Sodium nitroprusside-induced phosphorylation by GMP · subjected to electrophoresis at 5,600-6,400...

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Proc. Natl Acad. Set. USA Vol. 79, pp. 6470-6474, November 1982 Biochemistry Sodium nitroprusside-induced protein phosphorylation in intact rat aorta is niincked by 8-bromo cyclic GMP (cyclic GMP/cyclic AMP/relaxation/smooth muscle/isoproterenol) ROBERT M. RAPOPORT, MARTIN B. DRAZNIN, AND FERID MURAD Department of Medicine and Pharmacology, Palo Alto Veterans Administration Medical Center, Stanford University, 3801 Miranda Avenue, Palo Alto, California 94304 Communicated by Alfred Gilman, July 30, 1982 ABSTRACT The effects of sodium nitroprusside, 8-bromo cyclic GMP, 8-bromoguanosine 5'-monophosphate, 8-bromo cyclic AMP, dibutyryl cyclic AMP, and isoproterenol on incorporation of 32P into proteins in intact rat thoracic aorta were studied. Aor- tas were incubated in [32P]orthophosphate in order to label en- dogenous adenosine triphosphate. Agents were then added for various times and the tissues were homogenized and fractionated (100,000 x g for 60 min) into soluble and particulate fractions. Soluble and particulate fractions were subjected to isoelectric fo- cusing followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiographs were made. Nitroprusside induced a concentration-dependent increase in incorporation of 32p into nine proteins and a decrease in 32P incorporation into two proteins. Some of these proteins appeared in both the soluble and particulate fractions of homogenates; others appeared only in the soluble fraction. The pattern of 32p incorporation was identical after 2- or 15-min exposure to nitroprusside and was mimicked by exposure to 50-500 p.M 8-bromo cyclic GMP. 8-Bromoguanosine 5'-monophosphate did not alter 32p incorporation. Dibutyryl cyclic AMP at 50 jaM had no effect upon 32p incorporation whereas a higher concentration (0.5 mM) caused increased or decreased 32P incorporation into some, but not all, of the same proteins. 8- Bromo cyclic AMP (5 mM) produced only small changes in 32P in- corporation. The pattern of 32P incorporation induced by a rela- tively high concentration of isoproterenol 0.1 mM was similar but not identical to that seen with 0.5 mM dibutyryl cyclic AMP. The present study indicates that the incorporation of 32p into endog- enous proteins of intact rat aorta can be regulated by nitroprus- side. These effects can be mimicked by cyclic GMP analogues and only partially by cyclic AMP analogues or isoproterenol. Presum- ably, these effects of nitroprusside are mediated through a cyclic GMP-dependent process (protein kinase or phosphatase) which may play a role in the relaxant properties of nitroprusside and cyclic GMP. The present study investigates the hypothesis that smooth mus- cle relaxation induced by sodium nitroprusside may be me- diated by cyclic GMP-dependent phosphorylation of endoge- nous proteins. Increased levels of cyclic GMP have been observed in response to sodium nitroprusside in various tissues including smooth muscle preparations (1-4). Cyclic GMP an- alogues have also been shown to cause relaxation or decreased contraction in smooth muscle (1, 5-7). Furthermore, agents that inhibit nitroprusside activation of guanylate cyclase in broken cell preparations have been shown to inhibit relaxation as well as nitroprusside-induced increased levels of cyclic GMP (6-9). However, the molecular mechanism by which nitroprusside and cyclic GMP induce relaxation is not known. Protein phos- phorylation is thought to be a final common pathway for many biological processes (10). The addition of cyclic GMP to crude particulate fractions of various smooth muscle preparations spe- cifically increased 32p incorporation into several proteins with molecular weights of 75,000, 85,000, 100,000, 130,000, and 250,000 (11-13). In these studies the soluble fraction did not contain any substrates specifically phosphorylated by cyclic GMP. However, to date, no studies have been reported on the effect of nitroprusside or cyclic GMP analogues on 32p incor- poration into- proteins in intact cell preparations including smooth muscle. The present study with intact rat thoracic aorta demonstrates that nitroprusside can alter the incorporation of 32P into pro- teins from soluble and particulate fractions. Furthermore, the pattern of altered protein phosphorylation evaluated with two- dimensional gel electrophoresis was mimicked by 8-bromo cyclic GMP. Approximately 100- and 10-fold greater concen- trations of 8-bromo cyclic AMP and dibutyryl cyclic AMP, re- spectively, were required in order to increase 32P incorporation into certain proteins whose phosphorylation was altered by 8- bromo cyclic GMP or nitroprusside. Isoproterenol also in- creased 32p incorporation into some proteins but not as much as nitroprusside or 8-bromo cyclic GMP did. The incorporation of 32P into some proteins was preferentially increased by di- butyryl cyclic AMP. The changes we observed with protein phosphorylation in fractions obtained from intact rat thoracic aorta could result from altered activities of a protein kinase or a phosphoprotein phos- phatase or both. Although the results presented cannot distin- guish between these mechanisms, it can be concluded that ni- troprusside-induced 32p incorporation into various proteins is mimicked by cyclic GMP analogues and only partially by cyclic AMP analogues or isoproterenol. Presumably these nitroprus- side effects are cyclic GMP-dependent. Furthermore, the phosphorylation state of one or more of these proteins may be coupled to the relaxant effects of nitrovasodilators and cyclic nucleotides. Some of these observations have been presented in abstract form (14). METHODS Preparation of Rat Thoracic Aorta Strips. Thoracic aortas were rapidly removed from decapitated male 250- to 300-g Sprague-Dawley rats and placed in Krebs-Ringer bicarbonate solution which was gassed with 95% 02/5% CO2 and had the following composition (mM): NaCl, 118.5; KC1, 4.74; MgSO4, 1.18; KH2PO4, 1.18; CaCl2, 2.5; NaHCO3, 24.9; and glucose, 10. Spiral strips were prepared by the method of Furchgott and Bhadrakom (15). The intimal surface was rubbed to remove the endothelial layer, and strips were cut into approximately 1.5-cm segments. Segments derived from three aortas were incubated in 2.5 ml of low-phosphate (0.1 mM KH2PO4) Krebs-Ringer bicarbonate solution under an atmosphere of 95% 02/5% C02. 6470 The publication costs of this article were defrayed in part, by page charge payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Downloaded by guest on August 1, 2020

