Increased PKA activity and its influence on isoprenaline-stimulated L-type Ca 2 þ channels in the heart from ovariectomized rats

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1 British Journl of Phrmcology (25) 44, & 25 Nture Pulishing Group All rights reserved 7 88/5 $3. Incresed PKA ctivity nd its influence on isoprenline-stimulted L-type C 2 þ chnnels in the hert from ovriectomized rts Kenneth W.L. Km, Genndi M. Krvtsov, Jing Liu & *, Tk Ming Wong Deprtment of Physiology, Fculty of Medicine, the University of Hong Kong, Pokfulm, Hong Kong SAR, Chin We previously showed tht oestrogen confers crdioprotection y downregulting the crdic - drenoceptor ( -AR). The present study exmined the effect of oestrogen on the post -AR signlling cscde, with prticulr emphsis on the ctivity of protein kinse A (PKA) nd its influence on the L-type C 2 þ chnnel. 2 Three groups of dult femle Sprgue Dwley rts were used: shm-operted controls, ilterlly ovriectomized (Ovx) rts, nd Ovx rts with oestrogen replcement (Ovx þ E 2 ), which restored the oestrogen concentrtion to norml. 3 The electriclly induced intrcellulr C 2 þ trnsient (E[C 2 þ ] i ), 45 C 2 þ -uptke through crdic L- type C 2 þ chnnels (C 2 þ chnnels), hert rte nd force of contrction in response to -AR stimultion with nm isoprenline (Iso) in herts from Ovx rts were significntly greter thn those of control nd Ovx þ E 2 rts. The sl nd Iso-induced PKA ctivities were lso higher in herts from Ovx rts. KT572, selective PKA inhiitor, completely inhiited its potentiting effect on sl C 2 þ chnnel ctivity in the Ovx rt hert. On the other hnd, expression of G proteins (G s nd G i 3 ), sl nd forskolin-stimulted camp ccumultion, nd responsiveness of PKA to camp, were not ltered y Ovx. 4 Interestingly, the PKA inhiitor t the sme concentrtion significntly reduced the increses in PKA ctivity nd C 2 þ chnnel ctivity upon -ARstimultion in ll three groups of rts nd the inhiitions were significntly greter in the Ovx rt thn in the other two groups of rts. 5 This study provides the first evidence tht, in ddition to downregultion of -ARshown previously, suppression of PKA ctivity, which is prtly responsile for the suppressed C 2 þ chnnel ctivity, lso determines the E[C 2 þ ] i nd crdic contrctility following -ARstimultion in the femle rt. British Journl of Phrmcology (25) 44, doi:.38/sj.jp.7623 Pulished online 3 Jnury 25 Keywords: Oestrogen; -drenoceptor; protein kinse A; ovriectomy; L-type C 2 þ chnnel; electriclly induced C 2 þ trnsient; 45 C 2 þ uptke Arevitions: -AR, -drenoceptor; C 2 þ chnnel, L-type C 2 þ chnnel; camp, cyclic denosine monophosphte; E C, excittion contrction; E[C 2 þ ] i, electriclly induced intrcellulr C 2 þ trnsient; I c, L-type C 2 þ current; Iso, isoprenline; Ovx, ovriectomy; Ovx þ E 2, ovriectomy with oestrogen replcement; PKA, protein kinse A Introduction In 98, Ciric nd Susic oserved tht isoprenline (Iso), -drenoceptor (-AR) gonist, ffects hert rte to different extents in femle rts in different phses of the estrous cycle, nd in ovriectomized rts with nd without oestrogen replcement. These oservtions suggest tht oestrogen regultes the crdiovsculr system through modultion of -ARin the hert. It ws lso found tht oestrogen deficiency produced y ovriectomy (Ovx) cuses upregultion of -AR, n effect restored to the norml level y oestrogen replcement (Thwornkiwong et l., 23). This result suggests tht oestrogen suppresses the expression of crdic -AR. Recently, we extended this finding y demonstrting tht incution of ventriculr myocytes from Ovx rts with 9 M 7-estrdiol for 24 h, which suppressed the enhnced expression of -AR, ws ccompnied y crdioprotection ginst Iso-induced ischemic insult, wheres incution of ventriculr *Author for correspondence; E-mil: wongtkm@hkucc.hku.hk Pulished online 3 Jnury 25 myocytes with 7-estrdiol for 2 h neither suppressed the expression of -ARnor conferred crdioprotection ginst ischemic insult nd -ARstimultion. These oservtions were tken to indicte tht the crdioprotective effect of oestrogen is due, t lest prtly, to downregulting -ARin the rt hert (Km et l., 24). Studies in other lortories hve shown incresed ctivity nd overexpression of the crdic L-type C 2 þ chnnel (C 2 þ chnnel) in oestrogen receptor (ER)-knockout mice (Johnson et l., 997). In ddition, ltered C 2 þ sensitivity of crdic myofilments in ovriectomized rts hs een oserved (Wttnpermpool, 998). However, the influence of oestrogen on events downstrem from the crdic -ARsignlling mechnism leding to chnges in C 2 þ chnnel ctivity is not yet understood. The sympthetic nervous system is one of the most importnt extrinsic mechnisms regulting crdic function, minly through ctivtion of -AR. There re three -AR sutypes, nmely -, 2 - nd 3 -ARs. The roles of - nd 2 - ARs re resonly well defined; oth re involved in contrctile functions nd poptosis vi different signlling

