guanosine-triphosphate and Ischemic-Attack--Transient

In the canine basilar artery during chronic vasospasm following subarachnoid hemorrhage, endothelium-dependent relaxations were diminished. Release of endothelium-derived relaxing factor (EDRF), as measured by a bioassay method, was unchanged. Relaxation to nitric oxide (NO) in preparations without endothelium was smaller in the spastic arteries. Production of cyclic GMP, measured by radioimmunoassay, was reduced in the spastic arteries; the impaired production was accompanied by decrease in GTP, the substrate for the production of cyclic nucleotide. The contents of other high-energy phosphates, such as creatine phosphate and ATP were also markedly reduced. Close temporal correlation between the metabolic failure and development of vasospasm was observed. Sarcolemmal regulation of intracellular calcium concentration was impaired in the pathological condition, suggesting a link between the metabolic failure and the pathological protracted contractions. Metabolic changes, and resultant affected viability of smooth muscle cells are likely to be an important factor in the pathogenesis of chronic vasospasm.

Topics: Adenosine Triphosphate; Animals; Basilar Artery; Calcium Channels; Cell Membrane Permeability; Chronic Disease; Cyclic GMP; Dogs; Guanosine Triphosphate; Ischemic Attack, Transient; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide

P-glycoprotein (P-gp) is expressed not only in tumour cells but also in some normal tissues including brain capillaries. We investigated whether or not P-gp was expressed in the capillary endothelial cells of a rat focal ischaemic brain. The brains were immunohistochemically studied for Factor VIII, glial fibrillary acidic protein (GFAP), and P-gp. Endothelial gamma-glutamyl transpeptidase (gamma-GTP) activity, which is thought to be induced by glial cells, was also studied histochemically. The P-gp positive endothelial cells disappeared in the ischaemic lesion by post-ischaemic day 3. Factor VIII-positive regenerating capillaries were first observed on day 3 without P-gp expression. The P-gp positive endothelial cells began to reappear on day 5, and were detected in all the endothelial cells by day 8. The P-gp expression in endothelial cells showed a similar pattern as that of gamma-GTP, and seemed to correlate with GFAP-positive reactive astrocytes. The newly-formed brain capillaries thus appeared to have a potential to express P-gp in abnormal pathogenic conditions as cerebral infarction, and our present study also suggested that P-gp in the brain capillaries might therefore be expressed in conjunction with glial cells.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Blood-Brain Barrier; Brain Chemistry; Capillaries; Capillary Permeability; Cerebral Arteries; Dogs; Endothelium, Vascular; Factor VIII; Glial Fibrillary Acidic Protein; Guanosine Triphosphate; Immunohistochemistry; Ischemic Attack, Transient; Male; Rats; Rats, Sprague-Dawley

We used quantitative in situ hybridization and receptor autoradiography to study changes in adenosine receptors following hypoxia-ischemia (H-I) in the neonatal rat brain. Seven-day-old rat pups were subjected to a unilateral ligation of the common carotid artery followed by a 2 h 15 min hypoxic period (7.7% O2 in N2). Adenosine A1 receptor mRNA in cortex and several parts of hippocampus, and A2a mRNA was decreased in the ligated hemisphere 0 h, 1 h and 2 h following hypoxia. The binding of the A1 receptor selective antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX) in the presence or in the absence of GTP decreased immediately after the hypoxic period in both hemispheres and returned thereafter gradually towards control. These results show that there are rapid changes in A1 receptor number on both sides of the brain, and of adenosine A1 and A2a receptor mRNA in the hemisphere that would later develop infarction. Decreases in adenosine receptors may worsen H-I brain damage and have consequences for the use of adenosine directed therapy.

Topics: Animals; Animals, Newborn; Brain; Cerebral Cortex; Down-Regulation; Female; Guanosine Triphosphate; Hippocampus; Hypoxia; Ischemic Attack, Transient; Male; Neurons; Pyramidal Cells; Rats; Rats, Wistar; Receptors, Purinergic P1; Theophylline; Tritium

