Pawels Kurian

Cholinergic and serotonergic stimulation of phosphoinositide hydrolysis is decreased in Alzheimer's disease

Agonist-stimulated phosphoinositide (PPI) hydrolysis is a major signal transduction pathway in brain. These studies investigated neurotransmitter stimulated PPI hydrolysis in postmortem human brain. Preliminary studies using rat brain suggested that moderate postmortem delay has little effect on PPI hydrolysis and that human tissue might be reliably studied for differences in receptor-PLC coupling. Studies in human brain membranes (frontal cortex) indicated that the time course for GTP gamma S and carbachol/GTP gamma S-stimulated PPI hydrolysis was linear for at least 20 min. GTP gamma S-stimulated [3H]inositol phosphate (InsP) formation was enhanced by carbachol (232%) and 5-Hydroxytryptamine (5HT-147%). SAX-HPLC separation of [3H]inositol polyphosphates indicated that the major isomer of inositol trisphosphate (InsP3) was Ins(1.4.5)P3, the expected product of PtdIns(4,5)P2 hydrolysis. Ca2+ increased PPI hydrolysis progressively from 100 nM through 50 microM and synergistically enhanced carbachol/GTP gamma S stimulation. Comparisons of age-matched controls with Alzheimers patients indicated that GTP gamma S, carbachol/GTP gamma S, and 5HT/GTP gamma S-stimulation of PPI hydrolysis is reduced approximately 50% in membranes prepared from Alzheimers patients. Ca2+ of PPI hydrolysis was not different between controls and Alzheimers patients suggesting that muscarinic cholinergic and serotonergic receptors are uncoupled from PLC in Alzheimers disease. These studies indicate that there are changes in cholinergic and serotonergic signal transduction in Alzheimers disease. Further, this method can be used to study signal transduction events in postmortem human brain.

Angiotensin II receptor subtypes play opposite roles in regulating phosphatidylinositol hydrolysis in rat skin slices

Among the many functions of angiotensin II (Ang II) it now appears that Ang II is a growth factor. The concentration of Ang II in rat skin has been shown to increase during wound healing. To investigate the intracellular effect of Ang II in skin we determined the levels of total cytoplasmic inositol phosphates after incubation of skin slices with different doses of Ang II. 10(-6) M of Ang II increased significantly the phosphatidylinositol (PI) hydrolysis, and the effect was dose dependent up to 10(-4) M Ang II. The majority of inositol phosphates yielded after 1 hour incubation in the presence of lithium was InsP1, with lesser amount of InsP2. Losartan, the Ang II AT1 antagonist, at a dose of 10(-4) M blocked the effect of Ang II, while PD123319, the Ang II AT2 antagonist, had no antagonistic action; PD123319 at the higher dose of 10(-3) M, however, potentiated the effect of Ang II on PI hydrolysis. The results suggest that PI hydrolysis is a second messenger system for Ang II in rat skin. Also, the two subtypes of Ang II receptors mediate opposite effects on PI hydrolysis: Ang II binding to AT1 receptors increases inositol phosphate production, while Ang II binding to AT2 receptors decreases inositol phosphate production.

Insulin stimulates phosphatidylinositol 3-kinase activity in rat neuronal primary cultures

Abstract: We investigated the effect of insulin on phosphatidylinositol (PtdIns) 3‐kinase (PtdIns 3‐kinase) activity in neuronal cultures to determine if this enzyme is involved with the neurotrophic actions of insulin. Insulin caused a concentration‐dependent increase in PtdIns 3‐kinase activity in anti‐phosphotyrosine immuno‐precipitates. The kinase activity was able to phosphorylate PtdIns, PtdIns 4‐phosphate, and PtdIns 4,5‐bisphosphate. In intact neurons, a 10‐min 1 mM insulin treatment in the presence of [32P]orthophosphate increased the levels of both 3‐[32P]PtdIns phosphate and 3,4‐[32P]PtdIns bisphosphate by 55 and 193%, respectively. This increase was associated with an increase in neurite outgrowth mediated by insulin. Our results indicate that insulin treatment of neuronal cells in primary culture increases PtdIns 3‐kinase activity and the formation of the unique d‐3‐phosphorylated phosphoinositides, suggesting that growth factor‐mediated neuronal growth may include the formation of novel phosphoinositide 3‐phosphate phospholipids.