2 K.W.L. Km et l Oestrogen, -drenoceptor nd PKA 973 mechnisms (see Xio et l., 24). 3 -ARis found in humn nd murine hert nd its role is not well understood other thn negtive inotropic ction (see Conrth & Opthof, 23). Stimultion of the -ARsutype increses developed contrction (inotropy) nd ccelertes relxtion (lusitropy) y ctivting the GTP-inding protein (G s )/denylyl cyclse (AC)/cAMP/protein kinse A (PKA) pthwy (Bers, 22). PKA phosphoryltes severl key regultory proteins in excittion contrction (E C) coupling; its inotropic effect is minly medited through phosphoryltion of the C 2 þ chnnels nd thus increses the pek L-type C 2 þ current (I c ). This cuses more C 2 þ relese during excittion (Beuckelmnn & Wier, 988; Nuer et l., 989; Sperelkis & Whler, 998), which in turn increses the developed contrction. Recently, it hs lso een shown tht stimultion of 2 -ARlso increses camp nd PKA, leding to incresed I c vi C 2 þ chnnels (Ytni et l., 999). On the other hnd, it hs lso een suggested tht -ARstimultion my increse the I c vi PKA-independent ctivtion of C 2 þ chnnels (Ytni & Brown, 989; Ytni et l., 999). In preliminry study, we determined the effect of oestrogen on the expression/ctivity of the intermedites in the G s /AC/cAMP/PKA pthwy nd found tht the expression of G s nd G i nd camp ccumultion were the sme in the herts from control, Ovx, nd Ovx þ E 2 rts. On the other hnd, the sl PKA ctivity ws incresed in herts from Ovx rts. We therefore hypothesized tht oestrogen might suppress PKA ctivity, thus decresing C 2 þ chnnel ctivity nd therey reduce the crdic response to -ARstimultion. To test this hypothesis, we first determined the effect of - ARstimultion with Iso on PKA ctivity. Secondly, we determined the effect of PKA lockde on sl nd Isostimulted C 2 þ chnnel ctivity in the herts from shmoperted control, Ovx, nd Ovx þ E 2 rts. The min finding ws tht Iso-induced PKA ctivity nd C 2 þ chnnel ctivity were significntly higher in the herts from Ovx rts. This ws ccompnied y significntly greter increses in the electriclly induced intrcellulr C 2 þ trnsient (E[C 2 þ ] i ), nd crdic contrctility in herts from Ovx rts over those from control nd Ovx þ E 2 rts. So, esides suppressing -ARexpression s oserved in our previous study (Km et l., 24), oestrogen lso suppressed PKA ctivity, thus decresing C 2 þ chnnel ctivity in response to -AR stimultion in the hert. Methods Experimentl nimls Femle Sprgue Dwley rts weighing 9 2 g were purchsed from Chrles River Breeding Lortories (Wilmington, MA, U.S.A.) nd rndomly divided into two groups. One group underwent shm opertion nd served s norml control. The other group underwent ilterl Ovx (Km et l., 24). A dorsl midline skin incision ws mde cudl to the posterior order of the ris. Using lunt dissection, the muscles of the posterior dominl wll were seprted nd the dominl cvity opened. The periovrin ft ws gently grsped with forceps to lift nd exteriorize the ovry, which ws then removed. The uterine horn ws returned into the domen, nd the process ws repeted on the other side. At week fter Ovx, sugroup of the rts (Ovx þ E 2 ) ws sucutneously implnted with 6-dy relese pellet contining.5 mg of 7-estrdiol (Innovtive Reserch of Americ, Toledo, OH, U.S.A.). These pellets mintin oestrogen concentrtion within the physiologicl rnge. All surgicl procedures were performed under nesthesi with sodium pentoritl (6 mg kg, i.p.; Aott Lortory, Chicgo, U.S.A.). The protocol ws pproved y the Committee on the Use of Experimentl Animls for Teching nd Reserch, The University of Hong Kong. Crdic memrne preprtion Crdic memrne ws prepred from the left ventricle s previously descried (Yu et l., 2) with some modifiction. The rts were nesthetized with sodium pentoritone (6 mg kg i.p.) nd given heprin (2 IU, i.v.) efore decpittion y guillotine. The hert ws excised rpidly nd mounted on the Lngendorff pprtus. The hert ws perfused with sline (.9% NCl) for 5 min to wsh out ll the lood. The isolted left ventricle ws minced into smll pieces in homogeniztion