Characterization of Cloned Mouse Na+/taurocholate Cotransporting Polypeptide by Transient Expression in COS-7 Cells

The mouse Na⁺/taurocholate cotransporting polypeptide transiently expressed in COS-7 cells caused sodium-dependent uptake of [³H]taurocholic acid with K_m and V_max values of 18 μM and 102 pmol/mg protein/min, respectively. This K_m value is comparable to that for rat NTCP and higher than that for human NTCP. Substrate specificity was evaluated by measuring inhibitory effects of unlabeled bile acids on [³H]taurocholic acid transport.     

Selective inhibition of long-chain fatty acid uptake in short-term cultured rat hepatocytes by an antibody to the rat liver plasma membrane fatty acid-binding protein

Uptake of long-chain fatty acids by short-term cultured hepatocytes was studied. Rat hepatocytes, which were cultured for 16 h on plastic dishes (3.6 X 10(6) cells/dish), were incubated with [3H]oleate in the presence of various concentrations of bovine serum albumin as a function of the concentration of unbound [3H]oleate in the medium. At 37 degrees C initial uptake velocity (V0) was saturable (Km = 9 X 10(-8) M; Vmax = 835 pmol/min per mg protein). V0 was temperature dependent with an optimum at 37 degrees C and markedly reduced at 4 degrees C and 70 degrees C. To evaluate the biologic significance of a previously isolated rat liver plasma membrane fatty acid-binding protein as putative carrier protein in the hepatocellular uptake of fatty acids, cultured hepatocytes were treated with a monospecific rabbit antibody (IgG-fraction) to this membrane protein or the IgG-fraction of the pre-immune serum as controls. Uptake kinetics of [3H]oleate in antibody pretreated short-term cultured hepatocytes revealed a depression of Vmax by 70%, while Km was only reduced by 16% compared to controls, indicating a predominant non-competitive type of inhibition. V0 of a variety of long-chain fatty acids (oleic acid, arachidonic acid, palmitic acid, stearic acid) was reduced by 56-69%, while V0 of [35S]sulfobromophthalein, [3H]cholic acid and [14C]taurocholic acid remained unaltered. These data support the concept that in the system of cultured hepatocytes, uptake of long-chain fatty acids is mediated by the rat liver plasma membrane fatty acid-binding protein.     

Partial purification and characterization of NAD-dependent 7alpha-hydroxysteroid dehydrogenase from Bacteroides thetaiotaomicron.

A NAD-dependent 7alpha-hydroxysteroid dehydrogenase was purified 18-fold over the activity in crude cell extracts prepared from Bacteroides thetaiotaomicron NCTC 10852 using Bio-Gel A 1.5-M column chromatography. A molecular weight of 320 000 was estimated for the partially purified intact enzyme. Substrate saturation kinetics were performed using the 18-fold purified enzyme and the lowest Km values were obtained for 3alpha,7alpha-dihydroxy bile acid and bile salt substrates including chenodeoxycholic acid (Km 0.048 mM), glycochenodeoxycholic acid (Km 0.083 mM) and taurochenodeoxycholic acid (Km 0.059 mM). In contrast, 3alpha,7alpha,12alpha-trihydroxy bile acid and bile salts had higher Km values, i.e. cholic acid (Km 0.22 mM), glycoholic acid Km 0.32 mM) and taurocholic acid Km 0.26 mM). NAD had a Km value of 0.20 mM. The possible physiological significance of 7alpha-hydroxy bile acid oxidation to intestinal bacteroides strains was accessed by determining the rate of conversion of [14C]-cholic acid to 7-ketodeoxy[14C]cholic acid by whole cell suspensions under different incubation conditions. The rate of biotransformation of bile acid to keto-bile acid incubated anaerobically under N2 gas increased markedly when potential electron acceptors such as fumarate (10 mM) or menadione (4 mM) was added exogenously. These results suggest that bile acid oxidation reactions may be linked to energy-generating systems in this bacterium.     