Transcript of Sodium nitroprusside-induced phosphorylation by GMP · subjected to electrophoresis at 5,600-6,400...

Page 1: Sodium nitroprusside-induced phosphorylation by GMP · subjected to electrophoresis at 5,600-6,400 Vhras described byO'Farrell (19). Nonequilibrium pHgradientelectrophoresis (20)

Proc. Natl Acad. Set. USAVol. 79, pp. 6470-6474, November 1982Biochemistry

Sodium nitroprusside-induced protein phosphorylation in intact rataorta is niincked by 8-bromo cyclic GMP

(cyclic GMP/cyclic AMP/relaxation/smooth muscle/isoproterenol)

ROBERT M. RAPOPORT, MARTIN B. DRAZNIN, AND FERID MURADDepartment of Medicine and Pharmacology, Palo Alto Veterans Administration Medical Center, Stanford University, 3801 Miranda Avenue, Palo Alto,California 94304

Communicated by Alfred Gilman, July 30, 1982

ABSTRACT The effects of sodium nitroprusside, 8-bromocyclic GMP, 8-bromoguanosine 5'-monophosphate, 8-bromo cyclicAMP, dibutyryl cyclic AMP, and isoproterenol on incorporationof 32P into proteins in intact rat thoracic aorta were studied. Aor-tas were incubated in [32P]orthophosphate in order to label en-dogenous adenosine triphosphate. Agents were then added forvarious times and the tissues were homogenized and fractionated(100,000 x g for 60 min) into soluble and particulate fractions.Soluble and particulate fractions were subjected to isoelectric fo-cusing followed by sodium dodecyl sulfate/polyacrylamide gelelectrophoresis and autoradiographs were made. Nitroprussideinduced a concentration-dependent increase in incorporation of32p into nine proteins and a decrease in 32P incorporation into twoproteins. Some of these proteins appeared in both the soluble andparticulate fractions of homogenates; others appeared only in thesoluble fraction. The pattern of 32p incorporation was identicalafter 2- or 15-min exposure to nitroprusside and was mimicked byexposure to 50-500 p.M 8-bromo cyclic GMP. 8-Bromoguanosine5'-monophosphate did not alter 32p incorporation. DibutyrylcyclicAMP at 50 jaM had no effect upon 32p incorporation whereasa higher concentration (0.5 mM) caused increased or decreased32P incorporation into some, but not all, of the same proteins. 8-Bromo cyclic AMP (5 mM) produced only small changes in 32P in-corporation. The pattern of 32P incorporation induced by a rela-tively high concentration of isoproterenol 0.1 mM was similar butnot identical to that seen with 0.5 mM dibutyryl cyclic AMP. Thepresent study indicates that the incorporation of 32p into endog-enous proteins of intact rat aorta can be regulated by nitroprus-side. These effects can be mimicked by cyclic GMP analogues andonly partially by cyclic AMP analogues or isoproterenol. Presum-ably, these effects of nitroprusside are mediated through a cyclicGMP-dependent process (protein kinase or phosphatase) whichmay play a role in the relaxant properties of nitroprusside andcyclic GMP.

