Binding sites for inositol trisphosphate in the bovine adrenal cortex

Binding sites for inositol trisphosphate (IP3) have been identified in bovine adrenal cortex, employing [32P]IP3 prepared from human erythrocytes radiolabeled with [32P]ATP. IP3 was bound to adrenal microsomes with high affinity (Kd = 5 nM) and low capacity (186 fmol/mg protein). During kinetic studies, half-maximal binding was reached in less than one min at 4 degrees C, and dissociation was even more rapid with t1/2 of about 10 sec. [32P]IP2 showed no binding to the microsomal sites, which represent putative receptors at which IP3 acts to elevate intracellular calcium concentration during the actions of peptide hormones such as angiotensin II.     

Binding of Rb86 by a microsomal fraction of bovine cerebral cortex cells]

The maximal binding of 86Rb was noted when ions of Mg, ATP and Na were present simultaneously. The availability of olytoriside (1-10(-4)M), CaCl2 (1-10(-3)M) and phospholipase A (1-10(-5)M) in the medium and the removal of Na and substitution of ATP for ADP decreased to a different extent the binding of 86Rb.     

Isomers of inositol trisphosphate in exocrine pancreas.

In rat pancreatic acinar cells, the Ca2+-mobilizing receptor-agonist, caerulein, at both maximal and submaximal concentrations, stimulated a rapid, transient, increase in [3H]inositol 1,4,5-trisphosphate [(1,4,5)IP3], followed by a slower, sustained, increase in [3H]inositol 1,3,4-trisphosphate [(1,3,4)IP3]. Neither activation of protein kinase C by phorbol dibutyrate nor prevention of the caerulein-stimulated elevation of cytosolic [Ca2+] significantly affected the pattern of formation of the two isomers of IP3. Although carbachol evoked an increase in cytosolic [Ca2+], it did not significantly stimulate [3H](1,4,5)IP3 accumulation, but did promote [3H](1,3,4)IP3 accumulation. Moreover, both carbachol and caerulein maintained hormone-sensitive intracellular Ca2+ pools in a Ca2+-depleted state after [3H](1,4,5)IP3 had returned to basal concentrations. One interpretation of these findings is that total cellular concentrations of [3H](1,4,5)IP3 may not accurately reflect the concentration of this putative mediator in biologically relevant compartments.     

Regulation of autophagy by the inositol trisphosphate receptor

The reduction of intracellular 1,4,5-inositol trisphosphate (IP(3)) levels stimulates autophagy, whereas the enhancement of IP(3) levels inhibits autophagy induced by nutrient depletion. Here, we show that knockdown of the IP(3) receptor (IP(3)R) with small interfering RNAs and pharmacological IP(3)R blockade is a strong stimulus for the induction of autophagy. The IP(3)R is known to reside in the membranes of the endoplasmic reticulum (ER) as well as within ER-mitochondrial contact sites, and IP(3)R blockade triggered the autophagy of both ER and mitochondria, as exactly observed in starvation-induced autophagy. ER stressors such as tunicamycin and thapsigargin also induced autophagy of ER and, to less extent, of mitochondria. Autophagy triggered by starvation or IP(3)R blockade was inhibited by Bcl-2 and Bcl-X(L) specifically targeted to ER but not Bcl-2 or Bcl-X(L) proteins targeted to mitochondria. In contrast, ER stress-induced autophagy was not inhibited by Bcl-2 and Bcl-X(L). Autophagy promoted by IP(3)R inhibition could not be attributed to a modulation of steady-state Ca(2+) levels in the ER or in the cytosol, yet involved the obligate contribution of Beclin-1, autophagy-related gene (Atg)5, Atg10, Atg12 and hVps34. Altogether, these results strongly suggest that IP(3)R exerts a major role in the physiological control of autophagy.     

Thrombin-induced inositol trisphosphate production by rabbit platelets is inhibited by ethanol.

Ethanol has an inhibitory effect on some platelet functions, but the mechanisms by which it exerts this effect are not known. Using suspensions of washed platelets, we observed that ethanol (1-9 mg/ml) did not affect the aggregation of rabbit platelets stimulated with ADP (0.5-10 microM). When platelets were prelabelled with 5-hydroxy[14C]tryptamine, aggregation and secretion of granule contents in response to thrombin (0.01-0.10 unit/ml) were not inhibited by ethanol, but these responses to thrombin at lower concentrations (less than 0.01 unit/ml) were inhibited by ethanol (2-4 mg/ml). Platelets were prelabelled with [3H]inositol so that increases in inositol phosphates upon stimulation could be assessed by measuring the amount of label in these compounds. ADP-induced increases in IP (inositol phosphate) and IP2 (inositol bisphosphate) were not affected by ethanol. IP3 (inositol trisphosphate) was not changed by ADP or ethanol. Although ethanol did not affect the increases in IP, IP2 and IP3 caused by stimulation of platelets with thrombin at concentrations greater than 0.01 unit/ml, ethanol did inhibit the increases observed at 2 and 3 min in these inositol phosphates caused by lower concentrations of thrombin (less than 0.01 unit/ml). Since ADP did not cause formation of IP3 in rabbit platelets, and since no thromboxane B2 was detected in platelets stimulated with the lower concentrations of thrombin, it is unlikely that the inhibitory effect of ethanol in IP3 formation was due to effects on further stimulation of platelets by released ADP or by thromboxane A2. Ethanol may inhibit platelet responses to thrombin by inhibiting the production of the second messenger, IP3.     

