Phorbol Ester- and Diacylglycerol-Mediated Desensitization of Cardiac /ft-adrenergic Receptors

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1 443 Phrbl Ester- and Diacylglycerl-Mediated Desensitizatin f Cardiac /ft-adrenergic Receptrs Cnstantins J. limas and Catherine limas Frm the Departments f Medicine (Cardivascular Divisin), and Labratry Medicine and Pathlgy, University f Minnesta Schl f Medicine, Minneaplis, Minnesta SUMMARY. There are specific phrbl ester receptrs n cardiac mycytes which may be identical with the calcium/phsphlipid-dependent prtein kinase (prtein kinase C). Incubatin f enzymatically dissciated rat cardiac mycytes with bilgically active phrbl esters (such as 40- phrbl-12,13-dibutyrate and 12-O-tetradecanyl phrbl- 13-acetate) leads t a time- and cncentratin-dependent lss f js-adrenergjc receptrs detectable with the hydrphilic ligand [ 3 H]- CGP This lss is attributable t a reductin in bth maximal /S-receptr numbers and their affinities. The synthetic diacylglycerl, l-leyl-2-acetyldiglycerl, which is knwn t activate prtein kinase C, als induces desensitizatin f 0-receptrs. Bth phrbl diburyrate and 1-leyl- 2-acetyldiglycerl have additive effects t isprterenl, suggesting a separate site f actin in prmting 0-receptr desensitizatin. The effects f phrbl diburyrate and l-leyl-2-acetyldiglycerl are prevented by clchicine (but nt its inactive analg, trimethylclchicinic acid), indicating a micrtubule dependence. The lss f membrane-bund /3-receptrs after phrbl dibutyrate- r l-leyl-2-acetyldiglycerl preincubatin is accmpanied by an increase in /3- receptrs assciated with a cytsl-derived vesicular fractin devid f plasma membrane markers, a finding cnsistent with an intemalizatin prcess. These results suggest that prtein kinase C activatin by diacylglycerls derived frm receptr-linked phsphinsitide hydrlysis may be a nvel mechanism f cardiac /3-receptr desensitizatin. (Circ Res 57: , 1985) Dwnladed frm by n January 7, 2019 CONSIDERABLE attentin has been fcused recently n the actins f phrbl esters n nnprliferating cells. Althugh fr many years these substances have been studied as prmters f tumr grwth, it has becme evident that they have imprtant additinal effects, including mdificatin f hrmne-receptr interactins (Garte and Belman, 1980; Jacbs et al., 1983; May et al., 1984; Kelleher et al., 1984; Sibley et al., 1984). It is thught that the bilgical effects f phrbl esters are mediated thrugh their binding t specific receptrs which cpurify with, and may be identical t, the Ca ++ / phsphlipid-dependent prtein kinase C (Leach et al., 1983; Kikkawa et al., 1983; Niedel et al., 1983). Phrbl esters substitute fr unsaturated diacylglycerls, the endgenus activatrs f prtein kinase C, in lwering the Ca ++ requirements fr enzymatic activity (Kikkawa et al., 1983). We have recently identified and characterized specific phrbl ester receptrs n islated rat cardiac mycytes (limas, 1985). The bilgical significance f these receptrs relates t the functinal cnsequences f prtein kinase C activatin. This enzyme is present in the mycardium (limas, 1980; Wise et al., 1982) and has been shwn t influence Ca ++ transprt by the cardiac sarcplasmic reticulum and the sarclemma thrugh phsphlamban phsphrylatin (limas, 1980; Mvsesian et al., 1984; Iwasa and Hsey, 1984). Since mst f the prtein kinase C activity in nnstimulated cardiac cells is fund in the cytslic fractin and is assciated with the membranes nly after activatin, e.g., by