Activation of Rabbit Liver High Affinity cAMP (Type IV

THE JOURNAL OF BIOLOGICAL CHEMISTRY (0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol . 266, No. 26, Issue of September 15. pp. 17011-17019.1991 Printed in U. S.A.

Activation of Rabbit Liver High Affinity cAMP (Type IV) Phosphodiesterase by a Vanadyl-Glutathione Complex CHARACTERIZATION OF THE ROLE OF THE SULFHYDRYL*

(Received for publication, December 24, 1990)

W. Joseph Thompson$, Boen H. Tan, and Samuel J. Strada From the Department of Pharmacology, University of South Alabama College of Medicine, Mobile, Alabama 36688

Activation of rabbit liver microsomal high affinity cAMP phosphodiesterase (Type IV PDE) by vanadyl- glutathione complexes was studied as a possible model of insulin stimulation of the enzyme in a cell-free system. The effect of VO*2GSH activation of PDE was a 21-fold decrease in the ICso value for cGMP inhibition and a 2.6-fold increase in the V,,, of the higher affinity cAMP catalytic site. Cyclic AMP and cGMP substrate affinities and cGMP hydrolysis were unaffected by VO-2GSH activation. Selective Type IV PDE inhibitors and cGMP analogs indicated that VO-2GSH complexes activated the cGMP-inhibitable form of the Type IV PDE activities which co-localized in hepatic microsomes.

The Type IV PDE activating complex appears to consist minimally of vanadyl ion and 2 oxidized electron donor compounds. The components of the electron donor required to achieve an enzyme activation complex are: 1) a free -SH group as the electron donor for vanadate reduction and 2) a minimum structure of cysteamine (NH2-CH2-CH,-SH). Maximal activation of the enzyme required near 2: 1 molar ratios of either glutathione or cysteamine mixed with sodium orthovanadate. Active vanadyl-cysteamine complexes were isolated by reverse- phase high performance liquid chromatography. Tungsten, niobium, and tantalum, but not manganese, chromium, or molybdenum, substi- tuted for vanadium to form enzyme-activating complexes with glutathione. VO-RSH complex activation occured rapidly upon addition to microsomes and was reversible. We conclude from these studies that VO-RSH complexes and insulin activate the same form of Type IV PDE in rabbit liver microsomes; our findings are discussed with respect to the involvement of a possible electron transfer enzyme oxidation in the activation mechanism.

Vanadium is a trace metal of undetermined biological sig- nificance (1, 2). Sodium vanadate displays many insulinomi- metic properties, including enhanced glucose transport, and is being considered as a potential therapy in the treatment of diabetes (3-7). The studies of Degani et al. (8), Dubyak and Kleinzeller (9), and Bruech et al. (10) indicate that the active intracellular species of the complex oxides of vanadium is the

*This research was supported by United States Public Health Service Grant GM 33538 and a grant from the Eli Lilly Company. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed.

vanadyl oxide ion (V02+), most likely complexed to glutathione. We reported previously that sodium vanadate incubated with rat adipocytes, or mixtures of sodium vanadate and reduced glutathione incubated with adipocyte particulate fractions, activated a high affinity (Type IV) cAMP phosphodiesterase (PDE)’ activity (11). Furthermore, vanadyl-glutathione complexes activated both basal and insulin-stimulated Type IV PDE to the same maximal level. The mechanism of PDE activation involved membrane components rather than a direct effect on the enzyme. The biological effects of sodium vanadate are proposed to involve mechanisms other than the insulin receptor-tyrosine kinase system (7,12). Both vanadate (V5+) and vanadyl (V“) ions reportedly affect calcium transport in adipocytes (13).

Activation of Type IV PDE activity by vanadyl-glutathione complexes is of interest because insulin also activates this membrane enzyme activity in intact cells and fat and hepatic tissues (14-20). The available evidence indicates that insulin increases the activity of the cGMP-inhibitable form of Type IV PDE (21-25), an isozyme of PDE whose activity is enhanced by protein phosphorylation (25-29). Cyclic nucleotide PDE activation mechanisms, like other actions of insulin, are difficult to study because intact cells are required to obtain enzyme activation. Mechanisms proposed for activation of hepatic Type IV PDE by insulin in addition to regulation by phosphorylation-dephosphorylation, include critical thiol oxidative stabilization (30, 31), release of phospholipids (32), and production of insulin-evoked mediators (33-35). The insulin-like actions of thiols were first studied by Lavis and Williams (36).

The studies reported here were undertaken to determine if insulin-sensitive hepatic, as well as adipocyte, Type IV cAMP PDE is activated by complexes of vanadate and glutathione in a cell-free system, and if, as with insulin in the intact cell, these complexes activate the cGMP-inhibitable form of Type IV PDE. Also, we attempted to define the essential requirements of the sulfhydryl and transition metal portions of the complex in the activation mechanism. The results of these studies suggest a model wherein vanadyl-glutathione complexes supply a precise electron donating capacity to hepatic membranes or act as allosteric activators to initiate enzyme activation by an oxidative mechanism. A preliminary report of these findings has been presented (37).