The present study investigates the hypothesis that smooth mus-cle relaxation induced by sodium nitroprusside may be me-diated by cyclic GMP-dependent phosphorylation of endoge-nous proteins. Increased levels of cyclic GMP have beenobserved in response to sodium nitroprusside in various tissuesincluding smooth muscle preparations (1-4). Cyclic GMP an-alogues have also been shown to cause relaxation or decreasedcontraction in smooth muscle (1, 5-7). Furthermore, agents thatinhibit nitroprusside activation of guanylate cyclase in brokencell preparations have been shown to inhibit relaxation as wellas nitroprusside-induced increased levels of cyclic GMP (6-9).

However, the molecular mechanism by which nitroprussideand cyclic GMP induce relaxation is not known. Protein phos-phorylation is thought to be a final common pathway for many

biological processes (10). The addition of cyclic GMP to crudeparticulate fractions ofvarious smooth muscle preparations spe-cifically increased 32p incorporation into several proteins withmolecular weights of 75,000, 85,000, 100,000, 130,000, and250,000 (11-13). In these studies the soluble fraction did notcontain any substrates specifically phosphorylated by cyclicGMP. However, to date, no studies have been reported on theeffect of nitroprusside or cyclic GMP analogues on 32p incor-poration into- proteins in intact cell preparations includingsmooth muscle.The present study with intact rat thoracic aorta demonstrates

that nitroprusside can alter the incorporation of 32P into pro-teins from soluble and particulate fractions. Furthermore, thepattern of altered protein phosphorylation evaluated with two-dimensional gel electrophoresis was mimicked by 8-bromocyclic GMP. Approximately 100- and 10-fold greater concen-trations of 8-bromo cyclic AMP and dibutyryl cyclic AMP, re-spectively, were required in order to increase 32P incorporationinto certain proteins whose phosphorylation was altered by 8-bromo cyclic GMP or nitroprusside. Isoproterenol also in-creased 32p incorporation into some proteins but not as muchas nitroprusside or 8-bromo cyclic GMP did. The incorporationof 32P into some proteins was preferentially increased by di-butyryl cyclic AMP.The changes we observed with protein phosphorylation in

fractions obtained from intact rat thoracic aorta could result fromaltered activities of a protein kinase or a phosphoprotein phos-phatase or both. Although the results presented cannot distin-guish between these mechanisms, it can be concluded that ni-troprusside-induced 32p incorporation into various proteins ismimicked by cyclic GMP analogues and only partially by cyclicAMP analogues or isoproterenol. Presumably these nitroprus-side effects are cyclic GMP-dependent. Furthermore, thephosphorylation state of one or more of these proteins may becoupled to the relaxant effects of nitrovasodilators and cyclicnucleotides. Some of these observations have been presentedin abstract form (14).

METHODSPreparation of Rat Thoracic Aorta Strips. Thoracic aortas

were rapidly removed from decapitated male 250- to 300-gSprague-Dawley rats and placed in Krebs-Ringer bicarbonatesolution which was gassed with 95% 02/5% CO2 and had thefollowing composition (mM): NaCl, 118.5; KC1, 4.74; MgSO4,1.18; KH2PO4, 1.18; CaCl2, 2.5; NaHCO3, 24.9; and glucose,10. Spiral strips were prepared by the method ofFurchgott andBhadrakom (15). The intimal surface was rubbed to remove theendothelial layer, andstrips were cut into approximately 1.5-cmsegments. Segments derived from three aortas were incubatedin 2.5 ml of low-phosphate (0.1 mM KH2PO4) Krebs-Ringerbicarbonate solution under an atmosphere of95% 02/5% C02.