Purification of phospholipid methyltransferase from rat liver microsomal fraction.

Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.     

The cell-free synthesis of cytochrome c by a microsomal fraction from rat liver

Conditions were investigated for demonstrating the synthesis in vitro of the complete molecule of cytochrome c by isolated liver microsomal systems from partially hepatectomized rats. It was first found that in vivo the early labelled cytochrome c associated with the microsomal fraction required, by comparison with the mitochondrial pool, more drastic conditions of extraction and its binding was less affected by freezing and thawing of the subcellular particles. The procedure of extraction and purification of cytochrome c had to be modified accordingly, to assure the recovery of the recently synthesized molecule. Several subcellular fractions were isolated from regenerating liver with a homogenization medium containing either 5 or 10mm-Mg(2+) and most of them were active in the synthesis of the cytochrome c apoprotein. The microsomal fraction, in the presence of either cell sap or pH5.0 fraction, was also able to incorporate [(59)Fe]haemin, delta-amino[(3)H]laevulic acid and (55)Fe into the prosthetic group of cytochrome c. These experiments confirm firmly the conclusions of our previous results obtained in vivo showing that both the apoprotein and the haem moieties are made and linked together on cytoplasmic ribosomes and only then is the complete molecule transferred to the mitochondria.     

Models of the inositol trisphosphate receptor

The inositol (1,4,5)-trisphosphate receptor (IPR) plays a crucial role in calcium dynamics in a wide range of cell types, and is often a central feature in quantitative models of calcium oscillations and waves. We review deterministic and stochastic mathematical models of the IPR, from the earliest ones of the 1970s and 1980s, to the most recent. The effects of IPR stochasticity on Ca2+ dynamics are briefly discussed.     

Metabolism of synthetic inositol trisphosphate analogs

A series of synthetic analogs was employed to explore structure-activity relationships in the metabolism of the second messenger inositol trisphosphate (IP3) in vascular tissue. Cytosolic IP3-5-phosphatase activity was purified approximately 240-fold from bovine aorta. All synthetic analogs tested were apparent competitive inhibitors of the 5-phosphatase activity. The order of potency was DL-1,3,4,5-IP3 greater than D-1,4,5-IP3 greater than DL-1,3,4-IP3 greater than L-1,4,5-IP3 greater than 1,3,5-IP3 greater than DL-6-methoxy-1,4,5-IP3 greater than DL-2,4,5-IP3 greater than DL-1,2,4-cyclohexane-P3. The least potent analogs had Ki values only 11 times higher than the apparent Km of the substrate D-1,4,5-[3H]IP3. However, only three synthetic compounds, DL-1,3,4,5-IP4, D-1,4,5-IP3, and DL-2,4,5-IP3, could serve as substrates for the 5-phosphatase. IP3 kinase activity in the same tissue exhibited considerably more selectivity with respect to inhibition by IP3 analogs. D-1,4,5-IP3 was about 30 times more potent than DL-1,3,4,5-IP4 and 100-1000 times more potent than the other compounds tested. The function of the IP3 receptor was evaluated by measuring labeled calcium mobilization in permeabilized bovine aortic smooth muscle cells in culture. While all analogs tested were full agonists, vast differences in potency were observed. D-1,4,5-IP3 was about 30 times more potent than DL-2,4,5-IP3 and 100-2000 times more potent than the other analogs tested. The results suggest that IP3-5-phosphatase activity is relatively nonselective in the binding of inositol polyphosphates, while IP3 kinase activity and the IP3 receptor exhibit great selectivity in the recognition of these compounds.     

Regenerative release of calcium from functionally discrete subcellular stores by inositol trisphosphate.

Fluorescence imaging was used to determine the spatial and temporal patterns of subcellular calcium (Ca2+) liberation induced in Xenopus oocytes by photorelease of inositol 1,4,5-trisphosphate (InsP3) from a caged precursor. Increasing levels of InsP3 evoked Ca2+ release that began in a graded manner but, at varying threshold levels of InsP3, localized sites then showed transient and asynchronous 'puffs' of Ca2+ release. With higher levels of InsP3, Ca2+ from adjacent sites formed a focus for initiation of a propagating Ca2+ wave. The results show that InsP3-sensitive Ca2+ stores are arranged as distinct and functionally independent units, and that Ca2+ is released in both graded and regenerative fashions.     