The abbreviations used are: Type I PDE, calmodulin/calcium- activated cyclic nucleotide phosphodiesterase (38); Type I1 PDE, cGMP-activated cyclic nucleotide phosphodiesterase; Type IV PDE, high affinity cAMP phosphodiesterase; BSA, bovine serum albumin; CAMP, 3’,5’-cyclic adenosine monophosphate; TLCK, N-p-tosyl-L- lysine chloromethyl ketone; MES, 2-(N-morpholino)ethanesulfonic acid TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid DTT, dithiothreitol; EGTA, [ethylenebis(oxyethylenenitrilo)] tetraacetic acid HPLC, high performance liquid chromatography.

17011

17012 VanadyllGlutathione Activatable Type I V PDE

EXPERIMENTAL PROCEDURES

Materials-S-Benzyl-1-cysteine, D,L-homocysteine, L-methionyl glycine, L-cysteine ethyl ester-HCL, cystamine-diHCL, DL,DL-allO- cystathione, L-cystinyl-bis-1-glycine, L-cystinyl-bis-1-tyrosine, and L- cystinyl-bis-l-alanine were purchased from Research Plus, Inc. Re- duced and oxidized glutathione were from Boehringer Mannheim and L-cysteine, N-acetyl-cysteine, cysteamine-HCL, L-cystine, DL-homocystine, taurine, and amino acids from Sigma. EDTA and EGTA were purchased from EM Laboratories. [3H]cGMP (sp. act. 8 Ci/ mmol) and [3H]cAMP (sp. act. 28 Ci/mmol) were from ICN; TES, MES, and Tris were from Research Organics, Inc.; Dowex 1-X8 (200- 400 mesh) from Aldrich; sodium orthovanadate and vanadyl sulfate were from Alfa Products; and tantalum and niobium pentachlorides were from Fluka, AG. Reagents for HPLC were from Burdick and Jackson Laboratories, Inc. (acetonitrile, UV), Aldrich (trifluoroacetic acid, 99%), and EM Science (methanol, OmniSolv). Sep-Pak CIS cartridges were from Waters Associates. Synchropak RP-P C,, HPLC columns were purchased from Synchrom, Inc. Bovine crystalline insulin was a gift from Burroughs-Wellcome, Inc. and was further purified (11). SQ-65442 was supplied by Dr. Charles A. Free of E. R. Squibb and Sons, Princeton, NJ; RO 20- 1724 by Dr. Herbert Shep- pard of F. Hoffman-LaRoche, Nutley, NJ; CI-914 (imazodan) by Dr. R. E. Weishaar of Warner-Lambert/Parke-Davis, Ann Arbor, MI; WIN-47203 by Dr. A. Soria of Sterling Winthrop Research Inst., Rensselaer, NY; OPC-3689 (cilostamide) by Dr. H. Hidaka, Mie University School of Medicine and Otsuka Pharmaceuticals, Japan; Y-590 by Dr. K. Goto of Yoshitomi Pharmaceuticals, Osaka, Japan; RS-82856 (lixazinone) by Dr. Robert Alvarez, Institute of Biological Sciences, Syntex Research, Palo Alto, CA; and LY-195115 (indolidan) by Dr. David W. Robertson, Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, IN.

Rabbit Liver Light Microsome Preparation-Subcellular fractions of rabbit liver were prepared by differential centrifugation according to the procedures of Fleischer and Kervina (39) for rat liver. Proce- dural details and data regarding the distribution and partial characterization of cyclic nucleotide PDE activities are provided in the Miniprint (Tables 1-3 and Figs. 1 and 2). Residue 4, the particulate fraction (microsomes) from the 130,000 X g (39,000 rpm), 45-min centrifugation step, was used for these studies. This microsomal fraction contained most of the VO. 2GSH-activatahle and cGMP- inhibitable Type IV PDE activity. Rabbit liver microsomal membranes were resuspended in 10 mM TES, 0.25 M sucrose, 10% glycerol, 0.1 mM DTT (20 mg/ml) for activity analysis or storage a t -20 "C. If prepared from fresh rabbit liver, these microsomal preparations could be stored up to 3 weeks at -20 "C without loss of basal Type IV PDE activity, cGMP-inhibitable enzyme activity, or activation of cAMP hydrolysis by vanadyl-glutathione complexes (Fig. 1, Miniprint).

Vanadyl-Sulfhydryl Complex Preparation-Complexes of sodium orthovanadate (Na3V0,) and reduced glutathione (GSH) or other sulfhydryl compounds (RSH) were prepared according to Souness et al. (11). Stock solutions of Na3V04 (112 mM) were made by dilution from a 2 M solution in 1 M Tris-HC1 (pH 7.0) using 80 mM Tris-HC1 (pH 7.0). Complexes were prepared for each experiment from stock solutions of Na3VO4 (112 mM) and RSH (224 mM in 80 mM Tris- HC1, pH 7.0) using 1:l mixtures. The mixture turned emerald green in seconds and grew darker over the next 24 h, a t which time the complex still retained the capacity to activate Type IV PDE activity. Ten pl of the mixture was added to a 400-pl enzyme assay immediately before the addition of liver microsomes to achieve the indicated concentrations. In one experiment the sulfhydryl stock solution was diluted before mixing 1:l with the stock vanadate solution to determine the ratio required for complex formation. Electron paramagnetic resonance spectra have shown that vanadate ion (VOi-; V 5 + ) was reduced to vanadyl ion (VO"; V + ) in a complex with oxidized glutathione (11). This complex is referred to as VO.2GSH or VO. 2RSH in the case where other sulfhydryl compounds were used. Na,VO, and GSH were inactive when used individually. Na3V0, mixed with oxidized glutathione was also inactive, as was VOSO, either alone or mixed with reduced glutathione.

