Ingo Rustenbeck

Lipid composition of glucose-stimulated pancreatic islets and insulin-secreting tumor cells

The effect of glucose stimulation (25 mM for 5 min) on the phospholipid and neutral lipid composition of isolated pancreatic islets was studied to find out whether there is a change in the mass of potential lipid mediators or modulators of insulin secretion. For comparison, the lipid compositions of homogenates and subcellular fractions from RINm5F insulin-secreting tumor cells and of glucose-stimulated streptozotocin/nicotinamide-induced islet cell tumors were analyzed. After separation of the lipid extract into a neutral and an acidic fraction by anion-exchange chromatography, lipids were separated by high-performance thin-layer chromatography and quantitated byin situ densitometry of the cupric sulfate-charred bands. In glucose-stimulated islets, the molar percentages of phosphatidic acid (PA) and of phosphatidylinositol were significantly increased (3.1 vs. 4.7 mol% and 8.6 vs. 11.8 mol%), while those of all other phospholipids and neutral lipids, including 1,2-diacylglycerol, were not significantly changed. In stimulated islet cell tumors, an increase of PA was visible in the microsomal fraction, and there was an increase of lysophosphatidylcholine in the mitochondrial fraction. However, in both tumoral tissues, particularly in RINm5F cells, the lipid distribution pattern showed abnormalities which can be regarded as a loss of differentiation and which limit the usefulness of these tissues for the study of the physiological regulation of lipid metabolism during glucose stimulation. In conclusion, the data are in accordance with a role of PA early in stimulus-secretion coupling. The well-known stimulation of phospholipid synthesis in pancreatic islets during glucose-induced insulin secretion does not result in an increase in the total phospholipid mass.

Energetic Requirement of Insulin Secretion Distal to Calcium Influx

A number of agents that inhibit oxidative phosphorylation by different mechanisms (carbonyl cyanide m-chlorophenylhydrazone [CCCP], sodium azide, oligomycin) induced an increase of cytoplasmic Ca2+ concentration ([Ca2+]i) in pancreatic β-cells, as measured by microfluorimetry with digital imaging. All three agents are known inhibitors of insulin secretion, and the secretory response to 20 mmol/l glucose was found to be abolished in spite of elevated [Ca2+]i. Two reasons could account for this dissociation between increase of [Ca2+]i and insulin secretion: 1) the increase did not take place at a site critical for exocytosis, 2) a threshold concentration of a metabolism-derived factor like ATP exists for the induction of exocytosis. The increase of [Ca2+]i by CCCP and sodium azide involved release of Ca2+ from internal stores, whereas oligomycin induced a slow D 600–inhibitable Ca2+ influx. Because CCCP and sodium azide, but not oligomycin, decreased the mitochondrial membrane potential concomitantly with the increase of [Ca2+]i, release of Ca2+ from the mitochondria most probably plays a decisive role for the internal mobilization. A Ca2+ influx induced by 40 mmol/l K+ or 250 μmol/l tolbutamide was unimpaired in the presence of oligomycin, but oligomycin completely abolished insulin secretion in response to these agents. While CCCP and sodium azide opened ATP-sensitive K+ channels, oligomycin was virtually ineffective, although it could be shown to significantly reduce β-cell ATP production. By comparison of the effects of different inhibitors of oxidative phosphorylation, we conclude that the initiation of exocytosis in β-cells is particularly sensitive to a decrease of energy metabolism, more than ATP-sensitive K+ channels or voltage-dependent Ca2+ channels. Thus, any increase of [Ca2+]i in β-cells that occurs in a situation of a decreased ATP supply is unlikely to elicit a secretory response.

