Resistance of the intact and reconstituted adipocyte hexose transport system to irreversible inhibition by sulfhydryl and amino reagents

Sensitivity of the adipocyte D-glucose transport system in intact plasma membranes or following solubilization and reconstitution into phospholipid vesicles to several protein-modifying reagents was investigated. When intact plasma membranes were incubated with N-ethylmaleimide (20 mM) or fluorodinitrobenzene (4 mM), D-glucose transport activity was virtually abolished. However, washing the membranes free of unreacted reagents restored transport activity, indicating that covalent interaction with the membranes did not mediate the transport inhibition. Reaction of [³H] N-ethylmaleimide with plasma membranes under similar conditions resulted in extensive labeling of all protein fractions resolved on dodecyl sulfate gels. Similarly, addition of N-ethyl-maleimide to cholate-solubilized membrane protein had no effect on transport activity in artifical phospholipid vesicles reconstituted under conditions where the membrane protein was free of unreacted N-ethylmaleimide. Transport activity in plasma membranes was also inhibited by both reduced and oxidized dithiothreitol or glutathione (15 mM) in a readily reversible manner, consistent with a noncovalent mode of inhibition. Thus, the insulin-responsive adipocyte D-glucose transport system differs from the red cell hexose transport system in its remarkable insensitivity to modulation by covalent blockade of sulfhydryal or amino groups by the reagents studied.     

Binding of cytochalasin B to platelets

The binding of cytochalasin B (CB) to human platelets and to isolated platelet cytosol and membranes has been analyzed with [3H]CB. High- and low affinity classes of saturable binding sites were associated with intact platelets. Binding at very low concentrations of CB (i.e., high-affinity binding) was partially prevented by 100 mM D-galactose or D-glucose and to a much lesser extent by L-glucose. Binding to platelet cytosol also involved two classes of sites with affinities and capacities similar to those observed with the whole cells. None of this binding, however, was affected by 100 mM D-galactose. Saturable binding to platelet membranes occurred at sites with a uniform binding affinity. Approximately 52% of this binding was prevented by 1 M D-galactose and another 15% by cytochalasin E (CE). We hypothesize that binding in the cytosol is to monomeric (low-affinity) and polymerized (high-affinity) actin, whereas membrane binding (high-affinity only) occurs primarily at sites involved with galactose transport.     

Comparison of bile acid binding to sinusoidal and bile canalicular membranes isolated from rat liver

Simon et al. (J. Clin. Invest., 70 (1982) 401) studied cholate binding to crude liver plasma membrane vesicles and suggested that the binding may represent mainly the binding to the receptor (carrier) on the canalicular membrane. This hypothesis was supported by finding a good correlation between the number of cholate binding sites on liver plasma membrane and the maximal rate of biliary secretion (Tm) for taurocholate. We studied bile acid binding to sinusoidal and canalicular membrane vesicles isolated from rat liver by a rapid filtration technique. Scatchard analysis of the saturation kinetics showed both [3H]cholate and [3H]chenodeoxycholate bind to two classes of binding site on each membrane. However, little difference was observed between the binding to sinusoidal and canalicular membrane vesicles for each bile acid (cholate, Kd1 = 10.4 and 19.8 microM, n1 = 31.0 23.6 pmol/mg protein, Kd2 = 1.32 and 1.73 mM, n2 = 13.1 and 23.4 nmol/mg protein; and chenodeoxycholate, Kd1 = 0.207 and 0.328 microM, n1 = 36.7 and 27.4 pmol/mg protein, Kd2 = 1.16 and 2.26 mM, and n2 = 20.6 and 24.2 nmol/mg protein; numbers show the mean values sinusoidal and canalicular membrane vesicles, respectively). Chenodeoxycholate binding to sinusoidal membrane vesicles was markedly inhibited by cholate but not by Rose bengal, an organic anion dye. These studies indicate that both membranes (sinusoidal and canalicular membrane vesicles) have two kinds of binding site for bile acids, although no clear difference in the binding properties was observed between the two membranes. Consequently, the cholate binding Simon detected may represent the binding not only to canalicular membrane vesicles but also to sinusoidal membrane vesicles.     

