L. Judson Chandler

Chronic ethanol exposure potentiates NMDA excitotoxicity in cerebral cortical neurons

Abstract: The effect of acute and chronic ethanol exposure on excitotoxicity in cultured rat cerebral cortical neurons was examined. Neuronal death was quantitated by measuring the accumulation of lactate dehydrogenase (LDH) in the culture media 20 h after exposure to NMDA. Addition of NMDA (25–100 μM) to the culture dishes for 25 min in Mg2+‐free buffer resulted in a dose‐dependent increase in LDH accumulation. Phase‐contrast microscopy revealed obvious signs of cellular injury as evidenced by granulation and disintegration of cell bodies and neuritic processes. Chronic exposure of neuronal cultures to ethanol (100 mM) for 96 h followed by its removal before NMDA exposure, significantly increased NMDA‐stimulated LDH release by 36 and 22% in response to 25 μM and 50 μM NMDA, respectively. Neither basal LDH release nor that in response to maximal NMDA (100 μM) stimulation was altered by chronic alcohol exposure. In contrast to the effects of chronic ethanol on NMDA neurotoxicity, inclusion of ethanol (100 mM) only during the NMDA exposure period significantly reduced LDH release by ∼ 50% in both control and chronically treated dishes. This reduction by acute ethanol was also observed under phase‐contrast microscopy as a lack of development of granulation and a sparing of disintegration of neuritic processes. These results indicate that chronic exposure of ethanol to cerebral cortical neurons in culture can sensitize neurons to excitotoxic NMDA receptor activation.

Calcium- Versus G Protein-Mediated Phosphoinositide Hydrolysis in Rat Cerebral Cortical Synaptoneurosomes

Abstract: The role of calcium and sodium in stimulating phosphoinositide hydrolysis in brain was investigated in rat cerebral cortical synaptoneurosomes. In buffer containing 136 mM sodium and various concentrations of added calcium (0–1.0 mM), basal, potassium‐stimulated, and norepinephrine ‐stimulated formation of 3H‐inositol phosphates decreased with decreasing extracellular calcium. Potassium‐ and norepinephrine‐stimulated formation of 3H‐inositol phosphates was reduced to basal levels by addition of EGTA. Isosmotically replacing sodium with choline chloride or N‐methyl‐d‐glucamine to disrupt Na+/Ca2+ exchange resulted in a large increase in the formation of 3H‐inositol phosphates. Measurement of cytosolic calcium with fura‐2 revealed that the cytosolic calcium concentration was sensitive to changes in the extracellular calcium concentration and increased on resuspension of synaptoneurosomes in sodium‐free rather than sodium‐containing medium. In the absence of sodium, potassium‐stimulated formation of 3H‐inositol phosphates was reduced or eliminated, depending on the extracellular calcium concentration. Subtraction of basal formation of 3H‐inositol phosphates from that in the presence of 1 mM carbachol or 100 μM norepinephrine revealed that the carbachol‐stimulated component was the same in the presence and absence of sodium, whereas the norepinephrine‐stimulated component was reduced in the absence of sodium. Addition of the protein kinase C activator 12‐O‐tetradecanoylphorbol 13‐acetate inhibited norepinephrine‐ and, to a lesser extent, carbachol but not basal or aluminum fluoride‐stimulated formation of 3H‐inositol phosphates in sodium‐free medium. These results suggest that an increase in intracellular calcium, via disruption of Na+/Ca2+ exchange or depolarization‐induced calcium influx, may explain previous demonstrations that agents that stimulate Na+ influx can also stimulate phosphoinositide hydrolysis. These results also support previous evidence of two separate and distinct pathways for stimulating phosphoinositide‐linked phospholipase C (PLC) activity in brain: One pathway appears to involve a phosphoinositide‐associated guanine nucleotide binding protein‐PLC coupling process, and the other a direct activation of PLC by an increase in intracellular calcium.

Protein kinase C modulates receptor-independent activation of endothelial nitric oxide synthase

The intracellular regulation of nitric oxide synthase has been the focus of intense investigation. Bioassay studies using vascular rings have suggested that protein kinase C inhibits endothelium-dependent vascular relaxation. However, information regarding the effects of protein kinase C on the synthesis of nitric oxide in endothelial cells is not available. Therefore, we investigated the effects of protein kinase C to regulate receptor-independent activation of nitric oxide synthase activity in cultured bovine pulmonary artery endothelial cells. Activation of protein kinase C by phorbol 12-myristate 13-acetate or 1,2-dioctanoyl-sn-glycerol inhibited receptor-dependent and receptor-independent nitric oxide synthase activity. The inhibition of nitric oxide synthase by protein kinase C was concentration dependent and markedly blunted by staurosporine. The inhibition of protein kinase C by staurosporine alone enhanced basal nitric oxide synthase activity. Furthermore, depletion of protein kinase C enhanced both basal and agonist-stimulated nitric oxide synthase activity. These studies indicate that protein kinase C modulates the activity of the constitutive Ca2+/calmodulin-dependent endothelial nitric oxide synthase in the basal state and following agonist stimulation through direct inhibition of the enzyme as well as receptor desensitization. These direct regulatory effects of protein kinase C on endothelial nitric oxide synthase activity may have important implications in the physiologic regulation of vascular tone.