HPLC Chromatography and Vanadyl-Sulfhydryl Complex Puriji- cation-Mixtures of sodium vanadate (1.4 mM) and cysteamine (2.8 mM) were analyzed by high pressure liquid chromatography and purified in larger quantity using Sep-Pak Cle cartridges. For HPLC, 0.02 ml of the mixture above (30 s after mixing) or each compound individually was injected to a Synchropak RP-P C, , (250 X 4.1 mm, 6.5 pm) column equilibrated with HPLC grade water (pH 3.5; flow rate 0.75 ml/min). The eluate was monitored a t 256 nm with a

Beckman 160 detector (flow cell 18.5 pl). The active VO.2RSH complex was eluted with a 20-min gradient of 0-0.1% trifluoroacetic acid beginning 50 min after sample injection. In separate experiments the column was monitored a t 214 nm with a zinc lamp (P/N 235959) in the Beckman 160 detector. Using absorbance a t 214 nm the vanadyl-cysteamine mixtures showed intense peaks a t 3.4 and 4.3 min and a broad peak at 12-21 min that lacked Type IV PDE activating properties (data not shown). For Sep-Pak C,, purification, cartridges were prewashed with 5 ml of acetonitrile (70,50, and 10%) water. One ml of Na3VOa (112 mM) and cysteamine (224 mM) were mixed for 1 min and applied to the prewashed cartridge. After washing with water and methanol (5 ml), the dark green active complex was eluted with 6 ml of 0.1% trifluoroacetic acid and lyophilized to dryness. The dried complex was very hygroscopic, requiring storage over desiccant to maintain dryness. The dry weight yield was 9.25 mg from a possible 46.1 mg (dry weight) of reactants. For enzyme activation experiments and spectral analysis, the dry powder was recon- stituted in 0.1% trifluoroacetic acid and neutralized with 2 M Tris. The concentration of the complex was calculated based on the dry weight and an assumed structure of NHz-CHz-CHz-S-VO-S-CHz-

Enzyme Assay and Miscellaneous Procedures-Cyclic AMP phosphodiesterase activity was determined by the modified two-step ra- dioisotopic procedure of Thompson et al. (40) using reaction condi- tions of 40 mM Tris-C1 (pH 8.0), 10 mM MgClZ, 3.75 mM 2-mercaptoethanol, 30 pg of BSA, 0.25 M cyclic [3H]AMP (-160,000 cpm) in 0.4 ml. To test VO'RSH complex effects, the order of addition of reaction constituents was buffer, cation, substrate, and VO.RSH complex. Catalysis was initiated by the addition of microsomes (-60 pg of protein). Reactions were terminated by boiling for 45 s. Vanadyl- glutathione complexes had no effect on snake venom nucleotidase or on the isolation of reaction products with Dowex 1-X8 (40-400 mesh) resin. Kinetic parameters of membrane activities were determined using 0.024-50 p~ cAMP and 0.18-20 p~ cGMP substrate ranges. Drugs were dissolved in 10% dimethyl sulfoxide or 100% ethanol and stock solutions diluted with 0.1% dimethyl sulfoxide or water to give final vehicle concentrations in the assay that did not effect enzyme activity. Kinetic parameters were determined according to Cleland (41). Inhibition and activation constants and their percent error based on the confidence limits of the full dose-response curve were determined using the IBM XT version (M. L. Jaffe, Assoc., 1982, Silver Spring, MD 20901) of Curvefit. The program uses a 2 + 2 linear regression approach developed by P. Munson, D. Rodbard, and M. L. Jaffe (National Institutes of Health, Bethesda, MD). Protein was determined by the method of Bradford (42). A Perkin-Elmer X3 UV/ VIS spectrophotometer was used to obtain absorbance spectra.

CHz-NH, (Mr = 219).

RESULTS?

Effect of VO. 2GSH Activation on cGMP Znhibition of Type ZV PDE-Rabbit liver microsomal Type IV PDE measured with 0.25 PM cAMP substrate was inhibited by cGMP to a maximal level of near 60% (Fig. 3). The IC5o value for cGMP inhibition was approximately 4 PM. The total activity of the membrane was activated approximately 250% at maximal VO. 2GSH (Fig. 3). The stimulated activity showed an IC50 value for cGMP inhibition 20-fold lower (near 0.2 WM) than that for unstimulated activity. However, the maximal inhibition of basal and stimulated Type IV PDE activities were similar. The differences in inhibitory sensitivity enabled com- parison of the potencies of cGMP analogs (Table 3, Miniprint) and PDE inhibitors on basal and stimulated microsomal enzyme activity.