Imidazoline/guanidinium binding sites and their relation to inhibition of KATP channels in pancreatic B-cells

Abstract To elucidate the β-cytotropic effect of imidazoline compounds their inhibitory effect on ATP-dependent K+ channels (KATP channels) in pancreatic B-cells was compared with their binding to membranes from insulin-secreting HIT T15 cells. KATP channels in inside-out patches from B-cells were closed with the following rank order of efficacy at 10 μM: guanabenz > phentolamine = alinidine > clonidine > idazoxan > rilmenidine = amiloride. The last four compounds achieved an incomplete inhibition only. In contrast to sulfonylureas, the inhibitory action of imidazolines was not enhanced by ADP. With intact cells the site which mediates inhibition is less easily accessible for protonated compounds, suggesting a location at the inner face of the plasma membrane. Competition binding experiments were performed by masking α-adrenoceptors and using [3H]clonidine as ligand. Homologous displacement of [3H]clonidine revealed two distinct binding sites in HIT cell membranes characterized by dissociation constants of 38 nM and 4,911 nM and maximal binding capacities of 118 fmol/mg protein and 18 pmol/ mg protein. Generally, ligands for I2 imidazoline receptors were more potent than ligands for I1 imidazoline receptors to displace [3H]clonidine from the high affinity site, which does not fit into the current classification of imidazoline receptors. Binding to the second site had affinities in the micromolar range, similar to the concentrations necessary to inhibit KATP channels in B-cells. However, alinidine and phentolamine inhibited KATP channels already at concentrations at which they displaced [3H] clonidine only from the high affinity site, but not yet from the low affinity site. Since the proportion of the low and high affinity site varied in dependence of the competitor, the imidazoline binding sites in HIT cells may not be independent, but may rather represent two interacting or interconvertible sites both of which may be involved in KATP channel closure.

Polyamine modulation of mitochondrial calcium transport: I. stimulatory and inhibitory effects of aliphatic polyamines, aminoglucosides and other polyamine analogues on mitochondrial calcium uptake

In this study, the regulation of mitochondrial Ca2+ transport by polyamines structurally related to spermine and by analogous polycationic compounds was characterized. Similar to spermine, a number of amino groups containing cationic compounds exerted a dual effect on Ca2+ transport of isolated rat liver mitochondria: a decrease in Ca2+ uptake velocity and an enhancement of Ca2+ accumulation. In contrast to the effects of spermine and other aliphatic polyamines, however, the accumulation-enhancing effect of aminoglucosides, basic polypeptides, and metal-amine complexes turned into an inhibition of Ca2+ accumulation at higher concentrations. Within groups of structurally related compounds, the potency to decrease Ca2+ uptake velocity and to enhance Ca2+ accumulation correlated with the number of cationic charges. The presence of multiple, distributed cationic charges was a necessary, but not sufficient criterion for effects on mitochondrial Ca2+ transport, because cationic polyamines and basic oligopeptides which did not enhance mitochondrial Ca2+ accumulation could be identified. Spermine was not able to antagonize the blocking of Ca2+ uptake by ruthenium red, but rather showed an apparent synergism, which can be explained as a displacement of membrane-bound Ca2+ by spermine. The aminoglucosides, gentamicin and neomycin, but not the inactive polyamine bis(hexamethylene)-triamine, inhibited the binding of spermine to intact mitochondria. Apparently, the binding of spermine, gentamicin, and a number of polyamine analogues to low-affinity binding sites at mitochondria, which have low, but distinct structural requirements and which may correspond to phospholipid headgroups, indirectly influences the activity state of the mitochondrial Ca2+ uniporter. The ability of aminoglucosides to displace spermine from the mitochondria and to inhibit mitochondrial Ca2+ accumulation may contribute to the mitochondrial lesions, which are known to occur early in the course of aminoglucoside-induced nephrotoxicity.