Rats were subjected to the standard four-vessel occlusion model of cerebral transient ischaemia (vertebral and carotid arteries) for 15 and 30 min. After a 30 min recirculation period, protein synthesis rate, initiation factor 2 (eIF-2) and guanine nucleotide exchange factor (GEF) activities, and the level of phosphorylation of the alpha subunit of eIF-2 (eIF-2 alpha) were determined in the neocortex region of the brain from sham-operated controls and ischaemic animals. Following reversible cerebral ischaemia, the protein synthesis rate, as measured in a cell-free system, was significantly inhibited (70%) in the ischaemic animals. eIF-2 activity, as measured by its ability to form a ternary complex, also decrease parallel to the decrease in protein synthesis. As eIF-2 activity was assayed in the presence of Mg2+ and GTP-regenerating capacity, the decrease in ternary-complex formation indicated the possible impairment of GEF activity. Since phosphorylated eIF-2 [eIF-2(alpha P)] is a powerful inhibitor of GEF, the levels of phosphorylated eIF-2 alpha were determined, and an increase from 7% phosphorylation in sham control rats to 20% phosphorylation in 15 min and 29% phosphorylation in 30 min in ischaemic rats was observed, providing evidence for a tight correlation of phosphorylation of eIF-2 alpha and inhibition of protein synthesis. Moreover, GEF activity measured in the GDP-exchange assay was in fact inhibited in the ischaemic animals, proving that protein synthesis is impaired by the presence of eIF-2(alpha P), which blocks eIF-2 recycling.

Topics: Animals; Cell-Free System; Cerebral Cortex; Eukaryotic Initiation Factor-2; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Ischemic Attack, Transient; Magnesium; Male; Phosphorylation; Protein Biosynthesis; Proteins; Rats; Rats, Wistar; RNA, Transfer, Met

To investigate the pathogenetic significance of metabolic failure observed in spastic cerebral arteries after subarachnoid hemorrhage (SAH), the temporal profile of alterations in the arterial content of high-energy phosphates was studied. A canine model of double hemorrhage was used. Constriction of the basilar artery was measured angiographically on Days 3, 5, 7, and 14 after SAH in separate groups of animals. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine triphosphate (GTP), guanosine diphosphate, creatine phosphate (CrP), and creatine (Cr) levels in the arteries were assayed using high-performance liquid chromatography. A time-dependent development of angiographic spasm was confirmed. A mild vasospasm was seen in the group studied 3 days after SAH, progressed in the Day 5 group, remained comparably severe in the Day 7 group, and resolved partially in the Day 14 group. The content of high-energy phosphates (ATP, GTP, and CrP) declined rapidly over the course of the study, and a significant reduction in ATP, GTP, and CrP was observed in the Day 3 group. Levels of ATP and CrP decreased further in the Day 5 and 7 groups. The decrement in GTP was completed in the early phase; a significant reduction took place in the Day 3 group, with no progression thereafter and no recovery through Day 14. Total adenylate (ATP + ADP + AMP) and total creatine (Cr + CrP) content diminished markedly over the course of the study. These results indicate that metabolic failure and trophic disturbance in the cerebral artery occurs with a rapid onset following SAH and progresses in close association with the development of vasospasm, suggesting a significant causal relationship with the pathogenesis.

Topics: Adenosine Triphosphate; Animals; Cerebral Arteries; Creatine; Dogs; Energy Metabolism; Guanosine Triphosphate; Ischemic Attack, Transient; Phosphocreatine; Subarachnoid Hemorrhage

High-energy phosphate levels were measured in the canine cerebral artery during chronic vasospasm. Subarachnoid hemorrhage and vasospasm were induced by percutaneous injections of autologous venous blood into the cisterna magna. Narrowing of the artery was confirmed by angiography 7 days later. Levels of adenosine phosphates (adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP)), guanosine phosphates (guanosine triphosphate (GTP) and guanosine diphosphate (GDP)), and creatine phosphate (CrP) in the basilar artery were quantified using high-performance liquid chromatography. The total creatine (Crtotal) content was measured by a spectrophotometric method after acid hydrolysis of CrP. Levels of ATP, GTP, and CrP were markedly reduced in the spastic arteries, and ratios of ATP:ADP, GTP:GDP, and CrP:Crtotal were significantly decreased. The results indicate a serious disturbance in the energy metabolism that takes place in the cerebral artery during chronic vasospasm.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Cerebral Arteries; Chronic Disease; Dogs; Female; Guanosine Diphosphate; Guanosine Triphosphate; Ischemic Attack, Transient; Male; Phosphates; Phosphocreatine; Subarachnoid Hemorrhage