Pharmacological Sensitivity of Type ZV PDE Activity of Residue 4-In the basal state, microsomal Type IV PDE activity was very sensitive to inhibition by the quinazoline derivatives (OPC-3689, Y-590, and RS-82856), less sensitive to the bipyridine cardiotonics agents (LY-195115, WIN- 47203, and CI-914), and least sensitive to RO-20-1724 or SQ-

Portions of this paper (including part of "Results," Figs. 1 and 2, and Tables 1-3) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

VanadyllGlutathione Activatable Type IV PDE 17013

VO/PGSH ACTIVATABLE LIVER MEMBRANE CYCLIC GMP INHIBITION OF BASAL AND

HIGH AFFINITY CAMP PDE

9 1 ”“_ ”“_

“”_

CYCLIC GMP, UM 0.0 10 m , , m , o . 10

CYCLIC GMP, UM

FIG. 3. Cyclic GMP inhibition of basal and VO.2GSH-activatable microsomal high affinity cAMP phosphodiesterase. Microsomal membranes (Residue 4) were prepared from fresh rabbit liver as described under “Experimental Procedures.” Cyclic AMP hydrolysis at 0.25 p~ substrate was determined in the presence (*) or absence (A) of 1.2 mM sodium orthovanadate/2.8 mM reduced glutathione complex with varying concentrations of cGMP (0.001-20 p ~ ) . Values are duplicate or triplicate determinations and IC,, values were determined using Curvefit as described under “Experimental Procedures.’’

DRUG SENSITIVITY OF V0/2GSH ACTNATABLE L M R MEMBRANE HIGH AFFINITY PDE

FIG. 4. Drug sensitivity of VO-2GSH-activatable microsomal high affinity cAMP phosphodiesterase activity. Microso- mal membranes (Residue 4) were prepared as described under “Ex- perimental Procedures.” Particulate enzyme activities were determined in the presence of VO. BGSH complex with 0.25 p~ cAMP substrate. Inhibition constants were determined using Curvefit as described under “Experimental Procedures.” Percent errors for these values ranged from 8 to 22%.

65442 (Fig. 4). These latter two compounds are among the most potent inhibitors of crude or highly purified dog kidney Type IV PDE, an enzyme form not inhibited by low concentrations of cGMP (43). A difference in inhibitor ICbo values of 1.9 x 106-fold between RS-82856 and SQ-65442 was evident when these drugs were tested as inhibitors of VO.2GSH- activated Type IV PDE (Fig. 4). Y-590 inhibition was competitive ( K = 0.12 p ~ ) with high affinity cAMP catalysis using methods of Dixon (44) or Cornish-Bowden (45) (data

not shown) to assess the kinetics of inhibition. Effect of VO.2GSH Activation on the Kinetics of cAMP

Hydrolysis by Residue 4-The effects of VO. BGSH activation on microsomal membrane Type IV PDE kinetics were studied using 0.02-50 p~ cAMP substrate concentrations. In agree- ment with previous studies, nonlinear kinetic behavior was observed (17) and apparent K , values of 0.4 and 30 p~ for cAMP were found. Fig. 5 shows the kinetic data in the low substrate range. The apparent K,,, for cAMP was unaffected by VO-2GSH activation. However, the apparent maximum velocity of the higher affinity catalytic site was increased 2.7- fold. In the substrate concentration range of 3-50 p ~ , the plots of activated and basal cAMP hydrolysis converged, indicating no change in the V,,, or K,,, of the lower affinity catalytic site (data not shown). Cyclic GMP hydrolysis by the liver membranes showed Michaelis-Menten type kinetic behavior with an apparent K , for cGMP of 10 p ~ . Cyclic GMP hydrolysis was not modified by VO. 2GSH complex treatment (data not shown). Cyclic GMP inhibition of cAMP hydrolysis at substrate concentrations above 1 p~ showed apparent competitive inhibition ( K ; = 8 p ~ ) using Dixon plots. Nonlin- ear Dixon plots were obtained using substrate concentrations below the apparent K , of the higher affinity catalytic site (0.05-0.65 pM). Plots of KJV, ratios for cAMP hydrolysis at various cGMP inhibitor concentrations were linear using the enzyme in the basal state and nonlinear in VO .2GSH-activated preparations.

Type IV PDE Activation Ratio of Glutathione and Vana- date-The ratio of reduced glutathione to sodium vanadate required for maximal activation of microsomal Type IV PDE was determined by varying the concentration of reduced glutathione mixed with stock sodium orthovanadate solutions before addition of the diluted complexes mixtures to achieve the concentrations shown in the enzyme assays (Fig. 6). Concentrations of GSH from 0.1 to 5.6 mM were studied. The ECbO value for GSH was 1.2 mM (4% error), and maximal activation required 2-3 mM.

