Thomas A. Rooney

Ethanol and signal transduction in the liver.

The liver is a major target for both short‐ and long‐term actions of ethanol. The mechanisms that mediate the response of cells and tissues to chronic intake of ethanol are unknown, but it is likely that both adaptive and deleterious responses are triggered by short‐term interactions of the cell with ethanol. Cellular signaling processes are candidates to mediate the connection between short‐ and long‐term actions of ethanol. Receptor‐coupled signal transduction systems in the plasma membrane of many different cell types are affected by ethanol. In the liver, the signaling processes associated with phospholipases C and D are particularly responsive to ethanol. In this review, we investigate the direct and indirect short‐term effects of ethanol on the signal transduction systems in liver and discuss the possible implications for the responses of the liver to chronic ethanol exposure.—Hoek, J. B.; Thomas, A. P.; Rooney, T. A.: Higashi, K.; Rubin, E. Ethanol and signal transduction in the liver. FASEB J. 6: 2386‐2396; 1992.

Spatial and temporal organization of calcium signalling in hepatocytes

Treatment of hepatocytes with agonists which act via the second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), results in increases of cytosolic free Ca2+ [( Ca2+]i) which are manifest as a series of discrete [Ca2+]i transients or oscillations. With increasing agonist dose [Ca2+]i oscillation frequency increases and the initial latent period decreases, but the amplitude of the [Ca2+]i oscillations remains constant. Studies of these [Ca2+]i oscillations at the subcellular level have indicated that the [Ca2+]i changes do not occur synchronously throughout the cell, but initiate at a specific subcellular domain, adjacent to a region of the plasma membrane, and then propagate through the cell as a [Ca2+]i wave. For a given ceil, the locus of [Ca2+]i wave initiation is constant for every oscillation in a series and is also identical when the cell is sequentially stimulated with different agonists or when the phospholipase C-linked G protein is activated directly using AIF4-. The kinetics of the [Ca2+]i waves indicate that a Ca(2+)-activated mechanism is involved in propagating the oscillatory [Ca2+]i increases throughout the cell, and the data appear to be most consistent with a process of Ca(2+)-induced Ca2+ release. It is proposed that the ability to propagate [Ca2+]i oscillations into regions of the cell distal to the region in which the signal transduction apparatus is localized could serve an important function in allowing all parts of the cell to respond to the stimulus.

Intracellular calcium waves generated by ins(1,4,5)P3-dependent mechanisms

Cellular oscillations of cytosolic free Ca2+ ([Ca2+]i) have been observed in many cell types in response to cell surface receptor agonists acting through inositol 1,4,5-trisphosphate (InsP3). In a number of cases where appropriate spatial and temporal resolution have been used to examine these [Ca2+]i oscillations, they have been found to be organized as repetitive waves of Ca2+ increase that propagate through the cytosol of individual cells. In some cases Ca2+ waves also occur as a single pass through stimulated cells. This review discusses the factors underlying the spatial organization of [Ca2+]i signals in the form of Ca2+ waves. In addition, potential mechanisms for the initiation and subsequent propagation of these Ca2+ waves are described.

N-Methyl-d-aspartate Inhibits Apoptosis through Activation of Phosphatidylinositol 3-Kinase in Cerebellar Granule Neurons A ROLE FOR INSULIN RECEPTOR SUBSTRATE-1 IN THE NEUROTROPHIC ACTION OF N-METHYL-d-ASPARTATE AND ITS INHIBITION BY ETHANOL

Primary cultured rat cerebellar granule neurons underwent apoptosis when switched from medium containing 25 mm K+ to one containing 5 mmK+. N-methyl-d-aspartate (NMDA) protected granule neurons from apoptosis in medium containing 5 mm K+. Inhibition of apoptosis by NMDA was blocked by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002, but it was unaffected by the mitogen-activated protein kinase kinase inhibitor PD 98059. The antiapoptotic action of NMDA was associated with an increase in the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), an increase in the binding of the regulatory subunit of PI 3-kinase to IRS-1, and a stimulation of PI 3-kinase activity. In the absence of extracellular Ca2+, NMDA was unable to prevent apoptosis or to phosphorylate IRS-1 and activate PI 3-kinase. Significant inhibition of NMDA-mediated neuronal survival by ethanol (10–15%) was observed at 1 mm, and inhibition was half-maximal at 45–50 mm. Inhibition of neuronal survival by ethanol corresponded with a marked reduction in the capacity of NMDA to increase the concentration of intracellular Ca2+, phosphorylate IRS-1, and activate PI 3-kinase. These data demonstrate that the neurotrophic action of NMDA and its inhibition by ethanol are mediated by alterations in the activity of a PI 3-kinase-dependent antiapoptotic signaling pathway.

