RESUMEN

We report here that S-100 beta, a protein with neurotrophic activity on central nervous system neurons, stimulates neuritic outgrowth from cultures of dorsal root ganglia (DRG). S-100 beta elicited neurites from explant and dissociated cell cultures of embryonic chick DRG, and the extent of the response varied with the age of the embryo. Specificity was demonstrated by the observation that incubation of S-100 beta with antibodies directed against S-100 beta reduced the neurite outgrowth, whereas incubation of S-100 beta with normal rabbit serum had little effect. S-100 beta also stimulated the area of neuritic outgrowth from organotypic cultures of fetal rat DRG, showing that the activity of the protein is not restricted to a particular species or culture condition. A mutant S-100 beta lacking neurotrophic activity on cerebral cortex neurons was unable to effectively stimulate neurite outgrowth from DRG cultures. These studies suggest that S-100 beta may play a role in neuronal growth and/or maintenance in the peripheral nervous system.

Asunto(s)

RESUMEN

Explants of canine substantia nigra (SN) and rat mesencephalon (MES), grown in organotypic culture, were incubated with 1-methyl-4-phenylpyridinium (MPP+) and examined for ultrastructural changes. Prolonged exposure (3 days) to doses ranging from 0.1 nM to 10 microM MPP+ resulted in total destruction of all constituents (neuronal and glial) of canine SN cultures. No association was noted between MPP+-induced toxicity and age of canine SN cultures. The first ultrastructural change observed in canine SN cultures incubated with 0.1 nM MPP+ was at 3 h. Grossly swollen mitochondria were noted in large nerve cells. Swollen mitochondria were present in all cells of canine SN cultures by 8 h of incubation with MPP+. Only those rat MES cultures with relatively high preincubation levels of homovanillac acid, determined as an index of viable dopaminergic neurons, incubated with MPP+ (10 microM) for up to 8 days exhibited ultrastructural changes, namely, a swelling of mitochondria within the cytoplasm of large nerve cells. These findings suggest that continual exposure to MPP+ in vitro results in a generalized, nonspecific toxicity in those species known to be susceptible to the parent compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in vivo. However, the initial ultrastructural change, i.e., a swelling of mitochondria, may be the same in all species regardless of sensitivity suggesting that the ultimate mechanism underlying MPP+-toxicity relates to mitochondrial function.

Asunto(s)

RESUMEN

The present study was an extension of earlier work regarding the role of cyclic nucleotides and related enzymes during cerebral ischemia in the gerbil. Following unilateral carotid occlusion, levels of cyclic AMP and cyclic GMP were measured in four rapidly inactivated brain regions at 3, 6, and 24 hr after permanent occlusion and at 2 hr of occlusion plus 1 hr of reflow. An analysis of variance indicated significant minor fluctuations in the steady-state levels of the two cyclic nucleotides within the frontal cortex, the hippocampus, the striatum, and especially the olfactory tubercle with respect to occlusion time (3 and 24 hr) but not when comparing control vs ischemic hemispheres (except at 3 hr). Changes occurred only in animals developing neurological symptoms of ischemia. At 24 hr postocclusion the specific activity of the low-Km form of cyclic AMP phosphodiesterase was elevated especially on the ischemic side when determined in homogenates of the four brain regions. Alternatively, the high-Km form of the enzyme in the presence or absence of Ca2+-calmodulin was unchanged. Guanylate cyclase activity in tissue homogenates was not influenced by the conditions of ischemia until 24 hr had elapsed, an event likewise unique to symptomatic gerbils. The sensitivity of the enzyme to hematin-catalase was decreased in the ischemic hemispheres of the hippocampus, striatum, and olfactory tubercle. In addition, further activation of the hematin-catalase response by NaN3 was depressed in the ischemic side of the hippocampus and striatum. Taken together these and previous studies indicate that fluctuations in the steady-state levels of cyclic nucleotides that occur rather prominently during acute and to a lesser degree during prolonged ischemia are not correlated with associated changes in enzymes responsible for their synthesis and/or degradation.

Asunto(s)

RESUMEN

The present study was initiated to determine whether pretreatment of gerbils with calcium channel blockers, flunarizine and verapamil would prevent injury to cerebral ATPases following secondary ischemia consisting of 60-min bilateral clamping of the carotids followed by 40 min of reperfusion. The sequence of ischemia produced a deficit in Na+,K+-ATPase activity without influencing Ca++,Mg++- or Mn++-sensitive ATPases. Pretreatment with flunarizine significantly prevented the damage to Na+,K+-ATPase while the effect of verapamil was marginal. Verapamil, along with dimethylsulfoxide (DMSO), reduced the mortality rate of gerbils subjected to the paradigm of ischemia. When added directly to the cerebral fractions in vitro the two calcium channel blockers inhibited Na+,K+-ATPase alone. Flunarizine was more potent in vitro than verapamil.