Astrocytes but not microglia express NADPH-diaphorase activity after motor neuron injury in the rat.

The purpose of this study was to identify cellular sources of nitric oxide (NO) after injury to rat facial motor neurons using NADPH-diaphorase histochemistry. We employed intraneural injections of either saline or toxic ricin, followed by nerve crush, in order to produce regeneration or degeneration of facial motor neurons (FMNs), respectively. Reactive astrocytes responding to ricin-induced degeneration of FMNs showed increased NADPH-diaphorase activity while reactive astrocytes responding to axotomy (saline injection) did not. Reactive microglial cells were found not to express NADPH-diaphorase in either one of these two paradigms. We conclude that irreversible neuron injury resulting in neurodegeneration causes increased production of NO by reactive astrocytes.

Molecular Mechanisms of Alcohol Neurotoxicity

Heavy alcohol consumption over a period of years can lead to cognitive and neurological impairments. Studies have indicated that alcoholics have greatly reduced brain volume. Computer tomographic studies of the brains of alcoholics have indicated that chronic alcoholism leads to enlargement of the ventricles, decreases in tissue volume and increases in cerebral spinal fluid volume, i.e. brain shrinkage (Pfefferbaum et al. 1992). Although changes are somewhat similar to those that occur during normal aging, the increase in size of the lateral ventricles and the increase in the cortical space between sulci clearly indicate that the brain of alcoholics have a decrease in cellular mass above that found in age matched non-alcoholics (Pfefferbaum et al., 1992; Jernigan et al., 1992). The increases in cerebral spinal fluid spaces are particularly associated with loss of the gray matter with some reduction also occurring in white matter. These losses in cortical tissue are possibly an acceleration of age-induced effects as well as the cumulative toxicity that occurs during a lifetime of chronic alcohol exposure.

Effects of Ethanol on Receptors Coupled to Phosphoinositide Hydrolysis in Brain

Many of the pharmacological actions of ethanol are related to the disruption of hormone and neurotransmitter signals in the brain. Signal transduction by hormones and neurotransmitters occur through receptors which alter intracellular levels of second messengers. Recent studies have indicated that a large number of hormones and neurotransmitter receptors stimulate phosphoinositide hydrolysis through the activation of a phosphoinositide specific phospholipase C (reviewed by Abdel-Latif [1]). Phosphoinositides are a group of phospholipids containing inositol and include phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5 bisphosphate (PIP2). Hydrolysis of PIP2 leads to the formation of two intracellular second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate {l(1,4,5)P3}. Diacylglycerol has been shown to activate a phospholipid-calcium dependent protein kinase commonly referred to as protein kinase C (PKC). Inositol 1,4,5 trisphosphate has been shown to release calcium from intracellular stores and to be metabolized to other active second messengers.

Unique Aspects of Muscarinic Receptor Stimulated Inositol Polyphosphate Formation in Brain: Changes in Senescence

The agonist dependent hydrolysis of membrane phosphoinositides is a major signal transduction pathway in brain (Berridge 1985; Crews et al. 1988a). A variety of receptors including muscarinic cholinergic, α1-adrenergic, serotonergic, and a variety of peptides, couple to phosphoinositide hydrolysis via activation of phospholipase C (Berridge 1985, Gonzales and Crews 1985). Hydrolysis of one of these phosphoinositides, phosphatidylinositol 4,5-bisphosphate [PtdIns(4, 5)P2] results in the formation of 1,2 diacylglycerol (DAG) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], both of which appear to have second messenger functions (Batty et al., 1989; Berridge and Irvine, 1989; Rana and Hokin, 1990). DAG remains in the membrane where it can activate protein kinase C (PKC), a family of calcium/phospholipid dependent kinases, that regulate numerous cellular functions and may play a role in neuronal plasticity and neuronal cell death. Ins(1,4,5)P3 is released into the cytoplasm where it binds to specific receptors on the endoplasmic reticulum and releases intracellular Ca2+ into the cytoplasm. Specific phosphomonoesterases can rapidly metabolize Ins(1,4,5)P3 to inositol 1,4-bisphosphate, inositol 4-monophosphate and finally to free inositol via sequential dephosphorylation (Fig. 1). Ins(1,4,5)P3 can be phosphorylated to Ins(1,3,4,5)P4 by a specific Ca2+/calmodulin sensitive 3-kinase (Batty et al., 1985; Irvine et al., 1986). Ins(1,3,4,5)P4 may also be a second messenger involved in a variety of functions including the Ca2+ influx (Irvine et al., 1986), release of intracellular Ca2+ (Gawler et al., 1990) and sequestration of Ca2+ released by Ins(1,4,5)P3 (Hill and Boynton, 1990; Boynton et al., 1990). Ins(1,3,4,5)P4 is dephosphorylated by a 5-phosphatase to inositol 1,3,4-trisphosphate, an inactive isomer. In addition, a variety of cyclic inositol phosphates are produced by the action of phospholipase C on phosphoinositides. The cyclic inositol phosphates accumulate on prolonged agonist stimulation but their cellular functions are not clear (Bansal and Majerus, 1990).