As an approach to understanding the molecular basis of the pathophysiology of cerebral ischemia, we examined qualitative and quantitative changes in pertussis toxin substrates, Gi1 and G0, in the membrane of rat cerebral cortex after decapitation. Within 1 min after decapitation, the extent of pertussis toxin-catalyzed [32P]ADP ribosylation of the G proteins in the cerebral cortex membrane was significantly decreased and the magnitude of the decrease became slightly larger upon further incubation of the decapitated brain. Addition of guanine nucleotides, GTP and GDP, or the purified beta gamma subunits of transducin to the membranes of control and ischemic cerebral cortex stimulated [32P]ADP ribosylation of the G proteins. The stimulation of [32P]ADP ribosylation in the control situation by guanine nucleotides was almost to the same extent as that in ischemia. However, the stimulation by transducin beta gamma subunits was different; the control stimulation was greater than that in ischemia. In immunoblots probed with antibodies against Gi1 alpha, G0 alpha, and T beta, the immunoreactivity of the corresponding proteins in ischemia was similar to that in control, suggesting that the amounts of G proteins were not changed in ischemia. These results suggest that ischemia accelerates the dissociation of alpha-GDP-beta gamma to alpha-GDP and free beta gamma and causes the denaturation of the dissociated alpha-GDP, thereby decreasing [32P]ADP ribosylation.

Topics: Adenosine Diphosphate Ribose; Animals; Cell Membrane; Cerebral Cortex; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Immunoblotting; Ischemic Attack, Transient; Macromolecular Substances; Male; Pertussis Toxin; Rats; Rats, Inbred Strains; Virulence Factors, Bordetella

Nucleotide metabolism was studied in rats during and following the induction of 10 min of forebrain ischemia (four-vessel occlusion model). Purine and pyrimidine nucleotides, nucleotides, and bases in forebrain extracts were quantitated by HPLC with an ultraviolet detector. Ischemia resulted in a severe reduction in the concentration of nucleoside triphosphates (ATP, GTP, UTP, and CTP) and an increase in the concentration of AMP, IMP, adenosine, inosine, hypoxanthine, and guanosine. During the recovery period, both the phosphocreatine level and adenylate energy charge were rapidly and completely restored to the normal range. ATP was only 78% of the control value at 180 min after ischemic reperfusion. Levels of nucleosides and bases were elevated during ischemia but decreased to values close to those of control animals following recirculation. Both the decrease in the adenine nucleotide pool and the incomplete ATP recovery were caused by insufficient reutilization of hypoxanthine via the purine salvage system. The content of cyclic AMP, which transiently accumulated during the early recirculation period, returned to the control level, paralleling the decrease of adenosine concentration, which suggested that adenylate cyclase activity during reperfusion is modulated by adenosine A2 receptors. The recovery of CTP was slow but greater than that of ATP, GTP, and UTP. The GTP/GDP ratio was higher than that of the control animals following recirculation.

Topics: Adenosine; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Brain; Chromatography, High Pressure Liquid; Cytidine Triphosphate; Guanine; Guanosine Diphosphate; Guanosine Triphosphate; Hypoxanthine; Hypoxanthines; Inosine; Inosine Monophosphate; Ischemic Attack, Transient; Male; Nucleotides; Phosphocreatine; Rats; Rats, Inbred Strains; Uridine Triphosphate

Five minutes of bilateral carotid occlusion in unanesthetized gerbils produced substantial changes in spontaneous locomotor activity. Behavior was decreased after 1 hr of reperfusion and was increased at 24 hrs post-ischemia. Adenylate cyclase activity was measured in homogenates of frontal cortex and hippocampus at 90 min and 24 hrs following 5 min of cerebral ischemia. Enzyme activity was determined in the absence and presence of the activators guanosine-5'-triphosphate (GTP), guanylyl-5'-imidodiphosphate (GppNHp), isoproterenol (Iso) plus GTP, and forskolin (Fors) plus GTP. Homogenates responded with expected increases over basal adenylate cyclase activity with addition of all activators. An additional small increase in isoproterenol-stimulated activity was observed in frontal cortex homogenates at 90 min post-ischemia. No other significant changes in adenylate cyclase activity were observed after either 90 min or 24 hrs of reperfusion. The substantial increases in locomotor activity evident at 24 hrs after transient ischemia are not associated with measurable changes in adenylate cyclase activity in homogenates of frontal cortex or hippocampus.

Topics: Adenylyl Cyclases; Animals; Carotid Arteries; Cerebral Cortex; Colforsin; Diterpenes; Gerbillinae; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hippocampus; Ischemic Attack, Transient; Isoproterenol; Ligation; Male; Motor Activity; Time Factors