Kinetic characterization and radiation-target sizing of the glucose transporter in cardiac sarcolemmal vesicles

Stereospecific glucose transport was assayed and characterized in bovine cardiac sarcolemmal vesicles. Sarcolemmal vesicles were incubated with D-[3H]glucose or L-[3H]glucose at 25 degrees C. The reaction was terminated by rapid addition of 4 mM HgCl2 and vesicles were immediately collected on glass fiber filters for quantification of accumulated [3H]glucose. Non-specific diffusion of L-[3H]glucose was never more than 11% of total D-[3H]glucose transport into the vesicles. Stereospecific uptake of D-[3H]glucose reached a maximum level by 20 s. Cytochalasin B (50 microM) inhibited specific transport of D-[3H]glucose to the level of that for non-specific diffusion. The vesicles exhibited saturable transport (Km = 9.3 mM; Vmax = 2.6 nmol/mg per s) and the transporter turnover number was 197 glucose molecules per transporter per s. The molecular sizes of the cytochalasin B binding protein and the D-glucose transport protein in sarcolemmal vesicles were estimated by radiation inactivation. These values were 77 and 101 kDa, respectively, and by the Wilcoxen Rank Sum Test were not significantly different from each other.     

Enzyme activities and sodium-dependent active D-glucose transport in apical membrane vesicles isolated from kidney epithelial cell line (LLC-PK1)

Apical membrane vesicles were isolated from the confluent LLC-PK1 cells by nitrogen cavitation and Mg/EGTA precipitation methods. The specific activities of marker enzymes for apical membranes were enriched 8- to 18-fold relative to those in the homogenate. D-[3H]Glucose uptake into the vesicles was stimulated in the presence of Na+ gradient (overshoot phenomenon), and the values of apparent Km and Vmax for Na+-dependent component of D-glucose uptake were 0.3 mM and 5.8 nmol/mg protein per min, respectively.     

Identification of the D-glucose-inhibitable cytochalasin B binding site as the glucose transporter in rat diaphragm plasma and microsomal membranes

[3H]Cytochalasin B binding and its competitive inhibition by D-glucose have been used to identify, the glucose transporter in plasma and microsomal membranes prepared from intact rat diaphragm. Scatchard plot analysis of [3H]cytochalasin B binding yields a binding site with a dissociation constant of roughly 110 nM. Since the inhibition constant of cytochalasin B for D-glucose uptake by diaphragm plasma membranes is similar to this value, this site is identified as the glucose transporter. Plasma membranes prepared from diaphragms bind approx. 17 pmol of cytochalasin B/mg of membrane protein to the D-glucose-inhibitable site. If 280 nM (40000 microunits/ml) insulin is present during incubation, cytochalasin B binding is increased roughly 2-fold without alteration in the dissociation constant of this site. In addition, membranes in the microsomal fraction contain 21 pmol of D-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. In the presence of insulin during incubation the number of these sites in the microsomal fraction is decreased to 9 pmol/mg of membrane protein. These results suggest that rat diaphragm contain glucose transporters with characteristics identical to those observed for the rat adipose cell glucose transporter. In addition, insulin stimulates glucose transport in rat diaphragm through a translocation of functionally identical glucose transporters from an intracellular membrane pool to the plasma membrane without an alteration in the characteristics of these sites.     