Regulation of transmembrane ion transport by reaction products of phospholipase A2. II. Effects of arachidonic acid and other fatty acids on mitochondrial Ca2+ transport

The effects of arachidonic acid and other fatty acids on mitochondrial Ca2+ transport were studied. Cis-unsaturated fatty acids generally strongly inhibited mitochondrial Ca2+ uptake, induced a net Ca2+ efflux, and thereby increased the extramitochondrial Ca2+ concentration, whereas trans-unsaturated fatty acids were ineffective. Saturated fatty acids exhibited slight activity at chain lengths from C(10) to C(14) only. The structure-activity relationship and the inability of some of the effective fatty acids such as palmitoleic and myristoleic acid to be metabolized to eicosanoids suggest that Ca2+ release was induced by the fatty acids themselves and resulted from changes in the mitochondrial membrane bilayer structure. There was a correlation between Ca2+-releasing potency and reduction of mitochondrial membrane potential, which is the main driving force for mitochondrial Ca2+ uptake. There were, however, considerable differences compared with the effects of lysophospholipids on the membrane potential. The mechanism of action of fatty acids may be that of a fluidizing effect on the hydrophobic core of the membrane, thereby modulating the activity of integral membrane proteins of the respiratory chain.

Polyamine modulation of mitochondrial calcium transport: II. inhibition of mitochondrial permeability transition by aliphatic polyamines but not by aminoglucosides

In this study, the effects of polyamines and analogous compounds on mitochondrial permeability transition were characterized to distinguish between these effects and those on mitochondrial Ca2+ uptake, which are described in an accompanying report (Rustenbeck et al., Biochem Pharmacol 8: 977-985, 1998). When a transitional Ca2+ release from Ca2+-loaded mitochondria was induced by an acute increase in Ca2+ concentration in a cytosol-adapted incubation medium (Ca2+ pulse), this process was inhibited, but not abolished by spermine in the concentration range of 0.4 to 20 mM. The aminoglucoside, gentamicin, and the basic polypeptide, poly-L-lysine, which like spermine are able to enhance mitochondrial Ca2+ accumulation (preceding paper), had no or only a minimal inhibitory effect, while the aliphatic polyamine, bis(hexamethylene)triamine, which is unable to enhance mitochondrial Ca2+ accumulation, achieved a complete inhibition at 4 mM. The conclusion that the Ca2+ efflux was due to opening of the permeability transition pore was supported by measurements of mitochondrial membrane potential, ATP production, and oxygen consumption. Mg2+, a known inhibitor of mitochondrial membrane permeability transition, did not mimic the effects of spermine on mitochondrial Ca2+ accumulation, while ADP, the main endogenous inhibitor, showed both effects. However, a combination of spermine and ADP was significantly more effective than ADP alone in restoring low Ca2+ concentrations after a Ca2+ pulse. Two different groups of spermine binding sites were found at intact liver mitochondria, characterized by dissociation constants of 0.5 or 4.7 mM and maximal binding capacities of 4.6 or 19.7 nmol/mg of protein, respectively. In contrast to aminoglucosides, the aliphatic polyamine bis(hexamethylene)triamine did not displace spermine from mitochondrial binding sites. The total intracellular concentration of spermine in hepatocytes was measured to be ca. 450 microM and the free cytoplasmic concentration was estimated to be in the range of 10-100 microM. In conclusion, the enhancement of mitochondrial Ca2+ uptake by spermine is not an epiphenomenon of the inhibition of permeability transition. The physiological role of spermine appears to be that of an enhancer of mitochondrial Ca2+ accumulation rather than an inhibitor of permeability transition.

Effects of IP3, Spermine, and Mg2+ on Regulation of Ca2+ Transport by Endoplasmic Reticulum and Mitochondria in Permeabilized Pancreatic Islets

Inositol 1,4,5-trisphosphate (IP3) increased the free-Ca2+ concentration in the incubation medium of permeabilized ob/ob mouse pancreatic islets. Spermine decreased the free-Ca2+ concentration through stimulation of mitochondrial Ca2+ uptake and attenuated the effect of IP3. Mg2+ antagonized the effects of spermine, thereby increasing the free-Ca2+ concentration and enhancing the effect of IP3 on the free-Ca2+ concentration. Because IP3 releases Ca2+ from endoplasmic reticulum, these results indicate that endoplasmic reticulum and mitochondria can interact in the regulation of the free-Ca2+ concentration in the cytosol of the pancreatic β-cell.

Regulation of transmembrane ion transport by reaction products of phospholipase A2. I. Effects of lysophospholipids on mitochondrial Ca2+ transport.

