Thomas L. Smith

Regulation of glutamate uptake in astrocytes continuously exposed to ethanol

CNS glutamatergic transmission is altered by chronic ethanol intake and may underlie the behavioral hyperactivity associated with ethanol withdrawal. Because astrocytes regulate extracellular glutamate levels, the aim of this investigation was to characterize the effects of in vitro ethanol exposure on Na+-dependent glutamate uptake parameters in astrocytes. Ethanol exposure elicited a time and concentration-dependent increase in the maximal uptake capacity, Vmax, for [3H] glutamate, which was reversed upon withdrawal of ethanol from the media. None of the ethanol exposures had any effect on the Km for this process. In addition, the ethanol-induced increase in Vmax for glutamate was reversed by the protein kinase C inhibitors, calphostin C and bisindolylmaleimide, and was not associated with an increase in the expression of either of the major glutamate transporter proteins, GLT-1 or GLAST.

Changes in lymphocyte subsets and macrophage functions from high, short-term dietary ethanol in C57/BL6 mice

Chronic administration of a diet containing 7% ethanol (36% of total calories) for 8 days to male C57/BL6 mice resulted in significant changes in functioning of macrophages. Peritoneal exudate macrophages from the ethanol-fed mice released more tumor cell cytotoxic materials upon culturing in vitro than cells from controls. However, peritoneal exudate cells continued to respond to exogenous beta carotene in vitro to produce additional cytotoxic materials. Phagocytosis of sheep red blood cells in vitro was suppressed in cells from ethanol treated mice. The number of splenic lymphocytes of various subsets was significantly changed by the ethanol exposure. Total T cells and T suppressor cells were lower, with a significant decrease in B cells containing IgM on their surface. The percentage of spleen cells showing markers for macrophage functions and their activation were significantly reduced. It is concluded that short-term chronic consumption of dietary ethanol, which was sufficient to produce physical dependence, results in significant alterations in lymphocyte subtypes and suppression of some macrophage functions.

The effect of experimentally induced diabetes mellitus on the lipid order and composition of rat cerebral microvessels

To determine whether diabetes mellitus alters the lipid order and composition of cerebral microvessels, streptozotocin-induced diabetic rats were studied after 5 weeks of chronic hyperglycemia. Diabetes did not affect membrane order or the thermotropic transition temperature of cerebral microvessels as determined from the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). The cholesterol, phospholipid and fatty acid composition of isolated cerebral microvessels was also not significantly affected in diabetic rats. On the other hand, the cerebral microvessels of diabetic rats had significantly increased concentration of lipid peroxidation byproducts, namely conjugated dienes (the calculated O.D./mg phospholipids was 6.98 +/- 0.52 in controls and 11.82 +/- 0.40 in diabetic rats (P < 0.01). The accumulation of conjugated dienes in cerebral microvessels of diabetic rats in the absence of an alteration in the availability of substrates for peroxidation is consistent with the hypothesis that diabetes mellitus is a disease state which is associated with increased free radical activity. Increased oxidative damage may contribute to the changes in the blood-brain barrier observed in animal models of diabetes.

Increased calcium/calmodulin protein kinase activity in astrocytes chronically exposed to ethanol: influences on glutamate transport

The effects of ethanol exposures on calcium/calmodulin-dependent protein kinase activity as well as its influence on glutamate uptake were determined in astrocytes prepared from neonatal rat cerebral cortex. Acute 15-min exposure to 100 mM ethanol had no effect on Ca2+/CaM-dependent protein kinase activity. However, chronic exposure to 100 mM ethanol for 4 days elicited a significant increase in the activity of this enzyme with no parallel increase in its expression. Ca2+/CaM-independent kinase activity was less than 1% of the Ca2+/CaM-dependent kinase activity and was unaffected by any of the ethanol exposures. Exposure to 100 mM ethanol for four days also resulted in a significant increase in Na+-dependent [3H]glutamate uptake which was reversed when ethanol-exposed astrocytes were co-incubated with KN-93, a specific inhibitor of Ca2+/CaM kinase. These results suggest that the effects of ethanol on glutamate transport may be mediated in part, by the level of Ca2+/CaM kinase activity.

Microvascular actions of calcium channel antagonists

The microvascular actions of three calcium channel antagonists were studied in intact spontaneously hypertensive rats (SHR) provided with a dorsal striated muscle microcirculatory chamber. Verapamil and the dihydropyridine derivatives nifedipine and felodipine reduced mean arterial blood pressure (MAP) in a dose-dependent manner. They dilated arterioles of different sizes, with the most pronounced effect being on the smallest precapillary arterioles. Venular diameters were not affected by the calcium antagonists. Approximately 60% of the small arterioles showed a rhythmic pattern of vasodilatation and constriction. This pattern of spontaneous vasomotion was completely blocked by the calcium channel antagonists, especially those of the dihydropyridine type. It is concluded that (a) small precapillary arterioles play an important role in the vasodilator action of calcium channel antagonists, and (b) arteriolar vasomotion depends on vascular smooth muscle cell calcium influx.