Characterization, cDNA cloning, and functional expression of mouse ileal sodium-dependent bile acid transporter

Mouse ileal sodium dependent bile acid transporter (ISBT) was characterized using isolated enterocytes. Only enterocytes from the most distal portion showed Na+-dependent [3H]taurocholate uptake. Northern blot analysis using a probe against mouse ISBT revealed the expression of mouse ISBT mRNA to be restricted to the distal ileum. The Km and Vmax for Na+-dependent [3H]taurocholate transport into isolated ileocytes were calculated as 27 microM and 360 pmol/mg protein/min, respectively. Uptake of [3H]taurocholate was inhibited by N-ethylmaleimide. We have cloned ISBT cDNA from mouse ileum. The cDNA included the entire open reading frame coding 348 amino acid protein with seven hydrophobic segments and two N-glycosylation sites. COS-7 cells transfected with the expression vector containing this cDNA expressed Na+-dependent [3H]taurocholate uptake activity with a Km of 34 microM.     

Characterization and separation of the arachidonic acid 5-lipoxygenase and linoleic acid omega-6 lipoxygenase (arachidonic acid 15-lipoxygenase) of human polymorphonuclear leukocytes

The cytosolic fraction of human polymorphonuclear leukocytes precipitated with 60% ammonium sulfate produced 5-lipoxygenase products from [14C]arachidonic acid and omega-6 lipoxygenase products from both [14C]linoleic acid and, to a lesser extent, [14C]- and [3H]arachidonic acid. The arachidonyl 5-lipoxygenase products 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) derived from [14C]arachidonic acid, and the omega-6 lipoxygenase products 13-hydroperoxy-9,11-octadecadienoic acid (13-OOH linoleic acid) and 13-hydroxy-9,11-octadecadienoic acid (13-OH linoleic acid) derived from [14C]linoleic acid and 15-hydroxyperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) derived from [14C]- and [3H]arachidonic acid were identified by TLC-autoradiography and by reverse-phase high-performance liquid chromatography (RP-HPLC). Products were quantitated by counting samples that had been scraped from replicate TLC plates and by determination of the integrated optical density during RP-HPLC. The arachidonyl 5-lipoxygenase had a pH optimum of 7.5 and was 50% maximally active at a Ca2+ concentration of 0.05 mM; the Km for production of 5-HPETE/5-HETE from arachidonic acid was 12.2 +/- 4.5 microM (mean +/- S.D., n = 3), and the Vmax was 2.8 +/- 0.9 nmol/min X mg protein (mean +/- S.D., n = 3). The omega-6 linoleic lipoxygenase had a pH optimum of 6.5 and was 50% maximally active at a Ca2+ concentration of 0.1 mM in the presence of 5 mM EGTA. When the arachidonyl 5-lipoxygenase and the omega-6 lipoxygenase were separated by DEAE-Sephadex ion exchange chromatography, the omega-6 lipoxygenase exhibited a Km of 77.2 microM and a Vmax of 9.5 nmol/min X mg protein (mean, n = 2) for conversion of linoleic acid to 13-OOH/13-OH linoleic acid and a Km of 63.1 microM and a Vmax of 5.3 nmol/min X mg protein (mean, n = 2) for formation of 15-HPETE/15-HETE from arachidonic acid.     

Mrp2/Abcc2 transport activity is stimulated by protein kinase Calpha in a baculo virus co-expression system

Cholestatic and choleretic effect are well known for protein kinase C activator and inhibitor, respectively. However, post-translational regulation, especially the effect of phosphorylation status of the biliary transporters on their intrinsic transport activity has not been fully understood. In this study, effect of phosphorylation on the transport activity of Mrp2, a biliary organic anion transporter, was examined in membrane vesicles isolated from Sf9 cells co-expressing excess amount of protein kinase Calpha (PKCalpha). Mrp2-mediated transport activity was enhanced to three-fold by co-expressing PKCalpha. At the same time, phosphorylation of Mrp2 was also detected. The Km and Vmax values for the transport of [3H]estradiol-17beta-D-glucuronide exhibited a 1.5-fold decrease and a 1.9-fold increase, respectively. Probenecid (100 microM) and benzylpenicillin (1 mM), both are activator of Mrp2, did not stimulated the transport activity of phosphorylated Mrp2. On the other hand, transport activity was further stimulated by Estron-3-sulfate and taurocholic acid. Similar mechanism that occurred in the presence of probenecid and benzylpenicillin, but different from that occurred in the presence of Estron-3-sulfate and taurocholic acid seems to be involved in the stimulation. Considering the discrepancy between the previous in vivo inhibitory effect of PKC activators and our in vitro stimulatory effect of PKCalpha on Mrp2 transport activity, direct modulation of Mrp2-transport activity may be minor if any under in vivo condition.     