Time Course of VO.2GSH Activation of Light Microsomal High Affinity cAMP PDE-Progress curves of cAMP hydrolysis at 0.25 p~ substrate by microsomal membranes in the presence and absence of VO .2GSH complexes were determined (Fig. 7). Residue 4 (230 pg) was incubated for 5 min in 3.4 ml of 40 mM Tris-Cl/BSA (0.5 mg/ml) at 30 “C before the addition (time zero) of [3H]cAMP substrate (1.6 X lo6 cpm), 40 mM Tris-C1, pH 8.0, 10 mM MgC12, and 4 mM 2-mercap-

/

CYCUC AMP (uM) l/CYCLIC AMP (uM)

FIG. 5. Effect of VO-2GSH activation on the kinetics of microsomal cAMP PDE. Microsomal fractions (Residue 4) were prepared as described under “Experimental Procedures.’’ Kinetic constants were determined in the presence (0) or absence (*) of VO. BGSH using cAMP substrate concentrations from 0.024 to 2.6 PM. The indicated apparent V,,, and K, values for the lower substrate concentrations shown graphically were determined according to the weighted analysis of Cleland (41).

17014 VanadyllGlutathione Actiuatable Type IV PDE

E y o L -I 1 10

GSH (mu) with 1.4 mM VANADATE FIG. 6. Determination of the vanadate and glutathione ratio

required for activation of microsomal Type IV PDE. Rabbit liver microsomes were prepared and enzyme activity determined as described under “Experimental Procedures.” Solutions of various stock concentrations of reduced glutathione were mixed (1:l v/v) with stock sodium orthovanadate solutions (112 mM) prepared in 1 M Tris- HCl (pH 7.0). The mixtures were added (10 ~ 1 ) to assays mixtures (400 pl) containing substrate, cation and buffer to achieve the concentrations indicated. Reactions were initiated with the addition of microsomes. EC,, was determined using Curvefit.

V -i 3.9 nrnol/rnin/rnl

.,.........,.~., . . . . . . . . . . . . . . . . . . . . . .

2 4 6 8 10 MINUTES

FIG. 7. Progress curves of cAMP hydrolysis by rabbit liver microsomes in the presence and absence of vanadyl-glutathione complexes. Microsomes (230 pg) prepared as under “Experi-

taining [3H]cAMP (0.25 p ~ ) , 40 mM Tris-C1 (pH 8.0), 10 mM MgCL, mental Procedures” were incubated in 4-ml reaction mixtures con-

and 4 mM 2-mercaptoethanol. Concentrated vanadyl-glutathione mixtures prepared as described under “Experimental Procedures” were added at the indicated time to one set of reaction mixtures to achieve a final conentration of 1.4 m ~ / 2 . 8 mM, respectively (X). Aliquots of the reaction were taken at intervals, boiled for 45 s, and cAMP hydrolysis determined as indicated under “Experimental Pro- cedures.” The solid lines are data fitted by linear regression.

toethanol (total volume = 4 ml). Aliquots of the reaction mixture were taken at various intervals and boiled for 45 s. “H-labeled 5’AMP produced by cAMP PDE hydrolysis was determined by the addition of 0.1 ml of snake venom (Ophio- phagus hannah, 0.5 mg/ml) and incubation for 10 min at 30 “C and Dowex 1-X8 separation of [3H]adenosine (40). Preformed VO. 2GSH complexes (1.4 and 2.8 mM, respec-

tively) were added to additional microsome incubation mixtures at 2 min and aliquots taken at 0.5 min thereafter (Fig. 7). By these procedures there was no apparent lag time required for VO.2GSH to increase the rate of the reaction (Fig. 7). The rate of cAMP hydrolysis was increased 2.6-fold by VO + 2GSH. In separate experiments the ratio of membrane protein to VO . GSH concentration was shown to be important for rapid enzyme activation (Fig. 2, Miniprint).

Sulfhydryl Requirements for Active VO. 2RSH Complex For- mation-Various compounds containing reduced or oxidized sulfur moieties and other chemical substitutions were mixed with sodium vanadate (2.4 and 1.2 mM, respectively) and tested as activators of microsomal, high affinity cAMP PDE (Table 4). L-Cysteine was found to be the active center of glutathione. Acetylation or esterification of the carboxyl group of L-cysteine did not affect complex activation. Decar- boxylated cysteine, i.e. cysteamine, also formed active complexes with sodium vanadate (Table 4). Dithiothreitol and 2- mercaptoethanol were unable to form active complexes with sodium vanadate. The juxtaposition of the amino and sulfhydryl groups appears essential for formation of active complexes since homocysteine, which contains an additional in- tervening carbon, was inactive (Table 4). Benzylation of the sulfhydryl group of cysteine and all other disulfide compounds tested was inactive indicating the importance of a free sulfhydryl group in the formation of an active complex. All of the active mixtures shown in Table 4 formed greenish or dark yellow colored solutions suggesting a V5+ to V4+ reduction by the active sulfhydryl compounds. Sodium vanadate and the inactive mixtures were colorless.

Since cysteamine represented the minimum structure of the sulfhydryl portion of the activating complex, the ratio of cysteamine to sodium vanadate required for maximal effect was determined in an analogous fashion to the ratio for GSH. Cysteamine solutions of concentrations from 0.06 to 5.6 mM were mixed with 1.4 mM sodium orthovanadate. Activation of microsomal Type IV PDE by the resultant complexes measured using 0.25 p~ substrate showed an ECso value of 0.9 mM for cysteamine. The concentration ratio that caused maximal enzyme activation (2-3 mM) was very similar to that for GSH (above and Ref. 11).