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The publication costs ofthis article were defrayed in part, by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Proc. NatL. Acad. Sci. USA 79 (1982) 6471

Incubation with [32P]Orthophosphate and Agents. Two to2.5 mCi (1 Ci = 3.7 X 1010 becquerels) of carrier free [32p]-orthophosphoric acid (New England Nuclear) was added to eachflask, and tissues were incubated for 2 hr at 370C in a shakingwater bath. Agents (all obtained from Sigma) were then addedand the incubations were continued for 2 or 15 min. Incubationswere terminated by pouring samples onto a nylon gauze on avacuum filtration apparatus in order to remove the buffer, andthen the tissues were rapidly frozen in liquid nitrogen.

Homogenization of Muscle Strips. The frozen segmentswere transferred to 0.5 ml of ice-cold stopping buffer (100 mMNaF/80 mM sucrose/10 mM EDTA/10 mM N-[tris(hydroxy-methyl)methyl]aminoethanesulfonic acid, pH 7.4). Strips werehomogenized in this stopping buffer to minimize protein phos-phorylation or dephosphorylation during homogenization andprocessing (16). The inclusion of 2 mM phenylmethylsulfonylfluoride as a protease inhibitor in the homogenization bufferdid not alter the autoradiogram profile and was not routinelyused. Homogenates were centrifuged at 105,000 X g for60 min.Supernatant fractions were removed and the pellets were re-suspended in 0.5 ml of fresh homogenization buffer. Prior toelectrophoresis, aliquots ofthe samples were assayed for 32P andprotein concentration by the method of Lowry et al. (17) andadjusted with homogenization buffer so that the 32P and proteinconcentrations were equivalent in all supernatant fractions orin all of the particulate fractions.Two-Dimensional Gel Electrophoresis and Autoradiogra-

phy. Samples were heated (980C) in 2% sodium dodecyl sulfate/5% 2-mercaptoethanol/10% (vol/vol) glycerol for 15 min,cooled, and brought to 9.5 M in urea (Schwarz/Mann, ultrapuregrade), 9% in Nonidet P-40 (Sigma), and 2% in Ampholines (Bio-Rad) after the method ofAnderson and Anderson (18). The ratioofpH 3-10 to 5-7 Ampholines was 1:1. Fifty to 150 ,ug of pro-tein was loaded onto each 2.5 x 130 mm tube gel containing3% polyacrylamide, 3% Nonidet P-40, and 2% Ampholines andsubjected to electrophoresis at 5,600-6,400 V hr as describedby O'Farrell (19). Nonequilibrium pH gradient electrophoresis(20) did not yield additional proteins of interest. The pH gra-dients (approximately pH 8 to 4) were measured with a directelectrode (Ingold) on gels run in parallel without sample. Thesecond dimension of electrophoresis was as described byO'Farrell (19) on 11% polyacrylamide separating gels. Silverstaining (21) of the two-dimensional gel slabs was performed toverify that the patterns and amounts of protein were the samewithin the supernatant or particulate samples. Gels were driedonto filter paper and autoradiographs were prepared (KodakMin-R).

Typically, four tissue incubations (control and three experi-mental conditions) were performed for each experiment. A totalof 35 experiments were performed and each condition was ex-amined in 3-35 individual incubations. The photographs pre-sented are representative of the changes observed. Unfortu-nately, in some instances the obvious changes in 32P incorporationvisualized on the autoradiograms were not as apparent on thephotographs. We have confirmed the changes in phosphoryl-ated proteins by using a video camera scanning system with ananalog-to-digital converter and a microcomputer as developedby Mariash et al. (22).