Cyclic GMP Induces Oscillatory Calcium Signals in Rat Hepatocytes

The ability of guanosine-3′,5′-cyclic monophosphate (cGMP) to induce increases in the intracellular free calcium ion concentration ([Ca2+]i) was studied at the single cell level in fura-2-loaded rat hepatocytes. Both 8-bromo-cGMP (Br-cGMP) and dibutyryl cGMP (db-cGMP) produced oscillatory [Ca2+]i increases in hepatocytes. In addition, Br-cGMP increased the frequency of agonist-induced spiking or converted [Ca2+]i oscillations into sustained nonoscillatory [Ca2+]i responses. Addition of the nitric oxide donor sodium nitroprusside also produced oscillatory [Ca2+]i increases similar to those generated by cGMP analogues. In the absence of extracellular Ca2+, cGMP-induced [Ca2+]i responses were significantly reduced and mainly appeared as single transient [Ca2+]i increases. The effects of cGMP analogues do not appear to be mediated by a secondary increase in cAMP or activation of cAMP-dependent protein kinase (PKA), since [Ca2+]i responses to cGMP analogues were inhibited by the G-kinase inhibitor 8-bromoguanosine-3′,5′-cyclic monophosphorothioate (Rp-Br-cGMP[S]). Both Br-cGMP and db-cGMP also increased [Ca2+]i in the presence of the PKA inhibitor 8-bromoadenosine-3′,5′-cyclic monophosphorothioate (Rp-Br-cAMP[S]) and when the cGMP-inhibitable cAMP phosphodiesterase activity was inhibited by pretreatment with siguazodan. Br-cGMP stimulated the Mn2+-induced quench of compartmentalized fura-2 in intact hepatocytes, indicating a site of action at the level of the Ca2+ stores. This locus was further supported by the finding that pretreatment of hepatocytes with Br-cGMP potentiated submaximal inositol 1,4,5-trisphosphate (InsP3)-induced Mn2+ quench in subsequently permeabilized hepatocytes. db-cGMP also decreased PKA-mediated back phosphorylation of the hepatic type-1 InsP3 receptor, indicating that G-kinase phosphorylates the InsP3 receptor at sites targeted by PKA. These data indicate that phosphorylation of the hepatic InsP3 receptor by G-kinase increases the sensitivity to InsP3 for [Ca2+]i release and is associated with the production of [Ca2+]i oscillations in single rat hepatocytes.

Organization of intracellular calcium signals generated by inositol lipid-dependent hormones

Recent studies at the single cell level have demonstrated hitherto unsuspected complexities in the organization of intracellular Ca2+ homeostasis in both the temporal and spatial domains. Activation of receptors coupled to the phosphoinositide signalling system has been shown to generate [Ca2+]i oscillations in many cell types. These oscillations display diverse patterns, with variations in oscillation amplitude, latency and frequency which are often tissue and/or agonist dose specific. Furthermore, increases in [Ca2+]i can either occur uniformly or originate from a specific region and propagate throughout the cell in the form of a Ca2+ wave. The significance and underlying mechanisms responsible for these phenomena are discussed.

Mechanistic and Functional Aspects of Oscillatory Calcium Signalling

Mobilization of calcium from intracellular stores and the extracellular medium to yield an increase in cytosolic free Ca2+ ([Ca2+]i) is one of the most common forms of signal transduction utilized by extracellular stimuli in the control of cell function. It is now clear that receptor-induced increases in [Ca2+]i are generated, in part, by an elevation in the level of the second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) which is produced by a phospholipase C (PLC)-mediated hydrolysis of phosphatidyl inositol 4,5-bisphosphate (Berridge and Irvine, 1984). Ins(1,4,5)P3 is released into the cytosol where it interacts with an intracellular receptor which functions as a release channel for lumenal Ca2+ (Berridge and Irvine, 1989; Joseph and Williamson, 1989). However, intracellular Ca2+ stores are not uniformly sensitive to Ins(1,4,5)P3, as demonstrated in permeabilized cell studies where Ins(1,4,5)P3 can only release a fraction (30–50%) of the calcium accumulated by non-mitochondrial stores (Berridge and Irvine, 1984; Williamson et al., 1985; Joseph and Williamson, 1989). There is some debate as to the intracellular location of the Ins(1,4,5)P3-sensitive Ca2+ store. In Purkinje cells antibodies to the receptor have revealed localized concentrations on the nuclear envelope and parts of the endoplasmic reticulum (E.R.) (Ross etal., 1989).

Synchronized Ca2+ Transients Induced by Glucagon in Fura2 Loaded Hepatocytes

A wide range of agonists exert their effects on liver cells by the receptor-mediated activation of phospholipase C, leading to the formation of diacylglycerol (DAG) and inositol-1,4,5-trisphosphate(Ins-1,4,5-P3). The, latter compound acts as a second messenger to release Ca2+ from intracellular storage sites, resulting in an elevation of cytosolic free Ca2+ levels (for reviews, see Williamson et a1,1985; Putney, 1989). Secondary adjustments in cellular Ca2+ homeostasis include the Ca2+ release from the cell by activation of the ATP-driven plasma membrane Ca2+ pump, the activation of Ca2+ influx into the cell required for maintaining the total cellular Ca2+ content and the reuptake of Ca2+ by intracellular organelles; other inositol phosphate derivatives may contribute to the control of some of these processes (Putney et al, 1989). A variety of agonists operate through this pathway in liver, including vasopressin, α1-adrenergic agonists and angiotensin II. Glucagon also causes an increase in cytosolic free Ca2+ levels, associated with a small increase in Ins-1,4,5,- P3 levels, although it is not clear whether it activates phospholipase C as a secondary consequence of the formation of CAMP and the activation of Akinase (Staddon and Hansford, 1989) or by interacting with a separate glucagon receptor (GR1 receptor) which is specifically coupled to the activation of phospholipase C (Wakelam et al, 1986).