phrbl esters and diacylglycerls, the latter substances are useful prbes f the bilgical actins f prtein kinase C in intact cells. In the present study, we reprt that phrbl esters and a synthetic diacylglycerl, l-leyl-2-acetyl diglycerl (OADG), induce heterlgus desensitizatin f cardiac /9-adrenergic receptrs by a mechanism that is micrtubule-dependent and may invlve intemalizatin f the receptrs. Methds Experiments were carried ut n cardiac mycytes islated frm adult Sprague-Dawley rats by a prcedure described previusly (limas and limas, 1984). Rat ventricles were minced fine, and washed free f bld with cld (4 C) dissciatin buffer cntaining (g/liter): NaCl, 6.8; KC1, 0.4; NaH 2 PO 4, 0.06; Na 2 HPO 4, 0.21; and glucse, 0.9. Tissues then were incubated in 5 ml f buffer with 0.05 mg/ml cllagenase (CLS II, Wrthingtn) and 0.02 mg/ml hyalurnidase (Type IV, Sigma) at 37 C fr 10 minutes with shaking. The first dissciatin gave mstly erythrcytes and fragmented cells, and was discarded. The next three dissciatins were cmbined, and mycytes were pelleted at 60 g fr 10 minutes. They were washed nce with excess buffer befre use. Cardiac /3-receptrs were rutinely identified using the hydrphilic ligand (±) [ 3 H]CGP (4-[3-tertiary butylamin-2-hydrxyprpxy]-benzamidazl-2-n hydrchlride), which labels specifically cell-surface bund re-

2 Dwnladed frm by n January 7, Circulatin Research/Vl. 57, N. 3, September 1985 ceptrs (Staehelin and Hertel, 1983). Mycyte suspensins in 0.25 M sucrse-50 mm Tris-HCl (ph 7.4)-10mM MgCl 2 cntaining mg prtein were incubated with varying cncentratins ( ITM) f [ 3 H]CGP (Amersham/Searle; specific activity, 42.7 Ci/mml) in a ttal vlume f 0.5 ml. At 4 C, ( 3 H) CGP binding t cardiac mycytes prceeds rapidly fr the first 2 hurs and then mre slwly fr 4 hurs, after which it remains stable fr at least 16 hurs. At the end f the incubatin, a 10-fld excess f ice-cld sucrse-tris-mgcl 2 buffer was added t each tube, and the samples were filtered thrugh Whatman GF/C filters. The filters were washed three times with 5 ml f ice-cld buffer and were cunted in 10 ml f Aquasl-2 (New England Nuclear). Nnspecific binding was that detected in the presence3 f 1 JIM prpranll and, in the presence f 6 nm [ 3 H]CGP (6xK D ), averaged 15% f the ttal. T study the pssibility that phrbl esters and OADG induced 'dwn'-regulatin f cardiac ^-receptrs, we first incubated suspensins f mycytes in 0.25 M sucrse-50 mm Tris-HCl (ph 7.4)-10 HIM MgCl 2 at 37 C fr 30 minutes in the presence r absence f 40 nm phrbl dibutyrate (PDBu) r 20 jig OADG, and then fr 15 minutes with r withut 1 /tm /-isprterenl. At the end f the incubatin, the test tubes were transferred t an ice-water bath, a 10-fld excess f cld buffer was added t each tube, and the cells were washed three times by centrifuging at 1000 g fr 10 minutes at 4 C. T the washed resuspended cells, we added 6 nm [ 3 H]CGP , and cntinued the incubatin at 4 C fr 16 hurs befre filtering as abve. In sme experiments, isprterenl-mediated generatin f cyclic adensine mnphsphate (camp) by cardiac mycytes was studied. Mycytes first were incubated with r withut 1 /IM /-isprterenl at 37 C fr 15 minutes, then were washed with excess buffer and resuspended in 0.25 M sucrse-50 HIM9 Tris- HC1-5 mm MgCl 2 (ph 7.4). Incubatin with 10" 9 t 10" 5 M /-isprterenl was carried ut at 37 C fr 10 minutes in the presence f 0.2 mm 3-isbutyl-l-methylxanthine. Reactins were stpped by the additin f 2 ml f cld 