RESULTSTypical autoradiographs of control supernatant and particulatefractions are illustrated in Fig. 1. Three regions (designatedgroup 1, group 2, and group 3) ofthe autoradiographs containedphosphorylated proteins of interest. The alterations in 32p in-corporation in the proteins of groups 1 and 3 were observed in

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FIG. 1. Autoradiographs of soluble (Upper) and particulate (Lower)fractions of rat thoracic aorta after two-dimensional gel electropho-resis. Numbered boxes refer to areas where alterations in incorpora-tion of 32P were observed.

both soluble and particulate fractions, even when the particu-late fraction was washed twice with homogenization buffer. Thechanges observed in the proteins of group 2 with nitroprussidewere only observed with soluble fractions. The phosphorylatedproteins of groups 1 and 2 represented in Figs. 2-6 are fromthe supernatant fraction; those of group 3 are from the partic-ulate fraction. For the high molecular weight regions ofthe gels,differences in 32P-incorporation into proteins under the ex-perimental conditions examined were not apparent because ofthe poor resolution achieved.

Exposure of rat thoracic aorta to 50 nM or 0.5 ,uM sodiumnitroprusside for 2 min resulted in a concentration-dependentincrease in 32p incorporation into various proteins designatedla, lb, and Ic and 2a-2f (Fig. 2). Smaller increases in 32p in-corporation into these proteins were also observed with 5 nMnitroprusside (data not shown). Incubation with nitroprussidefor 15 min resulted in an identical pattern of increased 32p in-corporation (Fig. 3). Nitroprusside also resulted in decreased32P incorporation into proteins designated 3a and 3b (Fig. 4).The patterns of 32p incorporation were not altered when frac-tions were precipitated with 6% trichloroacetic acid and thenresuspended prior to electrophoresis. The profiles ofaltered 32pincorporation into proteins of groups 1 and 2 induced by nitro-prusside, cyclic nucleotide analogues, or isoproterenol were notaltered when the tissues were contracted in response to either

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6472 Biochemistry: Rapoport etalP

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FIG. 2. Effect of sodium nitroprusside (SNP) on 32p incorporation.Tissues were incubated with or without nitroprusside for 2 min. Two-dimensional gel electrophoresis and autoradiography were then per-formed. "Group" represents an area of the autoradiograph' as desig-nated in Fig. 1.

0.3 1ttM norepinephrine or 30 mM KC1 (data not shown).8-Bromo cyclic GMP (50 to 500 /iM) produced a pattern of

32p incorporation qualitatively identical to that observed withnitroprusside (Figs. 3-5). Furthermore, the effects of 8-bromocyclic GMP were also concentration dependent (Fig. 5). 8-Bromoguanosine 5'-monophosphate (1 mM) was without effect(data not shown).

Dibutyryl cyclic AMP at 0.5mM increased 32p incorporationinto proteins la, lb, and lc and the effects were similar to thosewith 50 /iM 8-bromo cyclic GMP (Fig. 5). Dibutyryl cyclicAMPat 0.5 mM had no effect on protein 2a but the effects on 32pincorporation into proteins 2b and 2c were greater than with0.5 mM 8-bromo cyclic GMP. Increased 32p incorporation intoproteins 2d, 2e, and 2fwas similar with either cyclic nucleotide.Dibutyryl cyclic AMP resulted in decreased 32p incorporatedinto protein 2g (Fig. 5). Dibutyryl cyclic AMP at 50 /iM hadlittle or no effect on 32p incorporation (data not shown). 8-Bromo

FIG. 4. Effect of sodium nitroprusside (SNP) and 8-bromo cyclicGMP (8-BcGMP) on 32P incorporation in group 3 proteins. Tissues wereincubated for 15 min.

cyclic AMP at 5 mM slightly increased 32p incorporation intoproteins la, lb, 1c, 2b, and 2c; no effects were observed on theother proteins.

Isoproterenol at 10 AiM increased 32p incorporation into pro-teins 2b, 2c, 2d, 2e, 2f, and 2h (Fig. 6). At 10 or 100 uM, iso-proterenol did not alter 32p incorporation into proteins 2a and2g; the increased 32p in proteins la, lb, and lc was less thanthe effects of nitroprusside. The proteins with altered 32p in-corporation in these studies are summarized in Table 1.