Deuterium nuclear magnetic resonance study of the interaction of substrates and inhibitors with soybean lipoxygenase

Deuterium NMR spectra for a series of selectively deuterated substrates and inhibitors in the presence of lipoxygenase-1 (EC 1.13.11.12) are presented. Extrapolation of the 2H NMR line widths yielded transverse relaxation rates for the bound inhibitors [2H21]dodecanoic acid (protonated at the 2,2-position), [2,2-2H]dodecanoic acid, and [12,12,12-2H]dodecanoic acid which are 1/T2,bd = 5.0 X 10(3), 1.12 X 10(4), and 1.16 X 10(3) s-1, respectively. The substrates [9,10,12,13-2H]linoleic acid and [11,11-2H]linoleic acid had 1/T2,bd = 8.2 X 10(3) and 7.95 X 10(3) s-1, respectively. Kinetic measurements established Ki = 1.5 X 10(-3) M for dodecanoic acid (lauric acid) inhibition of lipoxygenase when the substrate is linoleic acid (Km = 2.6 X 10(-5) M). Lipoxygenase, with Mr 102,000, is predicted to have a rotational correlation time tau c - 1.2 X 10(-7) s, yielding a 1/T2,bd = 1.56 X 10(4) s-1 for tightly bound ligand. Hence, the correlation times of the selectively deuterated inhibitors indicate internal motions are present in the bound species.     

Triacylglycerol synthesis in isolated fat cells. An effect of insulin on microsomal fatty acid coenzyme A ligase activity.

Fatty acid CoA ligase (AMP) (EC 6.2.1.3) specific activity was increased approximately 2-fold in microsomes prepared from isolated rat fat cells incubated with 400 microunits of insulin/ml (2.9 nM) for 45 to 60 min compared to paired controls using an assay based on the conversion of [3H]oleic acid to [3H]oleoyl-CoA. Similar insulin-dependent increases in microsomal fatty acid CoA ligase specific activities were observed using an assay based on the conversion of [3H]CoA to fatty acyl-[3H]CoA. Fatty acid CoA ligase activity was predominately (about 80%) associated with the microsomal fraction. The insulin-dependent increase in microsomal fatty acid CoA ligase specific activity was maximal in 2 to 5 min at 400 microunits/ml. At 10 min, 80 to 100 microunits of insulin/ml caused a maximal increase in fatty acid CoA ligase specific activity. Similar apparent Km values for ATP, CoA, and fatty acid were observed for fatty acid CoA ligase activity in microsomal preparations from control and insulin-exposed cells. These data suggest that fatty acid CoA ligase activity is regulated in adipose tissue by insulin. Such regulation may serve to promote the capture of fatty acid and thereby, triacylglycerol synthesis in adipose tissue.     

γ-Aminobutyric Acid Function in the Rat Striatum Is Under the Double Influence of Nigrostriatal Dopaminergic and Thalamostriatal Inputs: Two Modes of Regulation?