Lysophospholipids inhibited mitochondrial Ca2+ uptake, induced a net Ca2+ efflux, and thereby increased the extramitochondrial Ca2+ concentration. The inhibitory potency decreased in the order lysophosphatidylcholine (LPC) = lysophosphatidylglycerol (LPG) greater than lysophosphatidylinositol (LPI) greater than lysophosphatidylserine (LPS) much greater than lysophosphatidylethanolamine (LPE). This relative order is in inverse relation to the ability of the various phospholipid head-groups to build up intermolecular hydrogen bonds with neighbouring membrane lipids. This indicates that changes in Ca2+ transport induced by lysophospholipids are mediated by the interaction of the lysophospholipids with the mitochondrial membrane bilayer structure. The mitochondrial membrane potential, which is the main driving force for mitochondrial Ca2+ uptake, was affected in the same order by the various lysophospholipids. This reduction of the mitochondrial membrane potential may be the underlying cause for the inhibition of the mitochondrial Ca2+ uniport and the resulting release of Ca2+ from the mitochondria.

Relation between accumulation of phospholipase A2 reaction products and Ca2+ release in isolated liver mitochondria.

A Ca(2+)-dependent stimulation of mitochondrial phospholipase A2 is often assumed to play a role in mitochondrial Ca2+ release. We sought to clarify this relation by measuring Ca2+ transport and determining phospholipase A2 reaction products from the same sample of isolated, incubated rat liver mitochondria. When mitochondria had accumulated and spontaneously released again Ca2+, most probably by membrane permeability transition, there was no increase of phospholipase A2 reaction products. However, when the incubation was continued after Ca2+ release, significant increases of the content of lysophosphatidylcholine and unesterified fatty acids could be seen. Quinacrine, an inhibitor of phospholipase A2 activity, prevented Ca2+ release and p-hydroxymercuribenzoic acid, an inhibitor of lysophospholipid reesterification, induced a fast release of Ca2+ from isolated mitochondria. Such effects are usually taken as indirect evidence for a participation of phospholipase A2 in mitochondrial Ca2+ release, but analysis of the mitochondrial lipids revealed that no significant changes of the mass of phospholipase A2 reaction products had occurred. These experiments suggest that the accumulation of phospholipase A2 reaction products in mitochondria is the consequence rather than the cause of the membrane permeability transition. Exogenous phospholipase A2 products, lysophosphatidylcholine and arachidonic acid, induced mitochondrial Ca2+ release after a time lag, which decreased with aging of the mitochondrial preparation. The amount of lysophosphatidylcholine taken up by the mitochondria from the incubation medium during these experiments was measured and compared to the amount of lysophosphatidylcholine produced endogenously by mitochondrial phospholipase A2. From these data it appears likely that the amount of lysophosphatidylcholine generated in the mitochondria after the permeability transition is sufficient to sustain the permeable state. An accumulation of mitochondrially generated phospholipase A2 reaction products after the permeability transition could thus be a decisive factor for the limited reversibility of the membrane permeability transition.

Structural requirements of lysophospholipid-regulated mitochondrial Ca2+ transport

Analogues of lysophosphatidylcholine, including PAF (platelet-activating-factor) and HePC (an experimental anticancer drug), were studied for their influence on mitochondrial Ca2+ transport and membrane potential. Lysophospholipids released Ca2+ from mitochondria and reduced the maximal Ca2+ uptake. The structure-activity relations indicate that deprotonated head groups like phosphocholines yield active compounds while partially protonated head groups like phosphoethanolamines are essentially inactive. Structural requirements for the apolar part of the molecules were acyl or alkyl chain lengths of less than 18 carbon atoms at the C1-position of the glycerol backbone and residues of small size and/or low polarity at the C2-position. Choline lysophospholipids, but not ethanolamine lysophospholipids, may therefore induce mitochondrial Ca2+ efflux and become mediators of ischaemic tissue damage where dysregulated phospholipase A2 activity and an impairment of mitochondrial function are supposed to play a crucial role.