Stephen B. Bocckino

Ca2 +-mobilizing hormones elicit phosphatidylethanol accumulation via phospholipase D activation

Vasopressin, angiotensin II and epinephrine elicited the accumulation of phosphatidylethanol in rat hepatocytes exposed to ethanol and of phosphatidate in the absence of ethanol. When isolated liver plasma membranes were exposed to ethanol, GTPγS stimulated the production of phosphatidylethanol whereas phosphatidate was formed in the absence of ethanol. With increasing ethanol concentrations, phosphatidate formation declined whereas phosphatidylethanol production increased. These findings suggest that rat hepatocytes possess a hormone‐dependent phospholipase D activity that can also catalyse the formation of phosphatidylethanol.

Cell signalling through phospholipid breakdown

There is much evidence that G-proteins transduce the signal from receptors for Ca2+-mobilizing agonists to the phospholipase C that catalyzes the hydrolysis of phosphoinositides. However, the specific G-proteins involved have not been identified. We have recently purified a 42 kDa protein from liver that activates phosphoinositide phospholipase C and cross-reacts with antisera to a peptide common to G-protein α-subunits. It is proposed that this protein is the α-subunit of the G-protein that regulates the phospholipase in this tissue.

An early elevation of diacylglycerol and phosphatidate in regenerating liver

Liver diacylglycerol and phosphatidate are elevated following partial hepatectomy. These increases precede those in DNA synthesis and triacylglycerol accumulation. Possible factors involved in the increase in the lipids and the possible role of the lipids in liver regeneration are discussed.

An enzymatic assay for picomole levels of phosphatidate

An assay for phosphatidate that is sensitive, specific, and highly reproducible is described. Phosphatidate is transacylated with methylamine and the glycerol 3-phosphate produced is measured enzymatically. The assay is linear from 50 to 1500 pmol of phosphatidate and can be used to quantitate phosphatidate in small amounts of biological materials.

Role of Guanine Nucleotide Regulatory Proteins and Inositol Phosphates in the Hormone Induced Mobilization of Hepatocyte Calcium

Treatment of isolated hepatocytes with F- produced a concentration-dependent activation of phosphorylase, efflux of Ca2+, rise in [Ca2+]i, increase in Ins 1,4,5-P3 levels, decrease in PI-4,5-P2 levels, and increase in DAG levels. The levels of intracellular cAMP were decreased by NaF. The effects of NaF were potentiated by AlCl3. This potentiation was abolished by the Al3+ chelator deferoxamine. These results illustrate that AlF4- can mimic the effects of Ca2+-mobilizing hormones in hepatocytes and suggest that the coupling of the receptors for these hormones to the hydrolysis of PI-4,5-P2 is through a guanine nucleotide-binding regulatory protein. This is because AlF4- is known to modulate the activity of other guanine nucleotide regulatory proteins (Gi, Gs, and transducin). Calcium-sensitive inositide release in a purified rat liver plasma membrane preparation was increased by calcium-mobilizing hormones in the presence of guanine nucleotides. Vasopressin-stimulated inositide release was evident in the presence of GTP or GTP gamma S. The guanine nucleotide and hormonal stimulation was evident on both inositide production and PI 4,5-P2 degradation. Treatment of plasma membranes with cholera toxin or islet activating protein or prior injection of animals with islet activating protein did not affect stimulation of inositide release by GTP gamma S or GTP gamma S plus vasopressin. The results suggest that calcium-mobilizing hormones stimulate polyphosphoinositide breakdown in rat liver plasma membranes through a novel guanine nucleotide binding protein. The GTPase activity of rat liver plasma membranes was stimulated 20% by 10(-8) M vasopressin. The vasopressin-stimulated GTPase activity was not inhibited in plasma membranes that had been ADP-ribosylated with either cholera toxin or pertussis toxin. When membranes that had been solubilized after preincubation with [3H]vasopressin were subjected to sucrose gradient centrifugation, most of the protein-bound [3H]vasopressin migrated as a single band, also, there was a GTPase activity that migrated with the bound [3H]vasopressin. This peak of bound [3H]vasopressin was decreased 90% when the sucrose gradient centrifugation was run in the presence of 10 M GTP gamma S. Direct evidence that a GTP-binding protein was present in the [3H]vasopressin peak was obtained by the immuno-detection of a 35 kDa beta subunit of a GTP-binding protein and a 40 kDa alpha subunit. These results support the conclusion that liver plasma membranes contain a GTP-binding protein that can complex with the vasopressin receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Phosphatidylcholine Metabolism in Signal Transduction

Publisher Summary This chapter provides an overview of phosphatidylcholine metabolism in signal transduction. Although the hepatocyte is not an inflammatory cell, there is reason to believe that many of the signaling pathways found in the hepatocyte are also present in the other types of cells. The chapter reviews a work on phosphatidylcholine breakdown and hormone action in the rat liver. These studies reflect the tremendous interest in the mechanisms of signal transduction in many laboratories today. The rat liver has many advantages as a system for elucidating the mechanisms of signal transduction. Approximately, 10 g of liver is available from a 250-gm rat; the collagenase perfusion method yields 3–4 gm of hepatocytes from each liver. These hepatocytes are 85–95% viable and respond to a wide variety of agonists, including epinephrine, norepinephrine, vasopressin, angiotensin II, glucagon, epidermal growth factor, insulin, and ATP.

HEPATIC VASOPRESSIN RECEPTOR: A KEY RECEPTOR OF PHOSPHOINOSITIDE METABOLISM

It is generally accepted that many drugs, hormones and neurotransmitters elicit responses in cells by interacting with specific receptors located in the plasma membrane. A number of receptors for hormones and neurotransmitters have been purified to homogeneity and functionally reconstituted. Most of them mediate agonist actions by interacting with effector systems to modulate the intracellular levels of second messengers via members of a family of guanine nucleotide binding proteins (G proteins).

Regulation of Hepatic Glycogenolysis by Calcium-Mobilizing Hormones

The hormonal regulation of glycogen metabolism in the liver has been a subject of investigation for more than 30 years. The intracellular mediators responsible for this regulation have been identified as cAMP and Ca2+ . In the liver, several hormones such as vasopressin, angiotensin II, epidermal growth factor (Boschet al., 1986), glucagon (Blackmore and Exton, 1986),α 1-adrenergic agonists and P2purinergic agonists (Charestet al., 1985a,b) increase free cytosolic Ca2+ ([Ca2+ ]i). Each of these hormones binds to specific cell surface receptors; this interaction then leads to the activation of a guanine nucleotide-binding protein (Gp) (e.g., Blackmoreet al., 1985; Uhinget al., 1986). In the case of glucagon and epidermal growth factor, the mechanism of activation of Gpis not known but probably involves phosphorylation of Gp(Boschet al., 1986; Johnsonet al., 1986; Blackmore and Exton, 1986). This coupling protein then activates a specific phospholipase C which catalyzes the breakdown of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) (Creba et al., 1983; Rhodeset al., 1983; Thomaset al., 1983; Litoschet al., 1983). The hydrolysis of PI-4,5-P2yields myoinositol-1,4,5-trisphosphate (Ins-1,4,5-P3) (Thomaset al., 1984) and 1,2-diacylglycerol (DAG) (Bocckinoet al., 1985). The Ins-1,4,5-P3releases Ca2+ from the endoplasmic reticulum into the cytoplasm (Josephet al., 1984), while DAG activates a Ca2+ -and phospholipid-dependent protein kinase (protein kinase C) in the plasma membrane (Nishizuka, 1984; Berridge, 1984).