Glutamic acid decarboxylase (GAD), gamma-[3H]-aminobutyric acid [( 3H]GABA) high-affinity uptake into synaptosomes, and endogenous GABA content were measured in the rat striatum 2-3 weeks following 6-hydroxydopamine injection in the ipsilateral substantia nigra to destroy the nigrostriatal dopaminergic pathway and after kainic acid injection into the centromedial-parafascicular complex of the ipsilateral thalamus to lesion the thalamostriatal input. Both lesions resulted in apparent GAD increase concomitant with a decreased [3H]GABA uptake into striatal synaptosomes. GABA content was increased selectively following the dopaminergic lesion. Kinetic analysis of the uptake process for [3H]GABA showed selectively a decreased Vmax following the dopaminergic lesion; in animals with thalamic lesion, however, the change only concerned the Km, which showed a decreased affinity of the transport sites for [3H]GABA. Determination of Km and Vmax for GAD action on its substrate glutamic acid showed an increased affinity of GAD for glutamic acid in the case of the dopaminergic lesion without any change in Vmax, whereas the thalamic lesion resulted in GAD increase concomitant with a selective increase in Vmax. These data suggest that striatal GABA neurons are under the influence of nigrostriatal dopaminergic neurons which may reduce the GABA turnover, whereas the exact nature of the powerful control also revealed on these neurons following thalamic lesion remains to be determined. Both lesions induced adaptive neurochemical responses of striatal GABA neurons, possibly reflecting in the case of the dopaminergic deprivation an increased GABA turnover.     

Characteristics of taurine transport system and its developmental pattern in mouse cerebral cortical neurons in primary culture

Developmental patterns and pharmacological and biochemical properties of taurine transport system were investigated using developing primary cultured neurons prepared from mouse cerebral cortex by trypsin treatment. [3H]Taurine was incorporated into neurons via a high-affinity transport system of which the Km value as well as the Vmax value increased during neuronal development in vitro. This transport system was also inhibited by sodium withdrawal from incubation medium and exposures for 15 h to several metabolic inhibitors such as 2,4-dinitrophenol and monoiodoacetate. In addition, [3H]taurine uptake in both neurons cultured for 3 and 14 days was competitively inhibited by beta-alanine, guanidinoethanesulfonate and hypotaurine. Cysteic acid and cysteine sulfinic acid, metabolic intermediates produced in the process of taurine biosynthesis in the brain from cysteine, induced significant reductions in [3H]taurine uptake in both types of cultured neurons, while cysteine, isethionic acid, cysteamine and cystamine exhibited no alterations in [3H]taurine transport. Moreover, non-competitive inhibition of [3H]taurine uptake by cysteic acid was observed in both neurons. These results clearly indicate that taurine uptake was mediated by the sodium- and energy-dependent transport system with high affinity in 14-day-old neurons as well as neurons cultured for 3 days and that both the Km and Vmax values of this transport system increase during neuronal development in vitro. The results described above suggest that the decrease in taurine content observed in developing brain is unlikely to be due to alteration in the capacity of the taurine transport system during neuronal development.     

Uptake and release of [3H]gamma-aminobutyric acid by embryonic spinal cord neurons in dissociated cell culture

We have investigated the uptake and release of [3H]gamma-aminobutyric acid (GABA) by embryonic chick spinal cord cells maintained in culture. Cells dissociated from 4- or 7-d-old embryos were studied between 1 and 3 wk after plating. At 3 degrees C, [3H]GABA was accumulated by a high affinity (Km approximately equal to 4 microM) and a low affinity (Km approximately equal to 100 microM) mechanism. The high affinity transport was markedly inhibited in low Na+ media, by ouabain, at 0 degrees C, and by 2,4-diaminobutyric acid. Autoradiography, after incubation in 0.1 microM [3H]GABA, showed that approximately 50% (range = 30-70%) of the multipolar cells were labeled. These cells were neurons rather than glia; action potentials and/or synaptic potentials were recorded in cells subsequently found to be labeled. Non-neuronal, fibroblast-like cells and co-cultured myotubes were not labeled under the same conditions. The fact that not all of the neurons were labeled is consistent with the suggestion, based on studies of intact adult tissue, that high affinity transport of [3H]GABA may be unique to neurons that use GABA as a neurotransmitter. Our finding that none of fifteen physiologically identified cholinergic neurons, i.e., cells that innervated nearby myotubes, were heavily labeled after incubation in 0.1 microM [3H]GABA is significant in this regard. The newly taken up [3H]GABA was not metabolized in the short run. It was stored in a form that could be released when the neurons were depolarized in a high K+ (100 mM) medium. As expected for a neurotransmitter, the K+-evoked release was reversibly inhibited by reducing the extracellular Ca++/Mg++ ratio.     