7% trichlracetic acid (TCA), fllwed by centrifugatin at 2000 g fr 10 minutes. After extractin with ether, the amunt f camp in the supernatant was determined with the prtein-binding assay (Gilman, 1974). T study the distributin f (3-receptrs after phrbl dibutyrate r OADG, cardiac mycytes were first incubated fr 30 minutes at 37 C with 40 nm phrbl dibutytate r 20 /*g OADG while cntrls were incubated with slvent (DMSO) alne. After the cells had been washed with excess buffer, they were lysed in 1 mm Tris (ph 7.4)- 2 mm EDTA-0.1 mm PMSF fr 30 minutes at 0 C and disrupted with a Tefln pestle hmgenizer. The lysed cells were taken up in 10 mm Tris (ph 7.5)-154 mm NaCl- 5 mm MgCl 2 and were centifuged at 700 g fr 5 minutes. The supernatant was centrifuged at 40,000 g fr 30 minutes at 4 C t btain the crude membrane pellet and at 158,000 g fr 1 hur at 4 C t btain the vesicular fractin (pellet). Bth pellets were resuspended in 0.25 M sucrse- 50 mm Tris-HCl (ph 7.4)-5 mm MgCl^ and /3-receptrs were determined using 5 nm [ 3 H]dihydralprenll (New England Nuclear, specific activity, 110 Ci/mml), as previusly described {limas and limas, 1984). In sme experiments, /3-receptrs in the membrane and vesicular fractins were als determined using [ 3 H]CGP-12177, as described abve. T characterize these fractins further, we assayed Na +,K + -ATPase and 5'-nudetidase activities as described previusly (Limas and limas, 1984). When the effects f micrtubule-disrupting agents were studied, clchicine r trimethyl clchirinic acid was added t mycyte suspensins (10 fig/ml) and incubated at 37 C fr 15 minutes. Phrbl dibutyrate (40 nm) r OADG (20 ng/ ml) was then added, and the incubatin was cntinued fr anther 30 minutes at 37 C. Mycytes then were assayed fr 0-adrenergic receptrs using 6 nm [ 3 H]CGP , as described abve. Results Incubatin f enzymatically dissciated mycytes with PDBu results in a time-dependent decline f cardiac /3-receptrs identifiable with [ 3 H]CGP (Fig. 1); mst f this decline ccurs within the first 10 minutes f expsure t the phrbl ester. The decrease in the number f /3-receptrs detectable n cardiac mycytes with [ 3 H]CGP depends n the cncentratin f PDBu in the preincubatin buffer (Fig. 2). Cmparisn f different diesters shws that TPA is the mst effective, while 4-aphrbl and 4-a-phrbl-12,13 didecanate are inactive (Table 1). This parallels the relative bilgical ptency f these cmpunds in several cell systems. Scatchard plts f the binding data (Fig. 3) reveal that pretreatment f the cardiac mycytes with PDBu results in a substantial decline in the affinity f the /3-receptrs, in additin t a decrease in their ttal number. The synthetic diacylglycerl, OADG, which is knwn t activate prtein kinase C, als induces a cncentratin-dependent decline f cardiac /3-receptrs assayable with [ 3 H]CGP (Fig. 4) similar t that mediated by PDBu. Preincubatin with 80 nm PDBu + 20 Mg OADG induces a /3- receptr lss (50% f untreated cntrls) which is I DMSO PDBu ThmGnki) FIGURE 1. Time-curse f phrbl dibutyrate (PDBu)-induced lss f ^-receptrs detectable n cardiac mycytes with fhjccp Mycyte suspensins ( mg prtein) were incubated with either 40 nu PDBu (9) r DMSO alne (Q) fr the indicated length f time at 37 C were then transferred t an ice-water bath, 6 nu fhjcgp was added, and incubatin was cntinued fr 16 hurs at 4 C prir t filtratin, as described in the text. Results are expressed as percent f zer time cntrls and represent the mean ± SE fr fur experiments.

