known to decrease 22Na movement into choroid plexus and CSF, to investigate their effect on 22Na transfer across the cerebral capillaries. Acidic salts, acetazolamide, or amiloride was injected intraperitoneally into bilaterally nephrectomized rats, and the rate of 22Na uptake into parietal cortex, pons-medulla, and CSF was determined at 12, 18, and 24 min. Severe acidosis (arterial pH 7.2), produced by HCl injection, decreased the rate of 22Na entry into both brain regions and CSF by 25%, whereas mild acidosis (pH 7.3) from NH4Cl injection reduced brain entry by 18%, but CSF entry by only 10%. Like HCl acidosis, amiloride reduced transport into both brain and CSF by 22%. Penetration of 22Na into parietal cortex was unchanged by acetazolamide, but that into CSF was slowed 30%. Since uptake of 22Na into cortical regions is primarily movement of tracer across the cerebral capillaries when tracer uptake time is less than 30 min, the results indicate that both metabolic acidosis and amiloride decrease Na+ permeativity at the cerebral capillaries as well as at the choroid plexus. Acetazolamide, on the other hand, alters Na+ movement only across the choroidal epithelium.     

Ischemic Modification of Cerebrocortical Membranes: 5-Hydroxytryptamine Receptors, Fluidity, and Inducible In Vitro Lipid Peroxidation

The effect of ischemia on the properties of 5-hydroxytryptamine1A + B (5-HT1A+B) and 5-hydroxytryptamine1B (5-HT1B) binding sites, physical-state "fluidity" of the membrane, and its susceptibility to peroxidation in vitro was investigated in the cerebral cortex of gerbils. Ischemia was induced by bilateral carotid artery occlusion for 15 min alone or with release for 1 h. Ischemia both with and without reflow decreased the number of 5-HT1A + B and 5-HT1B binding sites, whereas ischemia and reflow altered the affinity for 5-HT1B binding sites. Resistance to the temperature-dependent increase in "fluidity" of the membrane was detected (by fluorescence anisotropy using 1,6-diphenyl-1,3,5-hexatriene as a probe) after ischemia and reflow but not in ischemia alone. Susceptibility of the membranes to Fe2+- and ascorbic acid-stimulated lipid peroxidation in vitro was decreased following ischemia and recirculation only. These findings strongly suggest that the composition and the function of the membrane are markedly disturbed during recirculation after ischemia.     

Effects of Hypoglycaemia and Hypoxia on the Intracellular pH of Cerebral Tissue as Measured by 31P Nuclear Magnetic Resonance

Changes in high-energy phosphate metabolites and the intracellular pH (pHi) were monitored in cerebral tissue during periods of hypoglycaemia and hypoxia using 31P nuclear magnetic resonance spectroscopy. Superfused brain slices were loaded with deoxyglucose at a concentration shown not to impair cerebral metabolism, and the chemical shift of the resulting 2-deoxyglucose-6-phosphate (DOG6P) peak was used to monitor the pHi. In some experiments with low circulating levels of Pi, the intracellular Pi was visible and indicated a pH identical to that of DOG6P, an observation validating its use as an indicator of pHi in cerebral tissue. The pHi was found to be unchanged during moderate hypoglycaemia; however, mild hypoxia (PO2 = 16.4 kPa) and severe hypoglycaemia produced marked reductions from the normal of 7.2 to 6.8 and 7.0, respectively. Hypoglycaemia caused a fall in the level of both phosphocreatine (PCr) and ATP, whereas hypoxia affected PCr alone, as shown previously. However, the fall in pHi was similar during the two insults, thus indicating that the change in pH is not directly linked to lactate production or to the creatine kinase reaction.     

Somatostatin binding to dissociated cells from rat cerebral cortex

A method has been developed for the study of somatostatin (SS) binding to dissociated cells from rat cerebral cortex. Binding of [¹²⁵I][Tyr¹¹]SS to cells obtained by mechanical dissociation of rat cerebral cortex was dependent on time and temperature, saturable, reversible and highly specific. Under conditions of equilibrium, i.e., 60 min at 25°C, native SS inhibited tracer binding in a dose-dependent manner. The Scatchard analysis of binding data was linear and yielded a dissociation constant of 0.60±0.08 nM with a maximal binding capacity of 160±16 fmol/mg protein. The binding of [¹²⁵I][Tyr¹¹]SS was specific as shown in experiments on tracer displacement by the native peptide, SS analogues, and unrelated peptides.     