HPLC Purification of VO. 2CSH Complex-The behavior of the vanadyl-cysteamine complex (VO.2CSH) was compared with that of the reactant solutions by reverse-phase HPLC using a Synchropak RP-P CIS column (Fig. 8). The majority of the sodium vanadate solution eluted from the C18 column previously equilibrated in water in a broad peak from 20 to 40 min. After complex formation with cysteamine, no absorbance of the applied complex solution was found in this range. A peak with absorbance at 256 nm was eluted with a 0-0.1% trifluoroacetic acid gradient. The tight binding of the vanadyl-cysteamine complex to the CI8 column allowed a rapid purification of larger quantities of the complex using disposable Sep-Pak CI8 cartridges as described under “Exper- imental Procedures.” When 26 mg (1 ml of 112 mM) of sodium vanadate was mixed with 25.5 mg of cysteamine (1 ml of 224 mM) and applied to the Sep-Pak column, 9.3 mg of the complex was recovered in the 0.1% trifluoroacetic acid eluate.

The absorbance spectra of cysteamine, sodium vanadate, fresh mixtures of the two, and purified vanadyl-cysteamine complexes are shown in Fig. 9. The unpurified mixture dis- played a decreased absorbance at,near 230 nm compared with cysteamine alone. The complex mixture showed increased absorbance in a broad 215-250 range compared with sodium vanadate alone. The original mixture and the Sep-Pak-purified complex showed similar absorbance peaks at 258 nm and

VanudyllGlutathione Activatable Type IV PDE 17015

TABLE 4 Sulfhydryl requirements for active uanadyl-RSH complex formation

Addition (+ Na3V04)” Activation (% above basal)

R = -CH-CH2-NH2 None 100 Reduced glutathione 320 (green) Glu-Cys-Gly L-cysteine 270 (green) HOOC- R -SH N-acetylcysteine 258 (green) CHsCO- R -SH L-cysteine ethyl ester HCL 259 (green) CzHSCO- R -SH Cysteamine-HC1 246 (yellow) H- R -SH D,L-homocysteine 102 (white) HOOC- R -CHZ-SH L-methionyl glycine 109 (no color) Gly-N-OCO- R -CHz-S-CHs S-benzyl-L-cysteine 87 (white) HOOC- R -S-Benzyl Taurine 115 (no color) H- R -SOsH Cystamine-diHC1 113 (no color) R-S-S-R L-cysteine 105 (white) HOOC-R-S-S-R-COOH D,L-homocystine 105 (white) HOOC-R-CHz-S-S-CH2-R-COOH L-cystinyl-bis-L-glycine 146 (no color) Gly-Cystine-Gly L-cystinyl-bis-L-tyrosine 120 (yellow) Tyr-Cys-Tyr L-cystinyl-bis-L-alanine 110 (white) Ala-Cys-Ala D,L,D,L-allo-cystathione 112 (white) HOOC-R-CHz-S-R-COOH a Rabbit liver light microsomes were prepared and Type IV PDE activity was determined with 0.25 p M cAMP

as described under “Experimental Procedures.” Sulfhydryl compounds (2.8 mM) were tested after mixing with an equal volume of sodium orthovanadate (1.4 mM) which was inactive alone. Values are the average of triplicate determinations. The colors of the vanadate-sulfhydryl compound mixtures were noted approximately 1 min after mixing and just before addition to the membrane phosphodiesterase assay reaction.

VO/PCSH MIXTURE

I \ I \ 1 I 1 1

\ \ \ \ I 1 I

FIG. 8. HPLC purification of vanadyl-cysteamine Type IV PDE activating complex. HPLC absorbance profiles of sodium orthovanadate before and after mixing with cysteamine and applica- tion of each solution to a Synchropak RP-P C-18 column are shown using 256-nm detection. The procedures used for HPLC chromatography are given under “Experimental Procedures” and summarized in the caption on the figure.

214 nm. The Sep-Pak C18-purified vanadyl-cysteamine complex had an EC50 value of 40 PM to activate microsomal high affinity cAMP PDE (data not shown), a value in the same range required for glutathione and the unpurified cysteamine complex to activate the enzyme. Maximal stimulation of microsomal Type IV PDE by the purified complex was also similar to that of the unpurified mixtures (200-300%). The concentrations used were calculated from the dry weight of the complex and assumed a minimal ratio of 1 mol of vanadyl ion to 2 mol of oxidized cysteamine (Mr = 219).

Amino Acid Substitution for Cysteine in Vanadyl Complex Formation-Table 5 shows the results of mixing sodium orthovanadate with various amino acids other than cysteine

and testing them as activators of microsomal high affinity cAMP PDE using 0.25 PM cAMP substrate. Arginine, tryptophan, tyrosine, and asparagine had no effect on microsomal Type IV PDE, whereas histidine did form an active complex. As observed with the sulfhydryl compounds above, the active mixture with histidine was green, suggesting the presence of vanadyl ion in the active complex.