3 Limas and Limas/Phrbl Esters and ^-Receptrs 100 r 5.6 r *-. 60 OJ 1 "c 40 * * Dwnladed frm by n January 7, I I I I PDBu(nM) FIGURE 2. Dependence f ^-receptr lss frm rat cardiac mycytes n the cncentratin f phrbl dibutyrate (PDBu) in the preincubatin mixture. Mycyte suspensins were incubated at 37 C fr 30 minutes with the indicated PDBu cncentratins, and the lubes were then transferred t an ice-water bath, 6 nu fhjcgp-l 2177 was added, and incubatin was cntinued fr 16 hurs at 4 C Results are expressed as percent f cntrls cntaining DMSO alne and represent the mean ± SE fr fur experiments. similar in extent t that bserved with either cmpund alne, indicating a nn-additive effect. In additin t a cncentratin-dependent lss f cardiac /S-receptrs assayable with [ 3 H]CGP fllwing PDBu r OADG, there is a substantial lss TABLE 1 Effect f Phrbl Esters n the Number and Affinity f Cardiac /3-Adrenergic Receptrs f Islated Rat Cardiac Mycytes Additins DMSO 4-a-Phrbl 4-/J-Phrbl-12,13-didecanate 4-a-Phrbl-l 2,13-didecanate Phrbl-12,13-dibutyrate 12-0-Tetradecanyl Phrbl-13-acetate (fml/mg) 25±4 26 ±4 18 ±3 27 ±5 14 ± 3* 10 ±3* (nm) 3.2 ± ± ± 0.2* 3.6 ± ± 0.4* 6.1 ± 0.3* Mycyte suspensins were first incubated with 40 nw f the indicated esters at 37 C fr 30 minutes prir t j8-receptr determinatin with [ 3 H]CGP-12177, as described in the text; cntrls were preincubated with the slvent (DMSO) nly. Results represent mean ± SE fr five experiments. P < 0.01, cmpared t cntrls Bund (ftnl/mg) FIGURE 3. Scatchard plt f the [ s H]CGP binding data frm cntrl (DMSO alne) r PDBu-pretreated mycytes. Preincubatin was carried ut at 37 C fr 30 minutes prir t transferring t an ice-water bath fr ph]cgp binding (at 4 C fr 16 hurs). in the capacity f isprterenl t increase camp generatin by islated mycytes (Fig. 5), suggesting desensitizatin f the /3-receptr-cydase system. The influence f phrbl ester r OADG pretreatment n isprterenl-induced dwn-regulatin f cardiac y3-receptrs was then investigated. Cntrl, phrbl dibutyrate-, r OADG-expsed mycytes were incubated with 1 jtm /-isprterenl, and the lss* f /3-receptrs was determined. As shwn in 100 j 80 t 60 1* fig OADG FIGURE 4. Effect f l-leyl-2-acetyl-diglycerl (OADG) n phjcgp binding t cardiac mycytes. Mycyte suspensins were preincubated at 37 Cfr 30 minutes with the indicated OADG cncentratins and were then transferred t an ice-water bath fr determinatin f ^-receptr number. Results are expressed as percent f cntrls cntaining DMSO alne and represent the mean ± SE fr five experiments.

4 Dwnladed frm by n January 7, Circulatin Research/VJ. 57, N. 3, September 1985 c O 0) m a) k_ c c O h_ Q (-) isprterenl (M) FIGURE 5. Isprterenl-mediated generatin f camp by cardiac mycytes. DhASO-alne (0), PDBu (O) r OADG (A) pretreated mycytes (37 C 30 minutes) were expsed t varying cncentratins f l-isprterenl in the presence f 0.2 mta isbutylmethyhanthine fr 10 minutes at 37 C Results represent mean ± SB fr fur experiments and are expressed as percent increase ver values in the absence f isprterenl (44 ± 3 pml camp/mg prtein in cntrls, 45 ± 3 in PDBu and 43 ± 4 in OADG-pretreated mycytes). 100 I! 8 5«40 as s 1 2 a j rh Figure 6, phrbl dibutyrate and OADG induce a mdest further decline in cardiac /9-receptrs beynd that mediated by isprterenl alne. Pretreatment f cardiac mycytes with 10 Mg/ml clchicine prir t expsure t phrbl dibutyrate r OADG largely prevents /9-receptr desensitizatin, whereas trimethylclchicinic acid, which des nt affect micrtubule assembly, is inactive (Fig. 7). Nne f these cmpunds affected [ 3 H]phrbl-12,13-dibutyrate binding t its receptr sites n intact mycytes (data nt shwn); neither did they affect /3-receptr numbers when added directly t the assay medium. Clchicine pretreatment als inhibited