Human platelet dense granules: improved isolation and preliminary characterization of [3H]-serotonin uptake and tetrabenazine-displaceable [3H]-ketanserin binding

An improved method for the isolation of human platelet dense granules was developed. A good yield (45%) of highly enriched (69-fold, based on serotonin content) dense granules was obtained after mild sonication and Percoll gradient centrifugation. The method has facilitated characterization of the granule, permitting the first report of Km and Vmax values for [3H]-serotonin uptake, as well as the first determination of Kd and Bmax values for tetrabenazine-displaceable [3H]-ketanserin binding, in the human platelet dense granule. The rates and affinities (Vmax 1.45 nmol/mg/min, Km 0.93 uM) of [3H]-serotonin uptake were similar to those previously reported for porcine dense granules. Tetrabenazine-displaceable [3H]-ketanserin binding was observed with a Kd (9.4 nM) similar to, and a Bmax (5.4 pmol/mg) approximately 10-fold lower than, that previously seen in bovine chromaffin granules.     

ABCG2 transports sulfated conjugates of steroids and xenobiotics

The mechanism for the cellular extrusion of sulfated conjugates is still unknown. In the present study, we investigated whether human wild type ABCG2 transports estrone 3-sulfate (E1S) using membrane vesicles from cDNA-transfected mouse lymphoma cell line (P388 cells). The uptake of [3H]E1S into ABCG2-expressing membrane vesicles was stimulated by ATP, and the Km value for [3H]E1S was determined to be 16.6 microm. The ABCG2-mediated transport of [3H]E1S was potently inhibited by SN-38 and many sulfate conjugates but not by glucuronide and glutathione conjugates or other anionic compounds. Other sulfate conjugates such as [3H]dehydroepiandrosterone sulfate (DHEAS) and [35S]4-methylumbelliferone sulfate (Km = 12.9 microm) and [35S]6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl)benzothiazole (E3040) sulfate (Km = 26.9 microm) were also transported by ABCG2. Although [3H]methotrexate, [3H]17beta-estradiol-17beta-D-glucuronide, [3H]2,4-dinitrophenyl-S-glutathione, and [14C]4-methylumbelliferone glucuronide were transported by ABCG2, this took place to a much lesser extent compared with [3H]E1S. It was suggested that ABCG2 preferentially transports sulfate conjugates and that E1S and DHEAS are the potential physiological substrates for this transporter.     

Substrate specificities of rat oatp1 and ntcp: implications for hepatic organic anion uptake

Transport of a series of 3H-radiolabeled C23, C24, and C27 bile acid derivatives was compared and contrasted in HeLa cell lines stably transfected with rat Na+/taurocholate cotransporting polypeptide (ntcp) or organic anion transporting polypeptide 1 (oatp1) in which expression was under regulation of a zinc-inducible promoter. Similar uptake patterns were observed for both ntcp and oatp1, except that unconjugated hyodeoxycholate was a substrate of oatp1 but not ntcp. Conjugated bile acids were transported better than nonconjugated bile acids, and the configuration of the hydroxyl groups (alpha or beta) had little influence on uptake. Although cholic and 23 norcholic acids were transported by ntcp and oatp1, other unconjugated bile acids (chenodeoxycholic, ursodeoxycholic) were not. In contrast to ntcp, oatp1-mediated uptake of the trihydroxy bile acids taurocholate and glycocholate was four- to eightfold below that of the corresponding dihydroxy conjugates. Ntcp mediated high affinity, sodium-dependent transport of [35S]sulfobromophthalein with a Km similar to that of oatp1-mediated transport of [35S]sulfobromophthalein (Km = 3.7 vs. 3.3 muM, respectively). In addition, for both transporters, uptake of sulfobromophthalein and taurocholic acid showed mutual competitive inhibition. These results indicate that the substrate specificity of ntcp is considerably broader than previously suspected and caution the extrapolation of transport data obtained in vitro to physiological function in vivo.     