Decreased carbachol-stimulated inositol 1,3,4,5-tetrakisphosphate formation in senescent rat cerebral cortical slices

It is well established that muscarinic cholinergic receptors are linked to phosphoinositide hydrolysis in brain. Previous studies of muscarinic responses used Li+ to increase inositol phosphate accumulation and suggested little or no change during aging. Li+ disrupts certain aspects of the inositol phosphate metabolism and inhibits the formation of inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. Ins(1,3,4,5)P4 appears to have second messenger functions. To investigate the effects of aging on agonist stimulated Ins(1,3,4,5)P4 formation, young (6-8 months) and old (28-30 months) Fischer 344 rat cerebral cortical or hippocampal slices were challenged with various agonists known to stimulate phosphoinositide hydrolysis in brain using a recently developed assay that does not use Li+. Carbachol and quisqualate stimulated [3H]inositol trisphosphate ([3H]InsP3) and [3H]Ins(1,3,4,5)P4 formation in young and old rat cerebral cortical slices. Norepinephrine, 5-hydroxytryptamine, and vasopressin failed to stimulate [3H]Ins(1,3,4,5)P4 or [3H]InsP3 formation in either young or old rat cerebral cortical slices. In old rat cerebral cortical slices, the carbachol-stimulated [3H]Ins(1,3,4,5)P4 formation was reduced by 44%. Angiotensin II stimulated [3H]InsP3 was increased (219%) in old rats. There was no influence of aging either on the basal level or on the maximal response to carbachol or quisqualate in hippocampal slices. These studies suggest region-specific changes in phosphoinositide hydrolysis during aging.

Synthesis and binding studies of an optically pure hexadeoxy-1,4,5-tris(methylenesulfonic acid) analogue of IP3

The synthesis of the (−)-sodium salt of the hexadeoxy-1, 4, 5-tris(methylenesulfonic acid) analogue of IP3 by use of an asymmetric Diels-Alder reaction is described together with the effects of this compound in binding to the IP3 receptor.

Receptor Coupling to Phosphoinositide Signals

A number of neurotransmitters have been shown to activate phospholipase C (PLC) through a G protein and/or Ca2+ pathway resulting in hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], leading to the formation of inositol 1,4,5-trisphosphate [Ins(l,4,5)P3] and diacylglycerol (DAG). Growth factors stimulate phospholipase C γl (PLCγ) and phosphatidylinositol 3-kinase (PtdIns 3-kinase) by tyrosine phosphorylation of these enzymes. Ins(l,4,5)P3 mobilizes intracellular calcium from a non-mitochondrial, ATP-dependent pool. Ins(l,4,5)P3 can be phosphorylated to inositol 1,3,4,5-tetrakisphosphate [Ins(l,3,4,5)P4] by inositol polyphosphate 3-kinase (Bansal and Majerus, 1990). Ins(l,3,4,5)P4 modulates cytoplasmic Ca2+ levels by mobilizing Ca2+ or sequestering Ca2+ into storage pools mobilized by Ins(l,4,5)P3 (Gawler et al., 1990; Hill et al., 1988). Ins(l,4,5)P3 and Ins(l,3,4,5)P4 are further catabolized by a variety of inositol phosphate-specific phosphatases (Fig. 1). Ptdlns 3-kinase, which phosphorylates phosphoinositides at the 3-position, has been implicated in the transduction of mitogenic signals. The products of Ptdlns 3-kinase are the novel phosphoinositides, phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate, which are not hydrolyzed by PLC (Ruderman et al., 1990).

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).

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.

Expedient synthetic routes to [3H]-D-3 azido-3-deoxy-myo-inositol and D-3-azido-3-deoxy-myo-inositol 2,4,5-trisphosphate

Expedient routes to a tritiated derivative of the v-sis oncogene-transformed cell specific growth inhibitor 3-azido-3-deoxy-myo-inositol and the unlabelled 2,4,5-trisphosphate derivative are described together with the effects of the latter compound in binding to the IP3 receptor.