isprterenl-mediated dwn-regulatin f /9-receptrs (cntrl: 28 ± 3 fml/mg, isprterenl: 17 ± 3 fml/ mg, clchicine/isprterenl: 24 ± 2 fml/mg). This is analgus t the in viv attenuatin by clchicine f the isprterenl-induced lss f cardiac /9-receptrs (limas and limas, 1983). The nature f the lss f cardiac /9-receptrs in PDBu- r OADG-expsed mycytes was investigated in tw sets f experiments. First, cardiac mycytes were pretreated with either 40 nm PDBu r 20 ng OADG at 37 C fr 30 minutes and washed, and the /9-receptrs were assayed either with 5 nm [ 3 H] dihydralprenll at 37 C fr 15 minutes r 6 nm [ 3 H]CGP at 4 C fr 16 hurs. As expected, /9-receptr numbers declined in PDBu-treated (18 ± 4 fml/mg) r OADG-treated (16 ± 4 fml/mg) mycytes cmpared t cntrls (27 ± 4 fml/mg) when [ 3 H]CGP was used as the ligand. With [ 3 H] dihydralprenll, hwever, n such decline culd be detected (cntrls: 31 ± 3 fml/mg, PDBUtreated: 24 ± 2, OADG-treated: 28 ± 3 fml/mg). In the secnd set f experiments, we separated the crude membrane and vesicular fractins frm cell lysates, by a mdificatin f the prcedure described by Stadel et al. (1983). As shwn in Figure 8, the disappearance f [ 3 H]dihydralprenll-binding sites frm the membranes f PDBu- r OADGtreated cells is accmpanied by a significant increase in /9-receptrs recvered in the vesicular fractin, whereas the ttal number f /9-receptrs (membranes + vesicular fractin) was cmparable in all grups, suggesting that little /9-receptr degradatin ccurs during internalizatin. The fact that /9-receptrs in the cytspl can be pelleted by ultracentrifugatin and assayed by filtratin thrugh glass-fiber filters demnstrates that these receptrs are recvered in a particulate fractin. This fractin has nly 10% the specific activity f enzymes (Na +,K + - ATPase, 5'nucletidase) present in the membrane pellet. In additin, the increase in /9-receptrs assciated with the vesicular fractin is detectable with [ 3 H]dihydralprenll but nt with the hydrphilic ligand [ 3 H]CGP-12177, suggesting sequestratin f these /9-receptrs in a fractin inaccessible t [ 3 H]- CGP a> a a < 4 FIGURE 6. Cmparisn f isprterenl and PDBu- r OADG-induced lss f p-adrcnergic receptrs. Mycytes preincubated at 37 C with either 40 nst PDBu r 20 i±g OADG were incubated further at 37 C fr 15 minutes with r withut 1 IIM l-isprterenl. Results are expressed as percent f DMSO-alne cntrls and represent the mean ± SE fr five experiments.

5 l 0 Limas and Limas/Phrbl Esters and ^-Receptrs r T 1 a T g 2 u cl m e T 1 Ine C3 a i FIGURE 7. Inhibitin by clchicine f the PDBur OADG-mediatcd desensitizatin f cardiac ^-receptrs. Myq/tcs were incubated with 40 nt4 PDBu, 20 fig OADC alne, r after 15 minutes f preincubatin with 10 ng clchicine r trimethylckhicinic acid CTMCA) prir t determinatin f ^-receptrs with I*H]CGP , as described in the text. Results are expressed as percent f DMSO-nly cntrls and represent the mean ± SE fr five experiments. Dwnladed frm by n January 7, 2019 Discussin We have recently demnstrated the presence f specific and saturable phrbl diester-binding sites n enzymatically dissciated rat cardiac mycytes (limas, 1985). The extent f binding by different phrbls parallels their relative bilgical ptencies s that 12-O-tetradecanyl phrbl-13-acetate and phrbl-12,13-dibutyrate are effective, whereas 4- a-phrbl and 4-a-phrbl-12,13-dibutyrate are inactive. [ 3 H]Phrbl dibutyrate binding t cardiac mycytes depends n prtein-lipid interactins and is cmpetitively inhibited by unsaturated diacylglycerls. These prperties are cmpatible with the finding in ther cell types that the receptr fr phrbl esters c-purifies with and may be identical t the Ca ++ /phsphlipid-dependent prtein kinase C (Kikkawa et al., 1983). Phrbl esters are, therefre, useful prbes f the bilgical rles