Asunto(s)

RESUMEN

It has been previously established that prolonged bilateral carotid occlusion followed by recirculation produces damage to the synaptic enzyme adenylate cyclase in the frontal cortex of the gerbil. Since calcium entrance into the brain may account in part for the deleterious consequences of stroke, the present study examined whether pretreatment with calcium channel blockers would modify the effects of 60 min of bilateral ischemia plus 40 min of reflow on various parameters of cortical adenylate cyclase activation. In this context activation of cerebral homogenates by norepinephrine with or without 5'-guanylyl imidodiphosphate was preserved by pretreatment of ischemic gerbils with verapamil but worsened by flunarizine. In contrast, in particulate fractions (treated with EGTA to reduce metallic ion levels) the damage to the Mn2+-sensitive catalytic site of adenylate cyclase was prevented only by flunarizine. Pretreatment with the two calcium channel blockers resulted in an elevated basal activity of the enzyme, thereby reducing the response in the homogenate preparation to forskolin. Gerbils pretreated with verapamil tended to have an increased ability for survival resulting from the ischemic episode. Under in vitro conditions the enzyme preparations were not markedly influenced by either drug.

Asunto(s)

RESUMEN

A variety of pharmacological agents were used as experimental probes to determine with greater precision the site(s) of damage to cerebral adenylate cyclase as a consequence of postischemic reperfusion in the gerbil. A paradigm of 60-min bilateral ischemia followed by 40-min reperfusion results in a decreased sensitivity of the catalytic site of adenylate cyclase to Mn2+. Likewise, the GTP-transducer site (guanine nucleotide regulatory or G protein) revealed depressed responses to GTP in the absence or presence of norepinephrine, dopamine agonists, substance P, yohimbine, and cholera and pertussis toxins. Moreover, a crude preparation of GTPase disclosed that damage elicited by postischemic reperfusion was directed to the higher-affinity form of this enzyme, which is associated with the overall function of the guanine nucleotide regulatory protein. Injury to adenylate cyclase was unrelated either to the ability of adrenergic ligands to bind to associated receptor sites or to the capacity of the brain to generate visual evoked potentials in response to visual stimuli.

Asunto(s)

RESUMEN

A therapeutic role for naloxone during stroke has been suggested, but a neurochemical site of action remains to be determined. Previous work with the gerbil cerebral cortex has shown that either bilateral secondary ischemia (60-min occlusion of the carotid arteries followed by 40-min reflow) or unilateral primary ischemia (permanent ligation of one carotid artery for 6 hr in symptomatic animals) produced deficits in both Na+, K+-ATPase (EC 3.6.1.3) activity and various parameters of activation of adenylate cyclase (EC 4.6.1.1). Pretreatment of gerbils with either naloxone or morphine failed to ameliorate or exacerbate, respectively, the neurological signs of ischemia; however, morphine did reduce mortality. Infusion of naloxone prior to ischemia afforded varying degrees of protection to forskolin, GTP analogs, and NE (norepinephrine) activation of adenylate cyclase, as well as to Na+, K+-ATPase (bilateral ischemia only). Similarly, morphine inhibited damage to basal activity of adenylate cyclase and to stimulation by NE, forskolin, and Gpp (NH)p (5'-guanylyl imidodiphosphate). Under in vitro conditions morphine increased the basal activity of adenylate cyclase but reduced responses to NE and forskolin. Furthermore, morphine injected into control gerbils elevated basal- and forskolin-elicited activities but reduced the activation of adenylate cyclase by NE.

Asunto(s)

RESUMEN

This study was designed to correlate histopathological changes in gerbil brain following unilateral primary and secondary ischemia to enzymatic-adenylate cyclase damage. At three hrs permanent occlusion of the right common carotid artery only minimal histological changes were evident in cerebrum, hippocampus, striatum and olfactory tubercle while the enzyme responses were unremarkable. Severe histological and enzymatic alterations were present at one hour of recirculation subsequent to 3 hrs of unilateral occlusion. Similar damage was evident at 6 and 24 hrs permanent occlusion. Principal enzyme damage was directed toward basal activity, as well as stimulation of the catalytic (forskolin-sensitive) sites on the enzyme complex. For the most part the transducer (GTP-sensitive) site was unaffected by ischemia until 24 hr ligation. These changes were observed in only those gerbils developing severe symptoms of stroke.