Nicotinic Autoreceptors Mediating Enhancement of Acetylcholine Release Become Operative in Conditions of "Impaired" Cholinergic Presynaptic Function

Abstract: The existence in the mammalian CNS of release-inhibiting muscarinic autoreceptors is well established. In contrast, few reports have focused on nicotinic autoreceptors mediating enhancement of acetylcholine (ACh) release. Moreover, it is unclear under what conditions the function of one type of autoreceptor prevails over that of the other. Rat cerebrocortex slices, prelabeled with [³H]choline, were stimulated electrically at 3 or 0.1 Hz. The release of [³H]ACh evoked at both frequencies was inhibited by oxotremorine, a muscarinic receptor agonist, and stimulated by atropine, a muscarinic antagonist. Nicotine, ineffective at 3 Hz, enhanced [³H]ACh release at 0.1 Hz; mecamylamine, a nicotinic antagonist, had no effect at 3 Hz but inhibited [³H]ACh release at 0.1 Hz. The cholinesterase inhibitor neostigmine decreased [³H]ACh release at 3 Hz but not at 0.1 Hz; in the presence of atropine, neostigmine potentiated [³H]ACh release, an effect blocked by mecamylamine. In synaptosomes depolarized with 15 mM KCI, ACh inhibited [³H]ACh release; this inhibition was reversed to an enhancement when the external [Ca²⁺] was lowered. The same occurred when, at 1.2 mM Ca²⁺, external [K⁺] was decreased. Oxotremorine still inhibited [³H]ACh release at 0.1 mM Ca²⁺. When muscarinic receptors were inactivated with atropine, the K⁺ (15 mM)-evoked release of [³H]ACh (at 0.1 mM Ca²⁺) was potently enhanced by ACh acting at nicotinic receptors (EC₅₀? 0.6 µM). In conclusion, synaptic ACh concentration does not seem to determine whether muscarinic or nicotinic autoreceptors are activated. Although muscarinic autoreceptors prevail under normal conditions, nicotinic autoreceptors appear to become responsive to endogenous ACh and to exogenous nicotinic agents under conditions mimicking impairment of ACh release. Our data may explain in part the reported efficacy of cholinesterase inhibitors (and nicotinic agonists) in Alzheimer's disease.     

Global Brain Ischemia and Reperfusion: Modifications in Eukaryotic Initiation Factors Associated with Inhibition of Translation Initiation

Abstract: We used in vitro translation and antibodies against phosphoserine and the eukaryotic initiation factors eIF-4E, eIF-4G, and eIF-2α to examine the effects of global brain ischemia and reperfusion on translation initiation and its regulation in a rat model of 10 min of cardiac arrest followed by resuscitation and 90 min of reperfusion. Translation reactions were performed on postmitochondrial supernatants from brain homogenates with and without aurintricarboxylic acid to separate incorporation due to run-off from incorporation due to peptide synthesis initiated in vitro. The rate of leucine incorporation due to in vitro-initiated protein synthesis in normal forebrain homogenates was ∼0.4 fmol of leucine/min/µg of protein and was unaffected by 10 min of cardiac arrest, but 90 min of reperfusion reduced this rate 83%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blots of these homogenates showed that neither 10 min of global brain ischemia nor 90 min of reperfusion induced significant alterations in the quantity or serine phosphorylation of eIF-4E. However, we observed in all 90-min-reperfused samples eIF-4G fragments that also bound eIF-4E. The amount of eIF-2α was not altered by ischemia or reperfusion, and immunoblotting after isoelectric focusing did not detect serine-phosphorylated eIF-2α in normal samples or in those obtained after ischemia without reperfusion. However, serine-phosphorylated eIF-2α was uniformly present after 90 min of reperfusion and represented 24 ± 3% of the eIF-2α in these samples. The serine phosphorylation of eIF-2α and partial fragmentation of eIF-4G observed after 90 min of reperfusion offer an explanation for the inhibition of protein synthesis.     

Kinetics of tert-[35S]Butylbicyclophosphorothionate Binding in the Cerebral Cortex of Newborn and Adult Rats: Effects of GABA and Receptor Desensitization

Abstract: The effects of GABA on the kinetics of tert -[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding to the convulsant site of GABA_A receptors were studied in membrane suspensions from the cerebral cortex of newborn (1-day-old) and adult (90-day-old) rats. TBPS dissociation was biphasic in neonates and adults, indicating that more than one interconvertible state of [³⁵S]TBPS binding sites may be present in the cerebral cortex. In the absence of GABA, the fast ( t _1/2, 11 min) and slow ( t _1/2, 77 min) components of TBPS dissociation in newborn rats were approximately fourfold slower than in adults. The acceleration of the dissociation rates caused by 2 µ M GABA, however, was more robust in neonates than in adults (six- to ninefold vs. twofold increase, respectively). Moreover, the dissociation rates of TBPS in membranes preincubated with 2 µ M GABA (dissociation started by adding 40 µ M picrotoxin) were two- to fourfold slower than in membranes preincubated without GABA (dissociation started by adding 40 µ M picrotoxin plus 2 µ M GABA). Taken together, these results suggest that (1) the closed state of GABA_A receptors is associated with a more effective steric barrier for the binding of TBPS in neonates compared with adults, (2) GABA produces a larger acceleration of the binding kinetics of TBPS in neonates than in adults, and (3) long incubations with GABA may cause receptor desensitization, which in turn slows down the dissociation rates of TBPS.     