Biochemical characterization and subcellular distribution of the glucose transporter from rat brain microvessels

This study describes the biochemical characterization and subcellular distribution of glucose transporters from isolated rat brain cortical microvessels. The D-glucose inhibitable [3H]cytochalasin B binding assay was used to quantitate glucose transporter binding sites in plasma membranes, high-density microsomes and low-density microsomes prepared from basal and insulin-stimulated cells. Incubation with insulin for 30 min increased the number of glucose transporters in the high-density microsomes by around 33% but had no effect on the number of glucose transporters in the plasma membrane or low-density microsomes. Prolonged incubation with insulin (2 h), however, resulted in a small but significant redistribution of glucose transporters to the low-density microsomes. Preincubation of cells with cycloheximide blocked this insulin-induced increase in glucose transporter number, suggesting that this effect of insulin was due to the synthesis of new glucose transport proteins. Specific labeling of glucose transporters was achieved by photoincorporation of [3H]cytochalasin B. Labeled membranes from all fractions contained a single D-glucose inhibitable peak, migrating with a molecular size of 55 kDa on SDS-polyacrylamide gel electrophoresis. Isoelectric focusing of the 55 kDa protein revealed one major peak of D-glucose inhibitable radioactivity focusing at pH 6.0 in all fractions.     

Catecholamine binding to plasma membrane enriched fractions of heart and skeletal muscle.

Binding of [3H]epinephrine to plasma membrane enriched fractions from guinea pig heart and rabbit skeletal muscle was investigated using the micropore filtration technique. [3H]Epinephrine and [3H]norepinephrine were found to be degraded rapidly in aqueous buffer at pH 7.6 and 37 degrees C. Deterioration of the compounds could be prevented by low concentrations of dithiothreitol. Binding of [3H]epinephrine to both membrane preparations was a slow process requiring 60 min to approach equilibrium in the case of cardiac membranes at 37 degrees C, and 20 min for skeletal muscle membranes at O degrees C. Binding was antagonized by the unlabeled beta-agonists, isopropylnorepinephrine, epinephrine, and norepinephrine but all were equipotent. A variety of catechol compounds were as effective antagonists of binding as the catecholamines. The beta-adrenergic antagonists propranolol, pronethalol, and dichloroisoproterenol were not effective in inhibiting binding to either membrane preparation. D-Norepinephrine and L-norepinephrine were equi-effective in antagonizing binding of [3H]norephinephrine to skeletal muscle membranes. It was concluded that binding of labeled catecholamine to isolated tissue membranes using the micropore filtration technique does not represent interaction with the specific beta-adrenergic receptor, but more likely reflects a less specific binding of compounds having one or more hydroxyl groups on a ring.     

ATP-dependent transport of taurocholate across the hepatocyte canalicular membrane mediated by a 110-kDa glycoprotein binding ATP and bile salt

Direct photoaffinity labeling of liver plasma membrane subfractions enriched in sinusoidal and canalicular membranes using [35S]adenosine 5'-O-(thiotriphosphate) ([35S]ATP gamma S) allows the identification of ATP-binding proteins in these domains. Comparative photoaffinity labeling with [35S]ATP gamma S and with the photolabile bile salt derivative (7,7-azo-3 alpha, 12 alpha-dihydroxy-5 beta-[3 beta-3H]-cholan-24-oyl-2'- aminoethanesulfonate followed by immunoprecipitation with a monoclonal antibody (Be 9.2) revealed the identity of the ATP-binding and the bile salt-binding canalicular membrane glycoprotein with the apparent Mr of 110,000 (gp110). The isoelectric point of this glycoprotein was 3.7. Transport of bile salt was studied in vesicles enriched in canalicular and sinusoidal liver membranes. Incubation of canalicular membrane vesicles with [3H] taurocholate in the presence of ATP resulted in an uptake of the bile salt into the vesicles which was sensitive to vanadate. ATP-dependent taurocholate transport was also observed in membrane vesicles from mutant rats deficient in the ATP-dependent transport of cysteinyl leukotrienes and related amphiphilic anions. Substrates of the P-glycoprotein (gp170), such as verapamil and doxorubicin, did not interfere with the ATP-dependent transport of taurocholate. Reconstitution of purified gp110 into liposomes resulted in an ATP-dependent uptake of [3H]taurocholate. These results demonstrate that gp110 functions as carrier in the ATP-dependent transport of bile salts from the hepatocyte into bile. This export carrier is distinct from hitherto characterized ATP-dependent transport systems.     