f prtein "a. ID I Membranes Vesicles B. kinase C in intact cells. Previus experiments frm this and ther labratries have suggested the participatin f prtein kinase C in the regulatin f Ca transprt in the heart (limas, 1980; Mvsesian et al., 1984; Iwasa and Hsey, 1984). The present reprt identifies the cardiac /8-adrenergic receptr as anther ptential site fr phrbl ester-mediated regulatin. This regulatin is similar t the reprted mdificatin f transferrin (May et al., 1984), insulin (Jacbs et al., 1984), epidermal grwth factr (McCaffery et al., 1984), and /3-adrenergic (Garte and Belman, 1980; Kelleher et al., 1984; Sibley et al., 1984) receptrs in ther cell types. In these instances, receptr mdificatin has invlved either uncupling r reversible internalizatin f the receptr. These are nt necessarily mutually exclusive effects, since uncupling frequently precedes agnist-induced intemalizatin f receptrs (Tews et al., 1984). FIGURE 8. Redistributin f ^-receptrs frm the membrane t the vesicular fractin. After PDBu- r OADGpreincubatin, at 37 C fr 30 minutes, the membrane and vesicular fractins were prepared frm 4-6 x 10 6 cells as described in the text, whereas cntrl mycytes were incubated with DMSO alne. Panel A- shift in the relative distributin f ^-receptrs after 40 nu PDBu r 20 fig OADC. Results are expressed as mean ± SE fr fur experiments. Panel B: ttal ^-receptrs (membrane + vesicular fractins). DMSO PDBu OADG DMSO PDBu OADG

6 Dwnladed frm by n January 7, In the case f the cardiac /3-adrenergic receptrs, phrbl esters reduce bth their affinity and the ttal number detectable with [ 3 H]CGP n the cell surface. This is different frm the effects f isprterenl which, in ur mycyte preparatin, prmtes internalizatin f the /3-receptrs withut a change in their affinity (Limas and Limas, 1984). It is interesting t nte that expsure f cardiac mycytes t bth phrbl dibutyrate and isprterenl has an additive effect n [ 3 H]CGP binding, an bservatin cnsistent with different sites f actin, but different frm the effects reprted in duck erythrcytes (Sibley et al., 1984). Hwever, there is similarity in the mechanism f desensitizatin f PDBu, OADG, and isprterenl, in that they all appear t prmte internalizatin f the /3- receptr by a micrtubule-dependent prcess. In the present study, PDBu- r OADG-mediated desensitizatin was shwn t prmte sequestratin f the /3-receptrs in a vesicular fractin which is separate frm the cell membrane as judged by the distributin f marker enzymes. These results are cnsistent with the reprted mechanism f agnist-induced desensitizatin in bth the erythrcyte (Stadel et al., 1983) and cardiac mycyte (Limas and limas, 1984). We d nt have any direct evidence that prtein kinase C is invlved in the bserved desensitizatin f cardiac /3-receptrs. Tw bservatins, hwever, make such invlvement likely: (1) the effectiveness f different phrbls in desensitizing /3-receptrs parallels their relative ptency in stimulating prtein kinase C activity, and (2) OADG, which is an endgenus activatr f prtein kinase C as well as a ligand fr the phrbl ester receptr, mimics the effects f phrbls n cardiac /3-receptrs. Mre direct experiments n the phsphrylarin f cardiac /3-receptrs (identified thrugh phtaffinity labeling) are currently under way. The experiments described in this reprt identify phrbl ester- and diacylglycerl-mediated heterlgus desensitizatin f the cardiac /3-receptr. The effects f OADG and, by inference, f ther unsaturated diacylglycerls, are f particular imprtance, since, in cntrast t phrbl esters, they are naturally ccurring ligands and are metablically regulated, deriving frm the breakdwn f phsphtidylinsitides. Phsphatidylinsitl turnver is, in turn, linked t hrmnal activatin f cell surface receptrs (Berridge, 1984). Therefre, these