Asunto(s)

RESUMEN

The activity of three forms of ATPase were examined in fractions of the brain of the gerbil treated with ethylene glycol-N-N-tetra-acetic acid (EGTA) under a variety of conditions of primary and secondary (reflow) ischemia. In animals which were unilateral ischemic (ligation of the right common carotid), damage to Na+, K+-ATPase alone was observed only after at least 6 hr of ischemia had elapsed. The phenomenon occurred in only symptomatic gerbils and was absent in animals which were either asymptomatic or only displayed partial neurological symptoms. Under conditions of bilateral cerebral ischemia, in which both carotid arteries were clamped, only irreversible ischemia (60 min) followed by reflow, was associated with highly significant damage to cerebral Na+, K+-ATPase. In regional studies of the forebrain involving ischemia for 60 min plus 30 min reflow, damage to Na+, K+-ATPase was evident in the cerebrum, hippocampus, striatum and thalamus, while the hypothalamus and olfactory bulb were spared. Pretreatment of gerbils with allopurinol, clonazepam or combinations of thiopental plus either indomethacin or methylprednisolone offered protection to cerebral Na+, K+-ATPase subsequent to secondary ischemia. With only minor exceptions (striatum) neither Ca2+, Mg2+- nor Mn2+-ATPase were altered by stroke or treatment with drugs.

Asunto(s)

RESUMEN

Adrenergic-sensitive adenylate cyclase was found to be present in the nucleus accumbens and ventral caudate of the rabbit, but displayed less activity in the dorsal caudate. In general, stimulation of the enzyme by dopamine (DA) was most sensitive to inhibition by fluphenazine while norepinephrine (NE)-stimulated activity was highly sensitive to both fluphenazine and propranolol. Other selective adrenergic-receptor blocking agents (butoxamine, practolol, yohimbine and prazosine) were more effective in antagonizing the effect of NE as opposed to DA-activation. Activation of adenylate cyclase by NE in the dorsal caudate displayed less sensitivity to these adrenergic antagonists than in the other two areas. Horseradish peroxidase-positive cells were present in the locus coeruleus, following injection into the nucleus accumbens. Activity of basal and guanosine triphosphate (GTP)-sensitive adenylate cyclase was reduced by enkephalins in these three brain regions. This action was reversed by naloxone. Met-enkephalin did not affect either NE- or DA-mediated responses in any area.

Asunto(s)

RESUMEN

The effect of three modes of anesthesia was evaluated with regard to regional damage to central cyclic nucleotide systems in the gerbil brain as a consequence of bilateral ischemia (clamping the common carotids) followed by various periods of recirculation. The injection of thiopental as much as 90 min before stroke prevented damage to chemical activation [catecholamines, guanosine triphosphate (GTP), or forskolin] of adenylate cyclase. However, the basal enzyme activity was lower in all brain regions whether thiopental was administered to stroke or sham-operated animals. Injection of ketamine drastically shortened the survival times of gerbils undergoing stroke followed by recirculation. About 90% of the animals could tolerate a maximum of only 15 min stroke with 15 min recirculation. At this time frame the patterns of activation of adenylate cyclase in only the olfactory tubercle and hippocampus were altered. When procaine was used as a local anesthetic agent during surgery, damage to catecholamine-, GTP-, or forskolin-activated adenylate cyclase was evident to varying degrees in the frontal cortex, hippocampus or olfactory tubercle, but not in the nucleus accumbens and olfactory bulb of gerbils subjected to 60-min stroke followed by 15 or 150 min of recirculation. The degree of enzyme damage was neither correlated with the fed vs. fasted state of the animal nor with the whole blood concentration of glucose. A depression in the amplitude of visually evoked potentials correlated to neurological signs and to enzyme damage. During anesthesia, ketamine increased steady-state concentrations of cyclic AMP in the frontal cortex and hippocampus from gerbil brains that had been rapidly inactivated by microwave irradiation. Thiopental increased steady-state cyclic AMP in only the olfactory tubercle. Cyclic GMP concentrations were unchanged by any anesthetic agent. In animals completely recovering from anesthesia and occluded for a brief period followed by 10 min of reflow, steady-state concentrations of only cyclic AMP were augmented.

Asunto(s)

Asunto(s)