Transition Metal Substitution For Vanadium in Glutathione Complex Formation-Reduced glutathione (2.8 mM) was mixed with compounds containing transition metals from periodic groups 5 and 6 of 5+ and 6+ valences (1.4 mM). Activation of liver microsomal Type IV PDE by these complexes was compared with VO e 2GSH-stimulated activity (Table 6). Of the group 6 elements tested, tungsten (W“) activated Type IV PDE activity, but Mo6+ or Cr6+ compounds were inactive. Ta5+ and Nb5+ transition metals in periodic group 5 along with V5+ formed complexes that increased enzyme activity when mixed with GSH. However, tantalum and niobium pentachlorides had a significant inhibitory effect on basal activity in the absence of GSH. Vanadyl sulfate (V“) and manganese chloride (Mn2+) mixed with GSH were inactive and thus served as additional controls for possible arti- factual effects of the metals.

DISCUSSION

These studies show that complexes of vanadyl ion and glutathione or an appropriately structured electron donor activate rabbit liver microsomal high affinity (Type IV) cAMP PDE in a cell-free system. Characteristics of the activation are similar to those found previously with particulate Type IV PDE of adipocytes (11). VO. 2GSH activation has two predominant effects on membrane activity; the apparent V,,, of the higher affinity catalytic site is increased 2-3-fold without changing substrate affinity, and the sensitivity of the enzyme to inhibition by cGMP is increased 21-fold. The activating effect is relatively specific for cAMP hydrolysis. The effects of cGMP analogs compared with those of cGMP are also consistent with a specific effect of VO. 2GSH to activate a cGMP-inhibitable, high affinity cAMP PDE activity.

17016 Vanadyl/Glutathione Activatable Type I V PDE

FIG. 9. Absorbance spectra of vanadyl-cysteamine mixtures and purified complexes. Absorbance spectra obtained with 0.56 mM cyteamine, 0.28 mM sodium vanadate, and their mixtures are shown in the left portion of the figure. The absorbance spectrum of the Sep-Pak CIS, 0.1% trifluoroacetic acid-eluted complex estimated to be 0.08 mM is shown on the right portion of the figure. 40 mM Tris-C1 (pH 8.0) was used for sample dilution and reference.

ORIGINAL MIXTURE

4 I ‘pSTUNlNE (0.56 mU)

0.0

WAVELENGTH (nm)

0.0 J ”-

l o - 2 S WAVELENGTH (nm)

6

0.4

0.2

O*O 1 WAVELENGTH (nm)

TABLE 5 Amino acid substitutions for cysteine to form microsomal type IV

PDE activation complexes Additions Type IV activity”

pmollminlml Basal 0.89 Basal + Na3V04 0.92 Basal + 1-cysteine 0.43 Basal + 1-cysteine + Na3V04 2.26 Basal + d,l-histidine 0.97 Basal + d,l-histidine + Na3V04 2.07 Basal + 1-histidine 1.00 Basal + 1-histidine + Na3V04 2.08 Basal + d,l-asparagine 0.94 Basal + d,l-asparagine + Na3V04 0.97 Basal + 1-asparagine 0.85 Basal + 1-asparagine + Na3V04 1.03 Basal + d,l-tyrosine 0.87 Basal + d,l-tyrosine + Na3V04 1.00 Basal + 1-tyrosine 0.99 Basal + 1-tyrosine + Na3V04 0.95 Basal + 1-tryptophan 0.94 Basal + 1-tryptophan + Na3V04 0.99 Basal + 1-arginine 0.96 Basal + I-arginine + Na3V04 0.95

Rabbit liver microsomes were prepared as described under “Ex- perimental Procedures.” Cyclic AMP phosphodiesterase activity was determined with 0.25 PM cAMP in the presence of 2.8 mM amino acid mixed with 1.4 mM sodium orthovanadate before addition to the enzyme assay reaction.

Continuing studies of insulin activation of hepatic high affinity cAMP hydrolysis, first observed by Senft et al.(46), have revealed that liver membranes have a complex set of high affinity enzyme activities possessing diverse regulatory properties. Insulin stimulated the Vmax of a 120-kDa Type IV PDE isozyme with a K,,, for cAMP or cGMP of 0.3 FM in hepatocytes (22). In other studies, insulin has been shown to activate different high affinity PDE activities, one which resides in liver plasma membrane fractions and one in “dense vesicle” fractions (16, 25). Both Type IV PDE forms were subsequently purified and showed different physical, kinetic, and pharmacological properties. Cyclic GMP inhibition distinguished both the dense vesicle enzyme and an insulin activated-Type IV PDE purified from a high speed liver particulate fraction (24), confirming the earlier observations of Witson and Appleman (21). Soluble and particulate hepatic Type IV PDE forms may also be distinguished on the basis of their pharmacological properties (14). The insulin- and cGMP-sensitive Type IV PDE is also strongly inhibited by milrinone and weakly inhibited by RO 20-1724 (16, 47). The

TABLE 6 Transition metal substitutions for vanadium to form type IV PDE

activating complexes with reduced glutathione Additions Basal activity

% Basal“ 100 Basal + Na3V04 104 Basal + Na3V04 + GSH 266 Basal + VOSO, + GSH 111 Basal + MnC12 + GSH 84 Basal + Na2W04 105 Basal + Na2W04 + GSH 288 Basal + Na2W04 + Cysteine 268 Basal + K2Cr207 85 Basal + K2Cr207 + GSH 103 Basal + NbCl, 29 Basal + NbCl, + GSH 131 Basal + TaCl, 8 Basal + TaC15 + GSH 42 Basal + NazMo04 104 Basal + NazMo04 + GSH 114

Rabbit light microsomes were prepared as described under “Ex- perimental Procedures.” Type IV PDE activities were determined with 0.25 PM cAMP substrate. Transition metal compounds (1.4 mM) were mixed with reduced glutathione (2.8 mM) before addition to the membranes. Values are percentage of unstimulated activity calculated using the means from triplicate determinations.

