Cloning and characterization of Ca(2+)-dependent and Ca(2+)-independent PKCs expressed in Aplysia sensory cells

We isolated cDNA clones from an Aplysia sensory-cell library encoding two isoforms of protein kinase C (PKC). Several isozyme-specific regions are conserved in the Aplysia kinases, notably the variable regions V5 in the Ca(2+)-dependent PKC (Apl I) and V1 in the Ca(2+)-independent PKC (Apl II). Neuronal proteins with the properties expected of these two isoforms can be identified with antibodies raised against peptides synthesized from the amino acid sequences deduced from the clones. Sacktor and Schwartz (1990) measured the proportion of kinase activity that can be translocated to membrane in Aplysia sensory neurons and ganglia by stimuli that produce the presynaptic facilitation underlying behavioral sensitization. Much less Apl I and Apl II are translocated, suggesting that still other isoforms of PKC exist in these cells.     

Intracellular Ca2+ suppressed a transient potassium current in hippocampal neurons

The effects of intracellular Ca2+ (Ca2+i) on K+ currents in hippocampal cells were examined using acutely isolated cells obtained from adult guinea pigs. Whole-cell voltage-clamp recordings were carried out in a configuration that allowed a continuous perfusion of the intracellular medium. Recording media were made to block inward currents and allowed selective activation of K(+)-dependent outward currents. Voltage-dependent outward currents consisted of an initial rapidly decaying component followed by a sustained component. The time constant of decay of the transient current was about 25 msec, and previous studies (Numann et al., 1987) showed that the kinetic and pharmacological properties of this current closely resembled the A current recorded in invertebrate neurons (Connor and Stevens, 1971; Thompson, 1982). Intracellular perfusion of hippocampal cells with a solution containing elevated Ca2+ (about 4.5 x 10(-4) M) elicited outward currents at the holding potential (-45 to -55 mV) and produced changes in voltage-dependent K+ currents. The transient outward current (IA) activated by depolarization was suppressed with increases in Ca2+i. Delayed, sustained K+ currents were greatly potentiated. Data also showed that, among the 3 effects elicited by Ca2+i, suppression of IA was most sensitive to Ca2+i elevation. Previous results (Numann et al., 1987) showed that IA had a lower threshold (about -45 mV) than sustained currents (about -40 mV). By using low levels of depolarization (-40 mV), IA can be selectively activated, and the suppressive effect of Ca2+i on IA was confirmed on the kinetically isolated IA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stable xenon does not increase intracranial pressure in primates with freeze-injury-induced intracranial hypertension

Stable xenon (Xe)-enhanced computed tomography is a potentially valuable tool for high resolution, three-dimensional measurement of CBF in patients. However, reports that Xe causes cerebrovascular dilation and increases intracranial pressure (ICP) have tempered enthusiasm for its use. The effects of 5 min of 33% Xe inhalation on ICP (right and left hemispheres) were studied in eight fentanyl-anesthetized Rhesus monkeys after right-sided cortical freeze injury. ICP, CBF, and physiological variables were monitored for up to 6 h postinsult. The preinjury (control) right hemispheric ICP was 8 +/- 5 mm Hg (mean +/- SD) and left hemispheric ICP was 5 +/- 2 mm Hg. Postinjury observations were classified into low (less than 15 mm Hg) and high ICP (greater than or equal to 15 mm Hg) groups. Both right and left ICP values averaged 9 +/- 3 mm Hg in the low ICP group. In the high ICP group, the right ICP was 20 +/- 4 mm Hg and left ICP was 21 +/- 6 mm Hg. ICP was unchanged by Xe inhalation under control conditions as well as in both low and high ICP groups postinjury. Postinjury, the MABP decreased 10-15 mm Hg in the low ICP group and 10-17 mm Hg in the high ICP group 2-3 min after the start of Xe inhalation (p less than 0.05). These results show that 33% Xe inhalation does not increase ICP in fentanyl-anesthetized monkeys but could decrease MABP in stressed states, presumably because of the anesthetic effects of Xe.     

Regionally selective effects of NMDA receptor antagonists against ischemic brain damage in the gerbil

This study compared the ability of three N-methyl-D-aspartate (NMDA) receptor antagonists to prevent neuronal degeneration in an animal model of global cerebral ischemia. The model employed is characterized by damage to the striatum, hippocampus, and neocortex. Antagonists were administered to gerbils either before or after a 5-min bilateral carotid occlusion. The intraischemic rectal temperature was either maintained at 36-37 degrees C or allowed to fall passively to 28-32 degrees C. Antagonists and doses tested were 1 and 10 mg/kg of MK-801 (pre- or postischemia), 30 mg/kg of CGS 19755 preischemia, four 25 mg/kg doses of CGS 19755 administered between 0.5 and 6.5 h postischemia, and 40 mg/kg of MDL 27,266 (pre- or postischemia). All three NMDA receptor antagonists exhibited some degree of neuroprotective activity when the carotid occlusion was performed under normothermic conditions. Most of the treatments with antagonist markedly reduced striatal damage. CA1 hippocampal and neocortical pyramidal cells were spared by only three of the treatments, however, and the extent of neuroprotection varied widely from case to case. Toxic doses of antagonist were required to protect CA1 pyramidal cells from ischemic damage. Ischemic damage to hippocampal areas CA2-CA3a and CA4 appeared to be resistant to all of these treatments. Most CA1 pyramidal cells that were protected from degeneration by an NMDA receptor antagonist were histologically abnormal. The neuroprotective effects of MK-801 and intraischemic hypothermia appeared to be additive. MK-801 (10 mg/kg) consistently reduced the postischemic brain temperature, but only the magnitude of hypothermia produced soon after reperfusion correlated with its neuroprotective action. These results suggest that NMDA receptor antagonists are relatively poor neuroprotective agents against a moderately severe ischemic insult.     