The effect of age on lipid composition and order of rat cerebral microvessels

To determine if alterations in lipid composition and/or membrane order of cerebral microvessels may contribute to the age-related changes in blood-brain barrier (BBB) function, cerebral microvessels isolated from male Fischer 344 rats at 3 (young), 12 (intermediate age), and 24 (aged) months of age were studied. The steady state fluorescence polarization of diphenylhexatriene incorporated into isolated cerebral microvessel membranes at 35°C, in aged rats was not different compared to young rats (0.2787±0.0029 vs 0.2847±0.0049). In addition, the thermotropic transition temperature of these membranes was not altered with age. Moreover, the fatty acid composition, the double bond index as well as cholesterol to phospholipid molar ratios were not significantly altered with age. In contrast, the concentration of conjugated dienes in lipid extracts of cerebral microvessels of aged rats (10.04±1.10 O.D./mg phospholipids) was significantly increased compared to the concentration in young rats (6.98±0.52 O.D./mg phospholipids) (p<0.01). It is concluded that aging is not associated with significant changes in lipid composition or membrane order of cerebral microvessels. However, the increased concentration of conjugated dienes in cerebral microvessels of aged rats is indicative of ongoing free radical damage in these microvessels which may contribute to the age-related changes in BBB function.

Lipid order and composition of synaptic membranes in experimental diabetes mellitus

The effect of diabetes in rats on lipid composition and order of synaptosomal membranes (SM) was determined in streptozotocin-induced diabetic rats after 6 weeks of chronic hyperglycemia. The cholesterol content was slightly, but not significantly, higher in diabetic SM (0.287±0.042 vs. 0.209±0.061 μmol/mg protein). The phospholipid concentration in diabetic SM was significantly increased (0.515±0.042 vs. 0.305±0.041 μmol/mg protein;P<0.005). Neither the molar ratios of cholesterol to phospholipids in the SM nor the fatty acid composition of the SM was significantly altered with diabetes. Diabetes did not affect membrane order or the thermotropic transition temperature of the SM as determined fluorometrically. On the other hand, the SM of diabetic rats had significantly increased concentration of lipid peroxidation products, namely conjugated dienes (the calculated O.D./μmol phospholipids was 11.56±1.83 in controls and 19.95 ±4.1 in diabetic ratsP<0.01). Despite the accumulation of lipid peroxidation byproducts in SM of diabetic rats the overall membrane order and the cholesterol to phospholipid molar ratio do not appear to be significantly altered.

Selective effects of ethanol exposure on metabotropic glutamate receptor and guanine nucleotide stimulated phospholipase C activity in primary cultures of astrocytes

The effects of acute and chronic ethanol exposures on the stimulation of inositol specific phospholipase C by metabotropic glutamate receptor activation were determined in primary cultures of rat cortical astrocytes. Phospholipase C activity was monitored by the formation of [3H]inositol phosphates in the presence of lithium in cells prelabelled with [3H]inositol. Acute exposure to 200 mM ethanol had no significant effect on either basal or L-glutamate stimulated [3H]inositol phosphate formation. In cells chronically exposed to ethanol for 4 days, the [3H]inositol phosphate responses to L-glutamate, quisqualate, and the selective metabotropic receptor agonist, 1S,3R-1-amino-cyclopentane-1,3 dicarboxylic acid (trans-ACPD), were significantly inhibited when compared to control (untreated) cells. In contrast, chronic ethanol exposure had no significant effect on the [3H]inositol phosphate response to endothelin-1, a peptide structurally and functionally unrelated to L-glutamate. Similarly, the stimulation of [3H]inositol phosphate formation by the stable GTP analog, guanine 5-(gamma-thiotrisphosphate), was also unaffected by chronic ethanol exposure. The results suggest that chronic ethanol exposure does not affect the coupling of GTP binding proteins to phospholipase C, but rather acts in a selective manner to either alter the metabotropic receptor number or to disrupt the normal coupling of this receptor to its GTP binding protein, which may in turn affect receptor affinity.

Increased Na(+)-dependent high affinity uptake of glutamate in astrocytes chronically exposed to ethanol.

The effects of ethanol exposure on the Na(+)-dependent high affinity uptake of [3H]glutamate were determined in primary cultures of astrocytes prepared from neonatal rat cerebral cortex. Acute exposure to 100 mM ethanol had only marginal effects on [3H]glutamate uptake. However, chronic exposure of astrocytes to 50 or 100 mM ethanol for 4 days elicited a dose-dependent increase in the maximal uptake capacity, Vmax, for glutamate with no significant effect on the Km for this process. Because Na(+)-dependent glutamate uptake by astrocytes is considered a principal mechanism for the clearance of brain extracellular glutamate, the present results suggest that the ethanol-induced upregulation of glutamate transport may modulate glutamatergic transmission in certain disease states such as alcoholism.

Ethanol enhances the in situ phosphorylation of MARCKS and protein kinase C activity in primary cultures of astrocytes

Protein kinase C (PKC) plays an important regulatory role in astrocyte function. Chronic exposure to ethanol for 4 days resulted in an increase in Ca2+-dependent PKC activity in the supernatant fraction of astrocyte homogenates. Only Ca2+-independent PKC activity could be observed in the membrane fraction and this activity was unaffected by ethanol exposure. Chronic ethanol exposure also increased the in situ phosphorylation of MARCKS in permeabilized astrocytes both in the absence or presence of the PKC activator, phorbol 12 -myristate 13 -acetate (PMA). These results suggest an increase in the expression of one or more astrocytic PKC isoforms after chronic ethanol exposure.