DISCUSSIONThe present results demonstrate that sodium nitroprusside al-ters 32p incorporation into various soluble and particulate pro-teins in intact aorta preparations. The effects of nitroprussideon protein phosphorylation were similar at 2 and 15 min. Ourprevious studies showed that nitroprusside increases cyclicGMP accumulation in aorta at these times, when relaxation isalso observed (6, 7). Furthermore, the concentrations of nitro-prusside required for cyclic GMP accumulation, relaxation, and32P incorporation are similar (6, 7). The effects of nitroprussideon both relaxation (6, 7) and protein phosphorylation in thepresent study are mimicked by 8-bromo cyclic GMP. The al-

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FIG. 5. Effect of 8-bromo cyclic GMP (8-BcGMP) and dibutyrylcyclic AMP (DBcAMP) on 32p incorporation. Tissues were incubatedfor 15 min.

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Proc. Nad Acad. Sci. USA 79 (1982)

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Proc. NatL Acad. Sci. USA 79 (1982) 6473

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FIG. 6. Effect of 0.5 1uM sodium nitroprusside (SNP) and 10 ALMand 0.1 mM isoproterenol (ISO) on 32p incorporation. Tissues were in-cubated for 2 min. (Left) Group 1. (Right) Group 2.

tered protein phosphorylation with nitroprusside appears to bemediated through a cyclic GMP-dependent process because 8-bromo cyclic GMP was considerably more effective than cyclicAMP analogues or isoproterenol in reproducing the pattern of32p incorporation. A correlation exists between the potency ofthe agents examined to cause aorta relaxation and incorporation

Table 1. Effect of sodium nitroprusside (SNP), 8-bromo cyclicGMP (8-BcGMP), dibutyryl cyclic AMP (DBcAMP), andisoproterenol (Iso) on incorporation of 32P

32p incorporationMr SNP 8-BcGMP DBcAMP Iso

Protein x 10-3 pI (0.5 MM) (0.5 mM) (0.5 mM) (10 AtM)la 49.0 7.8 T tt T Tlb 49.0 7.4 T T t T Tlc 49.0 7.1 T T I -2a 28.0 6.8 T T - -2b 24.0 6.5 T T T T T2c 24.0 6.2 T T T 1 T2d 21.5 6.6 T T T T2e 21.7 6.4 1 T t T2f 21.3 6.4 1 T T2g 24.0 6.6 - - 42h 25.0 6.7 - - - T3a 24.0 5.3 4 43b 24.0 5.1 4 4 - -

Proteins whose 32p incorporation was altered are identified by thenumbered area in which they were observed in the autoradiographs(1, 2, or 3; see Fig. 1) and a letter assigned to each protein (see Figs.2-6). Increased, decreased, and no change in incorporation are rep-resented by l, 4, and -, respectively. The concentrations of agentsindicated were those that would be maximally effective in relaxing ratthoracic aorta contracted with 0.3 ,uM norepinephrine. However, in theabove experiments the tissues were not contracted.

of 32P into proteins la, lb, lc, and 2a. The rank order of po-tency (EC50) to induce relaxation is 8-bromoguanosine 5'-mono-phosphate >8-bromo cyclic AMP> dibutyryl cyclicAMP> 8-bromo cyclic GMP > nitroprusside (unpublished data). Theorder ofpotency is similar when 32p incorporation into proteinsla, lb, lc, and 2a is ranked.

Altered 32p incorporation into these and other proteins couldresult from changes in the activity of a kinase(s) (presumablycyclic nucleotide-dependent kinase) or phosphoprotein phos-phatase(s) and these alternatives cannot be distinguished fromthe present studies. Furthermore, each of these cyclic nucleo-tides (cyclic AMP or cyclic GMP) can activate both cyclic nu-cleotide-dependent protein kinases, although generally a 50-to 100-fold greater concentration is required with the nonpre-ferred kinase (for a review, see ref. 23). Although the mecha-nism of the nitroprusside effect on endogenous protein phos-phorylation is unknown, it can be concluded that the effects ofnitroprusside are best mimicked with cyclic GMP analogues.The ability of higher concentrations of cyclic AMP analoguesor isoproterenol to reproduce some but not all of the effects ofnitroprusside indicate that high concentrations of cyclic AMP(exogenous or generated) can mimic some of the effects ofcyclicGMP through either effects on cyclic AMP- or cyclic GMP-de-pendent protein kinase or effects ofcyclic AMP-dependent pro-tein kinase on common protein substrates.