Muscarinic Receptors, Phosphoinositide Hydrolysis, and Neuronal Plasticity in the Hippocampus

Previous studies have suggested that muscarinic cholinergic receptors in the hippocampus are associated with memory processes. Antimuscarinic drugs, well known for their amnestic effects, support the cholinergic hypothesis of memory (Bartus et al„ 1982). Several biochemical and electrophysiological responses to muscarinic agonist stimulation have been described in the hippocampus, including reduction of a calcium-dependent potassium current (IKca) that is responsible for after-hyperpolarization (AHP) (Bernado and Prince, 1982), reduction of a time- and voltage-dependent non-inactivating potassium current termed the M-current (IKm) (Halliwell and Adams, 1982), inhibition of cyclic-AMP (Olianas et al., 1983), stimulation of cyclic-GMP (Snyder et al., 1984), and stimulation of phosphoinositide (PI) turnover (Gonzales and Crews, 1985; Fisher and Bartus, 1985). This multiplicity of responses has obscured the relationship between biochemical and electrophysiological responses to muscarinic agonists. In addition, the relationship between muscarinic action on specific membrane ionic conductances, especially IKm, and neuronal action potential generation, is not clear.

Actions and Interactions of Cholinergic and Excitatory Aminoacid Receptors on Phosphoinositide Signals, Excitotoxicity and Neuroplasticity

The excitatory neurotransmitters acetylcholine and glutamate are involved in neuronal plasticity, which is thought to be an essential component of learning and memory. The loss of cognitive ability in Alzheimer’s disease and in age-associated memory impairment has been suggested to be secondary to a loss of central nervous system cholinergic transmission. Drugs which specifically disrupt cholinergic transmission have profound effects on learning and memory. A loss of cholinergic neurons clearly occurs early in the course of Alzheimer’s disease when memory loss is the only prominent symptom. In studies using experimental models of Alzheimer’s disease, lesioning cholinergic neurons also disrupts the ability of animals to learn. Glutamate has also been implicated in memory processes. Drugs that block glutamate receptors, particularly the N-methyl-D-aspartate (NMDA) receptor subtype, can produce cognitive deficits. An in vitro model of synaptic plasticity, long-term potentiation (LTP), is thought to be mediated in part through NMDA receptors. These studies suggest that both cholinergic and glutamatergic signals play an important role in memory processes and cognitive function.

Radio-label and mass determinations of inositol 1,3,4,5-tetrakisphosphate formation in rat cerebral cortical slices : differential effects of myo-inositol

To investigate the effects of increasing concentrations ofmyo-inositol (inositol) on receptor stimulated [3H]inositol polyphosphate formation in the absence of lithium, slices of rat cerebral cortex were incubated with various concentrations of [3H]inositol (1 to 30 μM). Carbachol stimulated formation of [3H]inositol trisphosphate (InsP3) and [3H]inositol 1,3,4,5-tetrakisphosphate {Ins(1,3,4,5)P4} increased several fold when the inositol concentration was increased reaching a plateau at approximately 12 μM inositol. Time course studies revealed that in the presence of low concentrations of inositol (1 μM), [3H]InsP3 and [3H]Ins(1,3,4,5)P4 formation in response to carbachol stimulation increased slowly over a 10 to 20 min time period, whereas in the presence of 4 and 12 μM inositol, carbachol stimulated [3H]InsP3 and [3H]Ins(1,3,4,5)P4 formation was rapid and essentially complete within 3 to 5 min after carbachol addition. Although the carbachol dose response in 12 μM inositol had a much greater maximal efficacy, there was no change in potency. Similar to the effects of carbachol on [3H]Ins(1,3,4,5)P4 formation from prelabeled phosphoinositides, muscarinic receptor stimulation increased Ins(1,3,4,5)P4 mass formation by seven fold. Furthermore, Li+ (8 mM) completely inhibited carbachol stimulated increases in Ins(1,3,4,5)P4 mass formation. In contrast to the effects of increasing inositol on carbachol stimulated formation of radiolabeled inositol phosphates, increasing inositol had no effect upon mass formation of Ins(1,3,4,5)P4. These results show that when measuring inositol polyphosphate formation by the radiolabeling technique in the absence of Li+, increasing the inositol concentration greatly increases the stimulated component of [3H]InsP3 and [3H]Ins(1,3,4,5)P4 formation. However, this inositol induced increase in agonist stimulated Ins(1,3,4,5)P4 formation is not reflected as an increase in mass formation.