Ticlopidine and clopidogrel (SR 25990C) selectively neutralize ADP inhibition of PGE1-activated platelet adenylate cyclase in rats and rabbits

Ticlopidine and its potent analogue, clopidogrel, are powerful inhibitors of ADP-induced platelet aggregation. In order to improve the understanding of this ADP-selectivity, we studied the effect of these compounds on PGE1-stimulated adenylate cyclase and on the inhibition of this enzyme by ADP, epinephrine and thrombin. Neither drug changed the basal cAMP levels nor the kinetics of cAMP accumulation upon PGE1-stimulation in rat or rabbit platelets, which excludes any direct effect on adenylate cyclase or on cyclic nucleotide phosphodiesterase. However, the drop in cAMP levels observed after addition of ADP to PGE1-stimulated control platelets was inhibited in platelets from treated animals. In contrast, the drop in cAMP levels produced by epinephrine was not prevented by either drug in rabbit platelets. In rat platelets, thrombin inhibited the PGE1-induced cAMP elevation but this effects seems to be entirely mediated by the released ADP. Under these conditions, it was not surprising to find that clopidogrel also potently inhibited that effect of thrombin on platelet adenylate cyclase. In conclusion, ticlopidine and clopidogrel selectively neutralize the ADP inhibition of PGE1-activated platelet adenylate cyclase in rats and rabbits.     

Sex effects in defensive behavior: Baseline differences and drug interactions

Female rats consistently show a pattern of differences in defensive behaviors compared to males which parallel the effects of exposure to a nonpainful threat stimulus (cat or cat odor) in the same tests and measures. These indications of greater defensiveness for females are particularly common in situations involving potential, as opposed to actual and present, threat, a factor which probably also reflects ceiling or floor effects in situations involving very intense defensiveness. In addition, pharmacological studies indicate sex differences in the effects of selective serotonin (5-HT) receptor agonists and antagonists on defensive responding. These findings indicate that sex effects must be considered in studies of the pharmacological control of defensive behaviors, and suggest that responsivity to sex effects may be an additional criterion for the suitability of animal models of anxiety.     

The use of rat brain slices as an in vitro model for mechanistic evaluation of neurotoxicity-studies with acrylamide

Biochemical mechanisms underlying acrylamide induced neurotoxicity were examined using an in vitro model consisting of sagittal slices of rat brain. Incubation of brain slices under oxygen in artificial cerebrospinal fluid containing acrylamide produced a dose and time dependent inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Lysosomal enzymes, acid phosphatase, N-acetyl glucosaminidase and beta-glucuronidase decreased in a similar manner, while no changes were observed in the activity of Na+K+ATPase, cytochrome c oxidase and lactate dehydrogenase. Incubation of slices with two structurally related compounds, acetamide (a non-neurotoxic amide) and methylene bis-acrylamide (a weak neurotoxin), indicated that acrylamide selectively inhibited GAPDH, enolase and N-acetyl glucosaminidase at low concentration; similar doses of acetamide and methylene bis-acrylamide did not have the same effect on brain slices. Incubation with acrylamide depleted glutathione levels in slices, and the addition of glutathione to the incubation medium prevented acrylamide induced inhibition of GAPDH and lysosomal enzymes. Time dependent inhibition of lysosomal enzymes was also observed in vivo, in the brain and sciatic nerve of rats following a single dose of acrylamide. These results demonstrate that both in vitro and in vivo, lysosomal enzymes are also inhibited following acrylamide exposure. The rat brain slice model exhibits both selectivity and sensitivity towards neurotoxicants and hence, may prove to be an useful in vitro model for the mechanistic evaluation of neurotoxicity.     

The effects of maternal ethanol exposure on neurotransmission and second messenger systems: a quantitative autoradiographic study in the rat brain.

The effects of maternal ethanol exposure on neurotransmission and second messenger systems were examined in rats using histochemistry and in vitro autoradiography. Thirty % ethanol was administered to pregnant rats from gestational day 7 to the day of delivery. Quantitative autoradiography was used to map muscarinic cholinergic, dopamine D2, adenosine A1, and inositol 1,4,5-trisphosphate binding sites, as well as to localize adenylate cyclase and protein kinase C. We found no difference in the patterns of staining with acetylcholinesterase and Timm's stain between control and prenatally ethanol-exposed rats on postnatal day (PN) 30. In the ethanol-exposed rats, [3H]forskolin binding sites were increased during early development in the CA1 subfield of the hippocampus and the occipital cortex; [3H]phorbol ester binding sites were increased in the cortex, striatum, and hippocampus; hippocampal muscarinic cholinergic sites were increased on PN4 and 30; adenosine A1 binding was reduced on PN10 in most regions examined, but was increased in the CA1 subfield on PN30; dopamine D2 receptor levels were significantly reduced on PN30 in the striatum; and IP3 receptors were decreased in most regions studied, but particularly in the cerebellum. Thus, some of these changes were transient and others were long-lasting. Although histopathological abnormalities were minimal, the alterations of binding sites in the cerebellum (the coordination center) and in the hippocampus (related to memory and learning) that were detected may contribute to the behavioral and mental deterioration seen in the fetal alcohol syndrome.