Chemical synthesis of bile acid acyl-adenylates and formation by a rat liver microsomal fraction

In mammals, unconjugated bile acids formed in the intestine by bacterial deconjugation are reconjugated (N-acylamidated) with taurine or glycine during hepatocyte transport. Activation of the carboxyl group of bile acids to form acyl-adenylates is a likely key intermediate step in bile acid N-acylamidation. To gain more insight into the process of bile acid adenylate formation, we first synthesized the adenylates of five common, natural bile acids (cholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, and lithocholic acid), and confirmed their structure by proton NMR. We then investigated adenylate formation by subcellular fractions of rat liver (microsomes, mitochondria, cytosol) using a newly developed LC method for quantifying adenylate formation. The highest activity was observed in the microsomal fraction. The reaction required Mg²⁺ and its optimum pH was about pH 7.0. In term of maximum velocity (V_max) and the Michaelis constant (K_m), the catalytic efficiency of the enzyme under the conditions used was highest with cholic acid of the bile acids tested. The formation of cholyl-adenylate was strongly inhibited by lithocholic and deoxycholic acid, as well as by palmitic acid; ibuprofen and valproic acid were weak inhibitors. In cholestatic disease, such adenylate formation might lead to subsequent bile acid conjugation with glutathione or proteins.     

Properties of energy-dependent calcium transport by rat liver microsomal fraction as revealed by initial-rate measurements.

Measurements of the initial rate of Ca2+ transport by rat liver microsomal preparations reveal the existence of two phases of transport activity. The first, a phase of rapid transport, is complete by 3-5 min, at which time the second (slower) phase begins; this remains linear for up to at least 40 min. The initial phase is minimal in the absence of MgATP. The initial rate of Ca2+ transport reaches values as high as 25 nmol/min per mg of protein; the Km for Ca2+total is 1-2 micrometer and that for MgATPtotal about 500 micrometer. Ruthenium Red (3-5 nmol/mg of protein) has little effect on the initial rate of transport, whereas tributylin (2 micrometer) inhibits equally in a KC1- or a KNO3-containing medium. Compunds that collapse components of the proton electrochemical gradient in mitochondria (valinomycin and carbonyl cyanide m-chlorophenylhydrazone) each inhibit by 70-80% the initial rate of microsomal Ca2+ transport.     

The inositol trisphosphate phosphomonoesterase of the human erythrocyte membrane.

Human erythrocyte ghosts exhibit an inositol trisphosphate phosphomonoesterase activity that rapidly converts inositol 1,4,5-trisphosphate into inositol 1,4-bisphosphate and Pi. Degradation of the released inositol 1,4-bisphosphate is not observed. This activity is dependent on Mg2+ (or Mn2+) and it is not activated by Ca2+. Optimum activity is around pH 7 and activity is abolished by heat denaturation. The Km for inositol trisphosphate is approx. 25 microM. 2,3-bisphosphoglycerate is a competitive inhibitor, with a Ki of approx. 0.35 mM. Glycerophosphoinositol 4,5-bisphosphate is attacked at about one-eighth of the rate for inositol trisphosphate, but glycerophosphoinositol 4-phosphate is not a substrate. Incubation of 32P-labelled erythrocyte membranes with Mg2+ causes little breakdown of phosphatidylinositol 4,5-bisphosphate, the parent compound from which both glycerophosphoinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate are derived. On the basis of its substrate specificity and the inhibition by 2,3-bisphosphoglycerate, we suggest that this enzyme is selective for the 5-phosphate in those water-soluble phosphate esters of inositol that possess the vicinal pair of 4,5-phosphates but that it may also interact less strongly with other water-soluble compounds that have pairs of vicinal phosphates.     

Secretagogue-induced formation of inositol phosphates in rat exocrine pancreas. Implications for a messenger role for inositol trisphosphate.

The formation of inositol phosphates in response to secretagogues was studied in rat pancreatic acini preincubated with [3H]inositol. Carbachol caused rapid increases in radioactive inositol phosphate, inositol bisphosphate and inositol trisphosphate . This effect was blocked by atropine, and also elicited by caerulein, but not by ionomycin or phorbol dibutyrate. Thus phospholipase C-mediated breakdown of polyphosphoinositides, with the resulting formation of inositol phosphates, may be an early step in the stimulus-secretion coupling pathway in exocrine pancreas. Inositol trisphosphate may function as a second messenger in the exocrine pancreas, coupling receptor activation to internal Ca2+ release.     

Yeast inositol mono- and trisphosphate levels are modulated by inositol monophosphatase activity and nutrients

Yeast lithium-sensitive inositol monophosphatase (IMPase) is encoded by a non-essential gene pair (IMP1 and IMP2). Inhibition of IMPase with either Li(+) or Na(+) or a double null mutation imp1 imp2 causes increased levels of inositol monophosphates and reduced level of inositol 1,4,5-trisphosphate. Overexpression of the IMP2 gene has the opposite effects and these results suggest that IMPase activity is limiting for the inositol cycle. Addition of ammonium to cells starved for this nutrient results in a decrease of inositol monophosphates and an increase of inositol 1,4,5-triphosphate, pointing to simultaneous regulation of both inositol 1,4,5-triphosphate production and IMPase activity.