Effects of brain ischemia on intermediate filaments of rat hippocampus

Neurofilaments subunits (NF-H, NF-M, NF-L) and glial fibrillary acidic protein (GFAP) were investigated in the hippocampus of rats after distinct periods of reperfusion (1 to 15 days) following 20 min of transient global forebrain ischemia in the rat. In vitro [¹⁴Ca]leucine incorporation was not altered until 48 h after the ischemic insult, however concentration of intermediate filament subunits significantly decreased in this period. Three days after the insult, leucine incorporation significantly increased while the concentration NF-H, NF-M, and NF-L were still diminished after 15 days of reperfusion. In vitro incorporation of³²P into NF-M and NF-L suffered immediately after ischemia, but returned to control values after two days of reperfusion. GFAP levels decreased immediately after ischemia but quickly recovered and significantly peaked from 7 to 10 days after the insult. These results suggest that transient ischemia followed by reperfusion causes proteolysis of intermediate filaments in the hippocampus, and that proteolysis could be facilitated by diminished phosphorylation levels of NF-M and NF-L.     

Is the Mg2+-ATP-dependent proton pumping activity of the synaptic vesicles a factor involved in the cerebral hypoxia?

The changes in the Mg²⁺-dependent V-type ATPase activity and the Mg²⁺-ATP-dependent H⁺ pumping activity of the synaptic vesicles from the cerebral cortex of rats submitted to intermittent chronic (4 weeks) mild or severe hypoxia were evaluated. The adaptation to the chronic severe hypoxia increases both the ATPase and the H⁺ pumping activities which are inhibited by NEM with an exponential relationship between the IC₅₀ values and the in vivo O₂ concentration. The Mg²⁺-dependent increase in H⁺ pumping activity of synaptic vesicles from the rats subjected to in vivo chronic hypoxia may be antagonized by nigericin (dissipating ΔpH) and by FCCP (dissipating ΔpH and Δ_ΨSV). In contrast, valinomycin (dissipating the Δ_ΨSV and facilitating an enhancement in ΔpH) increases in vitro the H⁺ pumping activity that is inhibited by the addition of high concentration of K gluconate (reducing the rate of K⁺ efflux). The preincubation of vesicles from hypoxic rats with FCCP, but not with nigericin, inhibits the valinomycin-increased H⁺ pumping activity.l-glutamate increases the H⁺ pumping activity in synaptic vesicles from the cerebral cortex of chronic hypoxic rats, whereas other amino acids (i.e.,l-aspartate andl-homocysteate) and glutamate analogs (i.e., quisqualate and ibotenate) are ineffective. The adaptation to both chronic intermittent severe hypoxia and in vivo treatment with posatireline causes a decrease in the Mg²⁺-ATPase activity consistent with the decrease in the H⁺ pumping one of the synaptic vesicles. The addition of nigericin into incubation medium magnifies the decrease in the H⁺ pumping activity, while the addition of FCCP is ineffective, suggesting that the treatment with posatireline interferes with the Δ_ΨSV component in the of the synaptic vesicles from rats submitted to chronic hypoxia. The results of the in vivo and in vitro experiments suggest that in the synaptic vesicles from hypoxic rats the Δ_ΨSV component in may be most effective in increasing the Mg²⁺-ATP-dependent H⁺ pumping activity.     

The guanine nucleotide-binding regulatory proteins (G proteins) in myocardium with ischemia

Guanine nucleotide-binding regulatory proteins (G proteins) play a major role in the regulation of a number of physiological processes, such as stimulation or Inhibition of adenylate cyclase activity or gaiting of ionic channels. Myocardial ischemia could induce the changes in receptor-G protein signal transduction system in the heart. Therefore, this article will focus on the role and alterations of G proteins (especially, Gs and Gi) in myocardial ischemia. The Gi protein rapidly loses functional activity during very early myocardial ischemia. In contrast to Gi protein, the function of Gs protein during this phase has not been evaluated. Moreover, the changes in Gs protein after 30 min of ischemia are contradictory. However, the sensitization of the adenylate cyclase activity in the very early phase of acute ischemia is gradually replaced by a decrease in adenylate cyclase activity with prolonged ischemia. The decrease in the function and amount of Gs protein may be one of the factors that induce these changes. The function of Gs protein was also decreased in the canine hearts with ischemia and reperfusion. In contrast to ischemia and reperfusion, there are no significant alterations in G proteins and modulation of adenylate cyclase in the stunned myocardium. It has become increasingly evident that Gi protein may play an important role in the cardioprotective effects of preconditioning. When -adrenoceptor densities are reduced in chronic myocardial ischemia, decreased in the amount and function of Gi protein and increased amount of Gs protein may play the role in preservation of the adenylate cyclase activity. These alterations in G proteins may play the important role in the myocardial function during myocardial ischemia.