results identify new metablic pathways thrugh which the prperties f the /3-adrenergic system and the functinal effectiveness f the /3-agnists can be mdified in health and disease. We have already btained preliminary evidence that phrbl ester binding is altered during the curse f cardiac hypertrphy (unpublished results). The relatinship f this t the well-knwn changes f /3-receptrs in the hypertrphied heart remains t be established. It is tempting t speculate, hwever, that prtein kinase C-dependent mdificatin f the cardiac /3-receptr has Circulatin Research/V/. 57, N. 3, September 1985 metablic cnsequences that are different frm thse resulting frm /8-agnist-induced desensitizatin. The latter results in decreased magnitude f agnist-mediated physilgical effects, whereas prtein kinase C prmtes sme /3-agnist-like actins (e.g., phsphlamban phsphrylarin). It is likely, therefre, that the functinal impact f /3- receptr desensitizatin mediated thrugh prtein kinase C is quite different frm that mediated thrugh /3-agnists. Supprted in part by Grant HL31993 frm the Natinal Heart, Lung, and Bld Institute, Natinal Institutes f Health, Bethesda, Maryland. Address fr reprints: Cnstantins }. Limas, MX)., University f Minnesta Hspitals, Bx 19 May Memrial Building, Minneaplis, Minnesta Received February 20, 1985; accepted fr publicatin June 27, References Berridge MJ (1984) Insitl triphsphate and diacylglycerl as secnd messengers. Bichem J 220: Garte SJ, Behnan S (1980) Tumur prmter uncuples betaadrenergjc receptr frm adenyl cyclase in muse epidermis. Nature 284: Gilman AG (1970) A prtein binding assay fr adensine 3',5'- cydic mnphsphate. Prc Natl Acad Sci USA 67: Iwasa Y, Hsey MM (1984) Phsphrylatin f cardiac sarclenuna prtein by the calcium-activated phsphlipid-dependent prtein kinase. J Bil Chem 259: Jacbs SJ, Sahyun NE, Saltiel AR, Cuatrecasas P (1983) Phrbl esters stimulate the phsphrylatin f receptrs fr insulin and smatmedin C. Prc Natl Acad Scd USA 80: KeUeher DJ, Pessin JE, Ruh AE, Jhnsn GL (1984) Phrbl ester induces desensitizatin f adenylate cyclase and phsphrylatin f the beta-adrenergic receptr in turkey erythrcytes. Prc Natl Acad Sci USA 81: Kikkawa U, Takai Y, Tanaka Y, Miyake R, Nishizuka Y (1983) Prtein kinase C as a pssible receptr prtein f tumrprmting phrbl esters. J Bil Chem 258: Leach KL, James ML, Blumberg PM (1983) Characterizatin f a specific phrbl ester apreceptr in muse brain cytsl. 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7 Limas and L/mas/Phrbl Esters and /J-Receptrs reticulum calcium uptake. J Bil Chem 259: Niedel JE, Kuhn LJ, Vandenbark GR (1983) Phrbl diester receptr cpurifies with prtein kinase C. Prc Natl Acad Sci USA 80: Sibley DR, Nambi P, Peters JR, Lefkwitz JR (1984) Phrbl diester prmte beta-adrenergic receptr phsphrylatin and adenylate cyclase desensitizatin in duck erythrcytes. Bichem Biphys Res Cmmun 121: Stadel JM, Strulvici B, Nambi P, Lavin TN, Briggs MM, Carn MG, Lefkwitz RJ (1983) Desensitizatin f the beta-adrenergic receptr f frg erythrcytes. Recvery and characterizatin f the dwn-regulated receptrs in sequestered vesicles. J Bil Chem 258: Staehelin M, Hertel (1983) [ 3 H]CGP-12177, a /3-adrenergic ligand 449 suitable fr measuring cell surface receptrs. J Receptr Res 3: Tews ML, Wald GL, Harden TK, Perkins JP (1984) Relatinship between an altered membrane frm and a lw affinity frm f the beta-adrenergic receptr ccurring during catechlamineinduced desensitizatin. Evidence fr receptr intemalizatin. J Bil Chem 259: Wise BC, Raynr RL, Ku JF (1982) Phsphlipid-sensitive Ca 2+ - dependent prtein kinase frm heart. I. Purificatin and general prperties. J Bil Chem 257: INDEX TERMS: Phrbl esters Diacylglycerls Mycytes Receptrs Desensitizatin Dwnladed frm by n January 7, 2019