Inhibition by nucleosides of glucose-transport activity in human erythrocytes.

The interaction of nucleosides with the glucose carrier of human erythrocytes was examined by studying the effect of nucleosides on reversible cytochalasin B-binding activity and glucose transport. Adenosine, inosine and thymidine were more potent inhibitors of cytochalasin B binding to human erythrocyte membranes than was D-glucose [IC50 (concentration causing 50% inhibition) values of 10, 24, 28 and 38 mM respectively]. Moreover, low concentrations of thymidine and adenosine inhibited D-glucose-sensitive cytochalasin B binding in an apparently competitive manner. Thymidine, a nucleoside not metabolized by human erythrocytes, inhibited glucose influx by intact cells with an IC50 value of 9 mM when preincubated with the erythrocytes. In contrast, thymidine was an order of magnitude less potent as an inhibitor of glucose influx when added simultaneously with the radioactive glucose. Consistent with this finding was the demonstration that glucose influx by inside-out vesicles prepared from human erythrocytes was more susceptible to thymidine inhibition than glucose influx by right-side-out vesicles. These data, together with previous suggestions that cytochalasin B binds to the glucose carrier at the inner face of the membrane, indicate that nucleosides are capable of inhibiting glucose-transport activity by interacting at the cytoplasmic surface of the glucose transporter. Nucleosides may also exhibit a low-affinity interaction at the extracellular face of the glucose transporter.     

Glucose transport by uterine plasma membranes

Uterine plasma membrane preparations were obtained by centrifugation on discontinuous sucrose gradients. The specific activity of the plasma membrane marker 5'-nucleotidase was increased 10-fold while the specific activity of glucose-6-phosphatase was increased 3-fold. Electron microscopy showed mainly closed vesicles having diameters mainly in the range of 0.1 to 0.4 micron and an absence of other recognizable organelles such as mitochondria. D-Glucose transport was inhibited by sulfhydryl reagents, phloretin, and cytochalasin B. Uptake was prevented at high osmotic pressures. The Km of glucose transport was 12.2 +/- 1.1 mM. Studies of the inhibition of [3H]cytochalasin B binding by D-glucose indicated that the value of the Kd of the cytochalasin B-transporter complex was larger than 1 microM. These data demonstrate the potential usefulness of these preparations in the study of glucose transport in rat uterus and its control by steroid hormones.     

Distribution of glucose transporters and insulin receptors in the plasma membrane and transverse tubules of skeletal muscle

The distribution of glucose transporters and of insulin receptors on the surface membranes of skeletal muscle was studied, using isolated plasma membranes and transverse tubule preparations. (i) Plasma membranes from rabbit skeletal muscle were prepared according to Seiler and Fleischer (1982, J. Biol. Chem. 257, 13862-13871), and transverse tubules from rabbit skeletal muscle were prepared according to Rosemblatt et al. (1981, J. Biol. Chem. 256, 8140-8148) as modified by Hidalgo et al. (1983, J. Biol. Chem. 258, 13937-13945). The membranes were identified by the abundance of nitrendipine receptors in the transverse tubules, and their relative absence from the plasma membranes. (ii) Plasma membranes and transverse tubules were also isolated from rat skeletal muscle, according to a novel procedure that isolates both fractions from the same common homogenate. (iii) Glucose transporters were detected by D-glucose protectable binding of the specific inhibitor [3H]cytochalasin B, and insulin receptors were detected by saturable binding of 125I-insulin. The concentration of glucose transporters was about threefold (rabbit) or fivefold (rat) higher in the transverse tubule membrane compared to the plasma membrane, whereas the insulin receptor concentration was about the same in both membranes. These results indicate that the glucose transporters on the surface of the muscle are preferentially segregated to the transverse tubules, and this poses interesting consequences on the functional response of glucose transport to insulin in skeletal muscle.     