Beta-glucosidase from Trichoderma reesei. Substrate-binding region and mode of action on [1-3H]cello-oligosaccharides

To determine the mode of action of the beta-glucosidase from Trichoderma reesei a method was developed for synthesizing [1-3H]cello-oligosaccharides with specific radioactivities of approximately 3000 Ci/mol. The beta-glucosidase removed glucosyl residues from the non-reducing end of the [1-3H]cello-oligosaccharides in a multiple attack mode with little tendency to attack the substrates repetitively. Values of Km were lower for longer cello-oligosaccharides, whereas values of V remained essentially constant. A subsite map, constructed using values of V/Km for the cello-oligosaccharides, showed that the substrate-binding region comprises primarily three subsites.     

The characterization of a sodium-dependent high affinity choline uptake system unassociated with acetylcholine biosynthesis

The cardiac ganglion of the horseshoe crab, Limulus polyphemus, was incubated in Chao's solution containing 0.01 microM [3H]choline at room temperature (25 +/- 2 degrees C) and the ganglion readily accumulated the radiolabel. The ganglion uptake of [3H]choline was linear over 60 min. Kinetic analysis revealed dual choline uptake systems within the cardiac ganglion, a high affinity uptake system (Km = 2.2 microM, Vmax = 0.16 pmoles/mg/min) and a low affinity system (Km = 92.3 microM, Vmax = 3.08 pmoles/mg/min). The high affinity uptake system was sodium-dependent and inhibited by micromolar concentrations of hemicholinium-3. A 15 min pre-exposure of the ganglion to Chao's solution containing 90 mM potassium stimulated a significant increase in choline uptake. There was no detectable synthesis of [3H]acetylcholine from the [3H]choline taken up by the cardiac ganglion. The major portion of the extractable label appeared in a fraction which co-electrophoresed with phosphorylcholine. These results suggest that the sodium-dependent high affinity [3H]choline uptake system of the cardiac ganglion subserves a specific requirement for choline which is unrelated to a cholinergic function.     

Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3).

We have previously cloned rat MRP3 as an inducible transporter in the liver (Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume, K., Shimizu, T., and Sugiyama, Y. (1998) Mol. Pharmacol. 53, 1068-1075). In the present study, the function of rat MRP3 was investigated using membrane vesicles isolated from LLC-PK1 and HeLa cell population transfected with corresponding cDNA. The ATP-dependent uptake of both 17beta estradiol 17-beta-D-glucuronide ([3H]E217betaG) and glucuronide of [14C] 6-hydroxy-5, 7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040), but not that of [3H]leukotriene C4 and [3H]2, 4-dinitrophenyl-S-glutathione, was markedly stimulated by MRP3 transfection in both cell lines. The Km and Vmax values for the uptake of [3H]E217betaG were 67 +/- 14 microM and 415 +/- 73 pmol/min/mg of protein, respectively, for MRP3-expressing membrane vesicles and 3.0 +/- 0.7 microM and 3.4 +/- 0.4 pmol/min/mg of protein, respectively, for the endogenous transporter expressed on HeLa cells. [3H]E217betaG had also a similar Km value for MRP3 when LLC-PK1 cells were used as the host. All glucuronide conjugates examined (E3040 glucuronide, 4-methylumbelliferone glucuronide, and naphthyl glucuronide) and methotrexate inhibited MRP3-mediated [3H]E217betaG transport in LLC-PK1 cells. Moreover, [3H]methotrexate was transported via MRP3. The inhibitory effect of estrone sulfate, [3H]2,4-dinitrophenyl-S-glutathione, and [3H]leukotriene C4 was moderate or minimal, whereas N-acetyl-2,4-dinitrophenylcysteine had no effect on the uptake of [3H]E217betaG. The uptake of [3H]E217betaG was enhanced by E3040 sulfate and 4-methylumbelliferone sulfate. Thus we were able to demonstrate that several kinds of organic anions are transported via MRP3, although the substrate specificity of MRP3 differs from that of MRP1 and cMOAT/MRP2 in that glutathione conjugates are poor substrates for MRP3.     