VO. 2GSH activated form in liver membranes showed drug sensitivity profiles and ICso values that are characteristics of the cGMP-inhibitable form of the enzyme in several tissues (48). These data support the conclusion that VO . 2GSH complex activation in a cell-free system and insulin treatment in intact cells lead to an activation of the same liver microsomal enzyme. VO. 2GSH complex activatability is another distin- guishing property of Type IV PDE enzyme forms in hepatic microsomal membranes.

Electron paramagnetic spectra have shown that vanadium is in the V4+ valence state or vanadyl ion when complexed with GSH (11). The results of the current studies indicate a requirement for a free -SH group in order to form an active complex. Thus, active compounds from glutathione to cysteamine served as electron donors in forming an active vanadyl complex. Since both mercaptoethanol and DTT do not form active complexes with sodium vanadate, the amino group of cysteine or cysteamine must also play some critical role in complex formation. Also, homocysteine, which contains an additional carbon between the -NH2 and -SH groups, does not form enzyme activating complexes, suggesting an important size requirement for an active complex. In this Tegard histidine may also serve as an electron donor for active

Vanadyl/Glutathione Activatable Type I V PDE 17017

vanadyl complex formation. With respect to the electron acceptor portion of the complex, transition metals in the vanadium element group 5 are tolerated. However, inhibition of basal activity by these substances complicates the interpre- tation of the results.

The ratio of vanadyl ion to oxidized glutathione or cysteamine in the complex as determined indirectly by enzyme activation analysis was 1:2. This value is consistent with the cationic V02+ chemistry of the vanadyl ion at physiological pH (1). A minimum structure of the active complex that would be consistent with the data from these studies and literature reports is NH2-CH2-CH2-S-VO-S-CH2-CH2- NH,. HPLC or Sep-Pak C18 purification of the complex reported here will allow further and more precise chemical characterization of these novel complexes.

Kono et al. (30) proposed a model for adipocyte high affinity cAMP PDE wherein the insulin-activated enzyme is stabilized by critical thiol oxidation. The data reported here on the activation of the liver enzyme by VO . RSH complexes suggest that it is the oxidation process itself that produces an active state of the enzyme. Loten and Redshaw-Loten (31) have reported stimulation of high affinity cAMP catalysis of liver membranes and adipocytes by low concentrations of organic mercurial compounds and inhibition of the enzyme activity by high concentrations of these compounds. In addition, particulate Type IV PDE stimulated by incubation of adipocytes with insulin is inhibited by DTT without affecting basal membrane cAMP hydrolysis (30). Since insulin and vanadyl- sulfhydryl complexes both activate the cGMP-inhibited form of Type IV PDE and their effects on catalysis are similar, VO. 2GSH complexes may activate Type IV PDE by a com- mon pathway with insulin-activated receptors as suggested for glucose transport, anti-lipolysis, and glucose oxidation in adipocytes (7, 8).

A working model to explain the data on activation of liver microsomal cGMP-inhibitable-Type IV PDE by vanadyl- sulfhydryl complexes in vitro is given in Fig. 10. The membrane intermediate component of the vanadyl-sulfhydryl complex Type IV PDE activation process may involve a sulfhydryl reductase system. This system would provide an electron acceptor system that promotes electron transfer from the complex to the enzyme and in the process a critical thiol structure of the enzyme is oxidized producing the active state of the enzyme with an associated increased maximum velocity and sensitivity to cGMP inhibition. Alternatively, the complex may serve as an allosteric activator of a flavin-based sulfhydryl reducing system to achieve the same end result. Further studies will be directed towards the isolation and

(:Ee Affinity Hi?

FIG. 10. A working model of the insulin-like vanadyl- sulfhydryl complex activation of rabbit liver microsome Type IV PDE activity. The model hypothesizes that microsomal Type IV PDE is activated by an electron transfer mechanism. A membrane sulfydryl reductase system serves as an electron acceptor from the complex or may be activated by an allosteric action of the complex to achieve an enzyme-critical thiol oxidation. The activation of this form of Type IV PDE results in an increased V,,, for high affinity cAMP hydrolysis and a 21-fold increased sensitivity to cGMP inhibition.

reconstitution of this intermediate component. Since phosphorylation-dephosphorylation mechanisms play an important role in insulin activation of liver and adipocyte membrane forms of Type IV PDE (25, 26, 29), understanding the role of cGMP-inhibitable Type IV PDE protein phosphorylation in this process will be important.

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Activation of Rabbit Liver High Affinity cAMP (Type IV