https://www.gastrojournal.org/article/S0016-5085(82)80339-9/pdf

Utilization of nutrients by isolated epithelial cells of the rat colon.

A variety of neurotransmitters, hormones, and other regulatory agents affect the phosphorylation of specific proteins in their target tissues. The types of stimuli that share this common effect on protein phosphorylation include numerous substances that do not act through cyclic AMP. These and other observations suggest that many different classes of regulatory substances achieve certain of their biological effects by altering the phosphorylation of specific proteins.

http://science.sciencemag.org/content/sci/199/4325/146.full.pdf

Phosphorylated proteins as physiological effectors

The colonic epithelium in ulcerative colitis: an energy-deficiency disease?

Calcium is an important signaling molecule involved in the regulation of many cellular functions. The large free energy in the Ca(2+) ion membrane gradients makes Ca(2+) signaling inherently sensitive to the available cellular free energy, primarily in the form of ATP. In addition, Ca(2+) regulates many cellular ATP-consuming reactions such as muscle contraction, exocytosis, biosynthesis, and neuronal signaling. Thus, Ca(2+) becomes a logical candidate as a signaling molecule for modulating ATP hydrolysis and synthesis during changes in numerous forms of cellular work. Mitochondria are the primary source of aerobic energy production in mammalian cells and also maintain a large Ca(2+) gradient across their inner membrane, providing a signaling potential for this molecule. The demonstrated link between cytosolic and mitochondrial Ca(2+) concentrations, identification of transport mechanisms, and the proximity of mitochondria to Ca(2+) release sites further supports the notion that Ca(2+) can be an important signaling molecule in the energy metabolism interplay of the cytosol with the mitochondria. Here we review sites within the mitochondria where Ca(2+) plays a role in the regulation of ATP generation and potentially contributes to the orchestration of cellular metabolic homeostasis. Early work on isolated enzymes pointed to several matrix dehydrogenases that are stimulated by Ca(2+), which were confirmed in the intact mitochondrion as well as cellular and in vivo systems. However, studies in these intact systems suggested a more expansive influence of Ca(2+) on mitochondrial energy conversion. Numerous noninvasive approaches monitoring NADH, mitochondrial membrane potential, oxygen consumption, and workloads suggest significant effects of Ca(2+) on other elements of NADH generation as well as downstream elements of oxidative phosphorylation, including the F(1)F(O)-ATPase and the cytochrome chain. These other potential elements of Ca(2+) modification of mitochondrial energy conversion will be the focus of this review. Though most specific molecular mechanisms have yet to be elucidated, it is clear that Ca(2+) provides a balanced activation of mitochondrial energy metabolism that exceeds the alteration of dehydrogenases alone.

/pdf/role-of-mitochondrial-ca2-in-the-regulation-of-cellular-2d0byw9kl2.pdf

Role of Mitochondrial Ca2+ in the Regulation of Cellular Energetics

Dichloroacetate (DCA) exerts multiple effects on pathways of intermediary metabolism. It stimulates peripheral glucose utilization and inhibits gluconeogeneis, thereby reducing hyperglycemia in animals and humans with diabetes mellitus. It inhibits lipogenesis and cholesterolgenesis, thereby decreasing circulating lipid and lipoprotein levels in short-term studies in patients with acquired or hereditary disorders of lipoprotein metabolism. By stimulating the activity of pyruvate dehydrogenase, DCA facilitates oxidation of lactate and decreases morbidity in acquired and congenital forms of lactic acidosis. The drug improves cardiac output and left ventricular mechanical efficiency under conditions of myocardial ischemia or failure, probably by facilitating myocardial metabolism of carbohydrate and lactate as opposed to fat. DCA may also enhance regional lactate removal and restoration of brain function in experimental states of cerebral ischemia. DCA appears to inhibit its own metabolism, which may influence the duration of its pharmacologic actions and lead to toxicity. DCA can cause a reversible peripheral neuropathy that may be related to thiamine deficiency and may be ameliorated or prevented with thiamine supplementation. Other toxic effects of DCA may be species-specific and reflect marked interspecies variation in pharmacokinetics. Despite its potential toxicity and limited clinical experience, DCA and its derivatives may prove to be useful in probing regulatory aspects of intermediary metabolism and in the acute or chronic treatment of several metabolic disorders.

The pharmacology of dichloroacetate.