Effects of fatty acids on activity of cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver

Effects of fatty acids, prostaglandins, and phospholipids on the activity of purified cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver were investigated. Prostaglandins A2, E1, E2, F1 alpha, and F2 alpha, thromboxane B2, and most phospholipids were without effect; lysophosphatidylcholine was a potent inhibitor. Several saturated fatty acids (carbon chain length 14-24), at concentrations up to 1 mM, had little or no effect on hydrolysis of 0.5 microM [3H]cGMP or 0.5 microM [3H]cAMP with or without 1 microM cGMP. In general, unsaturated fatty acids were inhibitory, except for myristoleic and palmitoleic acids which increased hydrolysis of 0.5 microM [3H]cAMP. The extent of inhibition by cis-isomers correlated with the number of double bonds. Increasing concentrations of palmitoleic acid from 10 to 100 microM increased hydrolysis of [3H]cAMP with maximal activation (60%) at 100 microM; higher concentrations were inhibitory. Palmitoleic acid inhibited cGMP hydrolysis and cGMP-stimulated cAMP hydrolysis with IC50 values of 110 and 75 microM, respectively. Inhibitory effects of palmitoleic acid were completely or partially prevented by equimolar alpha-tocopherol. Palmitelaidic acid, the trans isomer, had only slightly inhibitory effects. The effects of palmitoleic acid (100 microM) were dependent on substrate concentration. Activation was maximal with 1 microM [3H]cAMP and was reduced with increasing substrate; with greater than 10 microM cAMP, palmitoleic had no effect. Inhibition of cGMP hydrolysis was maximal at 2.5 microM cGMP and was reduced with increasing cGMP; at greater than 100 microM cGMP palmitoleic acid increased hydrolysis slightly. Palmitoleic acid did not affect apparent Km or Vmax for cAMP hydrolysis, but increased the apparent Km (from 17 to 60 microM) and Vmax for cGMP hydrolysis with little or no effect on the Hill coefficient for either substrate. These results suggest that certain hydrophobic domains play an important role in modifying the catalytic specificity of the cGMP-stimulated phosphodiesterase for cAMP and cGMP.     

Irreversible Binding and Recovery of the Norepinephrine Uptake System Using an Alkylating Derivative of Norepinephrine

The effects of bromoacetylaminomenthylnorepinephrine (BAAN) on the sodium-dependent, high-affinity norepinephrine (NE) uptake system in rat brain synaptosomes and CNS neuronal cultures were investigated. BAAN inhibited [3H]NE uptake into synaptosomes in a dose- and time-dependent manner (IC50, 6.5 microM). Pretreatment of cortical synaptosomes or neuronal cells with BAAN alone, followed by washing to remove free drug, reduced the Vmax but did not alter the Km value for [3H]NE uptake. The BAAN-induced reduction in Vmax was attenuated by concurrent pretreatment with desipramine and blocked by the reaction of BAAN with dithiothreitol or cysteine. In contrast, BAAN was 19-fold less potent at inhibiting [3H]dopamine uptake in striatal synaptosomes, and no change in the Vmax or Km value for [3H]dopamine uptake was observed after a pretreatment with BAAN followed by washing. Furthermore, the irreversible beta-antagonist, bromoacetylalprenololmentane, was equipotent to BAAN for inhibiting [3H]NE uptake into cortical synaptosomes, but did not alter the Vmax or Km for [3H]NE after pretreatment. In neuronal cultures, BAAN inhibited sodium-dependent uptake of [3H]NE (IC50, 5.6 microM) with no effect on sodium-independent uptake. After pretreatment of cultures with 30 microM BAAN followed by washing, there was a 74% decrease in the Vmax for [3H]NE uptake. Following a 24-h lag period, uptake recovered to the control level within 48 h; however, recovery was completely blocked by cycloheximide. The data indicate that BAAN irreversibly binds to the [3H]NE uptake system in both CNS synaptosomes and neuronal cultures and may be a useful probe for studying the turnover of the [3H]NE uptake system.