Isoproterenol at 10 gM causes maximal relaxation of nor-epinephrine-induced (0.3 p.M) contraction of rat thoracic aorta(unpublished data). Isoproterenol at 10 or 100 ,uM produced apattern of 32p incorporation different from that observed withconcentrations of nitroprusside or 8-bromo cyclic GMP whichalso produce maximal relaxation. These studies with intact aortaindicate that there are functionally different and specific proteinsubstrates for cyclic GMP-dependent protein kinase and cyclicAMP-dependent protein kinase. Some differences were alsonoted in the proteins phosphorylated with isoproterenol anddibutyryl cyclic AMP. Isoproterenol increased 32p incorpora-tion into protein 2h whereas dibutyryl-cyclic AMP decreased32P incorporation into protein 2g. The significance of these dif-ferences is not known. However, these experiments indicatethat the effects of generated or endogenous cyclic AMP andexogenous cyclic AMP can be strikingly different. These effectsmay be due to the analogue examined, but the difference maybe due to the compartmentation or availability of the cyclicAMP.The identities of the proteins whose 32p incorporation was

altered are not known. Preliminary studies with bovine trachealsmooth muscle show that several ofthe proteins phosphorylatedby nitroprusside and 8-bromo cyclic GMP have pI values andmolecular weights remarkably similar to some of these foundin the present study (unpublished data). Some of the proteinsdescribed here with the same molecular weight and somewhatdifferent isoelectric points may represent a single protein withvariable amounts of phosphate. Thus, the number of proteinswith altered phosphate incorporation is unknown at present.Others have described protein substrates from particulate frac-tions of various smooth muscle preparations specifically phos-phorylated by cyclic GMP (10-12). However, all of the phos-phorylated proteins observed by those investigators were of ahigher molecular weight than those observed in the presentstudy. Furthermore, in the present study, many ofthe proteinswhose incorporation of 32P was altered appeared only in thesoluble fraction. This apparent discrepancy may be explainedby the fact that the present study was performed with wholetissue, whereas other studies utilized broken cell preparations.A soluble 23,000-dalton protein has also been described in prep-arations ofcerebellum (24). This protein appears to be a specific

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6474 Biochemistry: Rapoport et al

substrate for cyclic GMP-dependent protein kinase (24-26).However, this protein has been found primarily in nervous tis-sue and is most abundent in Purkinje cells of cerebellum (27).

Nitroprusside and 8-bromo cyclic GMP decreased 32p in-corporation into proteins 3a and 3b. These proteins may bemyosin light chain, although this remains to be tested. Others(for a review, see ref. 28) have shown that relaxation is accom-panied by decreased phosphorylation of myosin light chain.Barron et al (29) have shown that theophylline decreased theamount of [32P]phosphate in myosin light chain in pig carotidartery under resting tension. Thus, one might speculate thatrelaxation induced by nitroprusside, 8-bromo cyclic GMP, di-butyryl cyclic AMP, and isoproterenol may also involve the ac-tivity ofmyosin light chain kinase and the phosphorylation stateof myosin light chain.

These studies support our hypothesis that the effects of ni-troprusside and probably other nitrovasodilators on smoothmuscle relaxation are due to guanylate cyclase activation andcyclic GMP accumulation (1, 2, 8). This hypothesis could beextended to involve the cyclic GMP-dependent phosphoryl-ation (or dephosphorylation) of one or more of the describedproteins which in some manner is involved in the contraction/relaxation process. However, which, if any, of the phosphoryl-ated proteins described in the present study is related to therelaxant effects ofnitroprusside and cyclic GMP is unknown andadditional experiments are required.

Note Added in Proof. In other experiments in which tissue was con-tracted with 0.3 AM norepinephrine, all of the relaxants used in thepresent study (nitroprusside, 8-bromo cyclic GMP, dibutyryl cyclicAMP, and isoproterenol) decreased phosphorylation of proteins 3a and3b.

We thank Gilberto A. Martinez for his skillful assistance. The adviceand help of Dr. James C. Garrison in developing the two-dimensionalgel methods and reviewing the findings are gratefully acknowledged.This work was supported by Research Grants AM 30787 and HL 28474from the National Institutes of Health, a grant from the VeteransAdministration, grants from the Council for Tobacco Research-USA,Inc., and National Research Service Award GM 07673 (to R. M.R.).

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Proc. Nad Acad. Sci. USA 79 (1982)

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