Dichloroacetate and other halogenated carboxylic acids can activate pyruvate dehydrogenase in rat heart and in rat heart mitochondria by effecting conversion of phosphorylated (inactive) pyruvate dehydrogenase into an active (dephosphorylated) form. These compounds are inhibitors of pig heart pyruvate dehydrogenase kinase. Pig heart pyruvate dehydrogenase phosphate phosphatase requires both Mg2+and Ca2+ for activity. Evidence is given for a dual metal requirement of the phosphatase in rat fat cell mitochondria. It is concluded that Mg2+ and Ca2+ are essential cofactors for the phosphatase but that their physiological significance as regulators remains to be established. Pig heart pyruvate dehydrogenase kinase is inhibited by high concentrations of magnesium and by low concentrations of calcium.

Regulation of mammalian pyruvate dehydrogenase.

The metal-ion requirement of extracted and partially purified pyruvate dehydrogenase phosphate phosphatase from rat epididymal fat-pads was investigated with pig heart pyruvate dehydrogenase [(32)P]phosphate as substrate. The enzyme required Mg(2+) (K(m) 0.5mm) and was activated additionally by Ca(2+) (K(m) 1mum) or Sr(2+) and inhibited by Ni(2+). Isolated fat-cell mitochondria, like liver mitochondria, possess a respiration- or ATP-linked Ca(2+)-uptake system which is inhibited by Ruthenium Red, by uncouplers when linked to respiration, and by oligomycin when linked to ATP. Depletion of fat-cell mitochondria of 75% of their total magnesium content and of 94% of their total calcium content by incubation with the bivalent-metal ionophore A23187 leads to complete loss of pyruvate dehydrogenase phosphate phosphatase activity. Restoration of full activity required addition of both MgCl(2) and CaCl(2). SrCl(2) could replace CaCl(2) (but not MgCl(2)) and NiCl(2) was inhibitory. The metal-ion requirement of the phosphatase within mitochondria was thus equivalent to that of the extracted enzyme. Insulin activation of pyruvate dehydrogenase in rat epididymal fat-pads was not accompanied by any measurable increase in the activity of the phosphatase in extracts of the tissue when either endogenous substrate or (32)P-labelled pig heart substrate was used for assay. The activation of pyruvate dehydrogenase in fat-pads by insulin was inhibited by Ruthenium Red (which may inhibit cell and mitochondrial uptake of Ca(2+)) and by MnCl(2) and NiCl(2) (which may inhibit cell uptake of Ca(2+)). It is concluded that Mg(2+) and Ca(2+) are cofactors for pyruvate dehydrogenase phosphate phosphatase and that an increased mitochondrial uptake of Ca(2+) might contribute to the activation of pyruvate dehydrogenase by insulin.

/pdf/calcium-and-magnesium-ions-as-effectors-of-adipose-tissue-3qtlx43eqw.pdf

Calcium and magnesium ions as effectors of adipose-tissue pyruvate dehydrogenase phosphate phosphatase.

1. Isolated fat-cells and intact epididymal fat-pads were incubated in medium containing 45Ca2+ and the incorporation of 45Ca into mitochondrial and extramitochondrial fractions was studied. Redistribution of 45Ca between these fractions was essentially prevented by the addition of EGTA [ethanedioxybis(ethylamine)tetra-acetate] and Ruthenium Red to the sucrose-based extraction medium. 2. Incorporation of 45Ca into mitochondrial fractions of both fat-cells and fat-pads was found to be complete within 2-5 min, suggesting that mitochondria contain a pool of calcium in rapid isotopic exchange with extracellular Ca2+. This pool was about 20 times larger in mitochondria within fat-cells than within fat-pads. In fat-cells, 45Ca incorporation into the mitochondrial fraction accounted for about 34% of the total 45Ca incorporation into cells after 20 min and about 50% of the total mitochondrial calcium content measured by atomic absorption; values in fat-pads were about 7 and 20% respectively.

/pdf/exchangeable-and-total-calcium-pools-in-mitochondria-of-rat-395qwk3wqr.pdf

Exchangeable and total calcium pools in mitochondria of rat epididymal fat-pads and isolated fat-cells. Role in the regulation of pyruvate dehydrogenase activity.

David L. Severson

Papers

Regulation of mammalian pyruvate dehydrogenase.

Calcium and magnesium ions as effectors of adipose-tissue pyruvate dehydrogenase phosphate phosphatase.

Exchangeable and total calcium pools in mitochondria of rat epididymal fat-pads and isolated fat-cells. Role in the regulation of pyruvate dehydrogenase activity.

Calcium ions and the regulation of pyruvate dehydrogenase.