1,25-Dihydroxycholecalciferol-related Na+/D-glucose transport in brush-border membrane vesicles from embryonic chick jejunum. Modulation by triiodothyronine

1,25-Dihydroxycholecalciferol, when present at and above 10 nM in an organ-culture system of embryonic chick jejunum, approximately doubled the rate of Na(+)-gradient-driven D-glucose uptake by brush-border membrane vesicles, but had no effect on Na(+)-independent D-glucose transfer. The sterol also had no effect on Na+ influx along an outside/inside Na+ gradient ([Na+]o = 100 mM; [Na+]i = 0 mM). This renders it unlikely that in embryonic intestine, calcitriol raises Na(+)-dependent D-glucose transport through changes in the electrochemical Na+ gradient. D-[U-14C]Glucose tracer exchange, measured under voltage-clamp condition at Na+/D-glucose equilibrium, revealed that addition of calcitriol to the culture medium approximately doubled the activity of the Na+/D-glucose transporter in the brush-border membrane. This was also reflected by an corresponding increase in the maximal velocity of the transfer process. Increased [3H]phlorizin binding after calcitriol treatment suggests that the steroid hormone activates Na+/D-glucose transport through increasing the number of carrier molecules in the brush-border membrane. 10 nM triiodothyronine, which by itself has no effect on Na(+)-dependent D-glucose transport, potentiated the effect of 1,25-dihydroxycholecalciferol such that in the presence of both hormones, Na+/D-glucose-carrier activity was increased fourfold above control levels.     

Sugar uptake by intestinal basolateral membrane vesicles

A high yield of membrane vesicles was prepared from the basolateral surface of rat intestinal cells using an N2 cavitation bomb and density gradient centrifugation. The membranes were enriched 10-fold and were free of significatn contamination by brush border membranes and mitochondria. The rate of D-E114C]glucose and L-E13H]glucose uptake into the vesicle was measured using a rapid filtration technique. D-Glucose equilibrated within the vesicles with a half-time 1/25th that for L-glucose. The stereospecific uptake exhibited saturation kinetics with a Km of approx. 44 mM and a V of approx. 110 nmol . mg-1 min-1 at 10 degrees C. The activation energy for the process was 14 kcal . mol-1 below 15 degrees C and it approached 3 kcal . mol-1 above 22 degrees C. Carrier-mediated uptake was eliminated in the presence of 1 mM HgCl2 and 0.5 mM phloretin. The rate of transport was unaffected by the absence or presence of sodium concentration gradients. Competition studies demonstrated that all sugars with the D-glucose pyranose ring chair conformation shared the transport system, and that, with the possible exception of the -OH group at carbon No. 1, there were no specific requirements for an equatorial -OH group at any position in the pyranose ring. In the case of alpha-methyl-D-glucoside its inability to share the D-glucose transport system may be due to steric hindrance posed by the -OCH3 group rather than by a specific requirement for a free hydroxyl group at the position in the ring. It is concluded that sugars are transported across the basolateral membrane of the intestinal epithelium by a facilitated diffusion system reminiscent of that in human red blood cells.     

Mechanism for binding of fatty acids to hepatocyte plasma membranes

The purpose of this study was to examine the interaction between fatty acids and plasma membranes from liver cells. We were unable to reproduce the reported effect of heating on the capacity of these membranes to bind [3H]oleate (Stremmel et al. 1985 Proc. Natl. Acad. Sci. USA. 82: 4-8). In fact, the distribution of [3H]oleate between plasma membranes and unilamellar vesicles of lipids extracted from these membranes was in favor of the lipids, indicating the absence of a detectable amount of binding to a putative fatty acid binding protein in plasma membranes. Radius of curvature of vesicles (125 A vs 475 A) had no effect on the partitioning of fatty acid. In addition, the distribution of [3H]oleate between plasma membranes and other phases had the properties of a partition coefficient over a 200-fold range of [3H]oleate. There was no evidence in this experiment for a binding isotherm, i.e., binding of [3H]oleate at a specific site, superimposed on the nonspecific partitioning of [3H]oleate into the lipids of the plasma membrane. There was no competition between [14C]oleate and [3H]palmitate for entry into plasma membranes. Finally, rates of uptake of [14C]oleate and [3H]palmitate by perfused rat liver were not affected by the presence of the other fatty acid in perfusates. These data indicate that the avidity of hepatocyte plasma membranes for [3H]oleate is a simple consequence of the physical chemical properties of oleate, lipids, and water. The data exclude the idea that the uptake of fatty acids into cells is the result of binding proteins and/or catalyzed reactions at the water-membrane interface of the cell or within the plane of the plasma membrane.     

Identification of an Mr 75000 component of the H+/D-glucose cotransporter from Zea mays with monoclonal antibodies directed against the mammalian Na+/D-glucose cotransporter

Monoclonal antibodies which interact with the mammalian Na+/D-glucose cotransporter and bind to Mr 75,000 and Mr 47,000 polypeptide components of this transporter have been described (Koepsell, H., Korn, K., Raszeja-Specht, A., Bernotat-Danielowski, S. and Ollig, D. (1988) J. Biol. Chem., 263, 18419-18429). The interaction of these antibodies with plasma membranes from Zea mays L. coleoptiles containing an H+/D-glucose cotransporter was studied. Four monoclonal antibodies cross-reacted with Mr 75,000 and Mr 33,000 polypeptides. One of these antibodies, which inhibits Na+/D-glucose cotransport in the kidney and stimulates Na+/D-glucose cotransport in intestine, stimulates electrogenic uptake of 3-O-methyl-D-[14C]glucose in plant membrane vesicles. The data indicate common epitopes in the mammalian Na+/D-glucose cotransporter and the H+/D-glucose cotransporter of plants and suggest that both transporters contain an Mr 75000 polypeptide component.     

A plasma membrane-enriched preparation from giant barnacle muscle fibers

We describe a procedure for obtaining a highly enriched plasma membrane (sarcolemmal) preparation from muscle fibers of the giant barnacle (Balanus nubilus). The sarcolemmal-enriched portion migrated as a light fraction (F1) at the 10-24% sucrose interface. This fraction displayed saturable ouabain binding (Kd = 0.119 microM) that was enriched 10 times compared to that in the original homogenate. F1 was also prepared using muscle fibers previously labeled with 1,2-ditritio-1,2(2,2'-disulfo-4,4'-diisothiocyano)diphenylet hane, disodium salt [( 3H]-H2DIDS). F1 was enriched 25-fold in [3H]H2DIDS binding sites with respect to the homogenate. Ca2+-ATPase and succinic dehydrogenase-activities were low in F1, as was oxalate-supported Ca2+ uptake. Compared to membranes of sarcoplasmic reticulum origin, F1 was enriched in sarcolemma membranes by about 45-fold while it was enriched approximately 30-fold over mitochondrial membranes. Thus, F1 provides an extremely pure source of external muscle membranes.     

Regulation of hexose transporters in chicken embryo fibroblasts: stimulation by the phorbol ester TPA leads to increased numbers of functioning transporters

As has been observed with many types of cultured cells, chicken embryo fibroblasts (CEF) when exposed to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) develop a 3- to 4-fold increase in hexose transport activity in 4 h. This increase in transport activity occurred despite a modest decline of 20% in [3H]leucine incorporation into acid insoluble fractions. Cycloheximide largely, but not completely, blocked the increase in transport activity during TPA exposure. The effects of TPA were somewhat similar to those of glucose starvation induced enhancement of hexose transport activity. Furthermore, with TPA there was no additive effect to that produced by glucose starvation. Plasma membrane enriched fractions were prepared from CEF treated with or without TPA. Membranes prepared from TPA exposed cells had a two-fold enhancement of stereospecific D-glucose transport activity as well as D-glucose inhibitable [3H]cytochalasin B binding as compared to the membranes from control CEF. There was no effect on transport when membranes were exposed to TPA in vitro. These results provide strong evidence that TPA exposure leads to an increase in the number of functioning transporters, an effect largely requiring protein synthesis.     

Involvement of Ni protein in the functional coupling of the atrial natriuretic factor (ANF) receptor to adenylate cyclase in rat lung plasma membranes

In the presence of 1 microM atrial natriuretic factor (ANF) and low (0.1 mM) Mg2+ concentrations, the initial rate of binding of [3H]guanosine 5'-[beta, gamma-imido)triphosphate [( 3H]p[NH]ppG) to rat lung plasma membranes was increased twofold to threefold. ANF-dependent stimulation of the initial rate of [3H]p[NH]ppG binding was reduced at high (5 mM) Mg2+ concentrations. Preincubation of membranes with p[NH]ppG (5 min at 37 degrees C) eliminated the ANF-dependent effect on [3H]p[NH]ppG binding whereas ANF-dependent [3H]p[NH]ppG binding was unaffected by similar pretreatment with guanosine 5'-[beta-thio]diphosphate (GDP[beta S]). An increase in ANF concentration from 10 pM to 1 microM caused a 40% decrease in forskolin-stimulated or isoproterenol-stimulated adenylate cyclase activities (IC50 5 nM) in rat lung plasma membranes. GTP (100 microM) was obligatory for the ANF-dependent inhibition of adenylate cyclase, which could be completely overcome by the presence of 100 microM GDP[beta S] or the addition of 10 mM Mn2+. Reduction of Na2+ concentration from 120 mM to 20 mM had the same effect. Pertussis toxin eliminated ANF-dependent inhibition of adenylate cyclase by catalyzing ADP-ribosylation of membrane-bound Ni protein (41-kDa alpha subunit of the inhibitory guanyl-nucleotide-binding protein of adenylate cyclase). The data support the notion that one of the ANF receptors in rat lung plasma membranes is negatively coupled to a hormone-sensitive adenylate cyclase complex via the GTP-binding Ni protein.     

Secretagogue-responsive and -unresponsive pools of phosphatidylinositol in pancreatic islets

The effect of glucose on phosphatidylinositol turnover was studied. Phosphatidylinositol of rat pancreatic islets was labeled with myo[2-3H]inositol in the presence of various secretagogues (16.7 mM D-glucose, 22 mM D-mannose, 20 mM D-glyceraldehyde) and nonsecretagogues (3.3 mM D-glucose, 20 mM pyruvate, 16.7 mM D-galactose, 16.7 mM L-glucose). Upon subsequent stimulation with 16.7 mM D-glucose, only the islets that were labeled in the presence of secretagogues showed a loss of radioactivity from phosphatidylinositol. No loss of radioactivity from phosphatidylinositol occurred in the presence of 3.3 mM D-glucose even after labeling in the presence of secretagogues. A comparison of the subcellular distribution of labeled phosphatidylinositol in islets before and after stimulation with insulinotropic glucose revealed a loss of radioactivity from the plasma membrane fraction as judged by subcellular fractionation with a sucrose gradient. These results support a hypothesis advanced previously that pancreatic islets contain a unique pool of phosphatidylinositol that undergoes rapid turnover only in the presence of insulinotropic concentrations of D-glucose or other secretagogues [R. S. Rana, R. J. Mertz, A. Kowlura, J. F. Dixon, L. E. Hokin, and M. J. MacDonald (1985) J. Biol. Chem. 260, 7861-7867]. On the basis of the subcellular fractionation studies reported here, the secretagogue-responsive phosphatidylinositol pool appears to be located primarily in the plasma membrane of pancreatic islets.