uffer contining 2 mm Tris-HCl, ph 7.5, 5 mm MgCl 2,mM EGTA, mm DTT, nd.4 mm PMSF. The minced left ventricle ws homogenized with Polytron PT 35 homogenizer for 3 s. The rest of the isolting procedure ws the sme s descried y Yu et l. (2). The finl pellet ws dispersed with Polytron in 4 ml of the memrne uffer. The resuspended pellets were stored t 8C until use. The protein content ws determined s descried previously (Lowry et l., 95). Western lotting nlysis In ll, 6 mg of memrne protein from the left ventricle ws diluted in loding uffer (3 mm Tris-HCl, ph 8., 2% (v v ) glycerol, 5% (w v ) sodium dodecyl sulphte (SDS),.2% romophenol lue, 2% DTT) nd dentured for 5 min t 95C. Smples were seprted y 2% polycrylmide gel electrophoresis in the presence of SDS nd then electrophoreticlly trnsferred onto polyvinylidene difluoride memrne t V for.5 h. The memrne ws then incuted with nti-g s, nti-g,2 nd nti-g i3 (ech t : ) overnight t 4C. After three -min wshes in TBST (Tris-uffered sline, ph 7.4,.% Tween-2) solution, HRP-linked nti-rit IgG t dilution rtio of : ws used s secondry ntiody nd incuted for h t room temperture, followed y three -min wshes in TBST solution. Protein nds were detected using n enhnced chemiluminescence detection system nd visulized y utordiogrphy. Densitometric nlysis ws conducted using the Syngene CCDBIO cquisition system nd its nlysis softwre (Hitchi Genetics System, Almed, CA, U.S.A.). Isolted perfused hert preprtion Rts were nesthetized with sodium pentoritone (6 mg kg i.p.) nd given heprin (2 IU, i.v.) efore decpittion y guillotine. The hert ws removed immeditely, mounted on Lngendorff pprtus nd perfused retrogrdely with Kres Henseleit solution (in mm: NCl 8, KCl 4.7, CCl 2.25, KH 2 PO 4.2, MgSO 4 British Journl of Phrmcology vol 44 (7)

3 974 K.W.L. Km et l Oestrogen, -drenoceptor nd PKA.2, NHCO 3 25, nd glucose ) equilirted with 95% O 2 þ 5% CO 2 t constnt pressure of 8 cm H 2 O nd temperture of 37C. A lloon inserted through the left trium into the left ventricle ws inflted y injecting. to.2 ml of sline to djust the end-distolic pressure to 6 mmhg. Crdic prmeters were monitored continuously y PowerL/ 4SD nlog-to-digitl converter (AD Instruments, Cstle Hill, Austrli). Isoltion of ventriculr myocytes Ventriculr myocytes were isolted from the herts of control, Ovx, nd Ovx þ E 2 rts, using the collgense perfusion method descried previously (Wu et l., 999). After isoltion, they were llowed to stilize for t lest 3 min efore experiments. Mesurement of [C 2 þ ] i A spectrofluorometric method with Fur-2/AM s the C 2 þ indictor ws used to lod cells nd mesure [C 2 þ ] i s previously descried (Wu et l., 999). The myocytes selected for the study were rod shped nd quiescent with cler stritions. They exhiited synchronous contrction in response to.2 Hz electricl field stimultion with 5 ms pulse t 6 V through two pltinum wires in the thing chmer. The myocytes were superfused with Kres icronte uffer contining (in mm) 8 NCl, 5 KCl,.2 MgSO 4,.2 KH 2 PO 4,.25 CCl 2, 25 NHCO 3, nd glucose nd gs phse of 95% O 2 5% CO 2, ph 7.4 throughout the study. After 5 min of stiliztion in the thing chmer, single ventriculr myocyte ws selected. The chnges in E[C 2 þ ] i ws recorded for min, which constituted the sl [C 2 þ ] i trnsient in response to electricl stimultion. Afterwrds, the perfuste ws chnged to vrious Kres icronte uffer contining different concentrtions of Iso nd the recording ws continued for nother 5 min to ensure tht equilirium with Iso hd een reched. Fluorescence signls otined t excittion wvelengths of 34 nm (F 34 ) nd 38 nm (F 38 ) were recorded nd stored in computer for susequent processing nd nlysis. The F 34 /F 38 rtio represented cytosolic [C 2 þ ] i in the ventriculr myocyte. The E[C 2 þ ] i, which represents the influx of C 2 þ vi C 2 þ chnnels nd the relese of C 2 þ from the srcoplsmic reticulum triggered y the C 2 þ influx, is directly correlted with crdic contrction in the experimentl conditions of the present study (Yu et l., 999). Mesurement of 45 C 2 þ uptke Freshly isolted cells (2 5 ml ) were equilirted for 5 min t room temperture (22C) in MEM solution, where the concentrtion of C 2 þ ws grdully incresed to. mm. In ll, 2 ml liquots were plced in plstic tues contining 2 ml of solution A (in mm): NCl 4, KCl 5, MgCl 2, CCl 2. ( 45 CCl 2 2 mci ml ) nd HEPES-Tris 2 (ph 7.4; 22C). The cell suspension (2 4 ml ) ws incuted for 2 2 min t room temperture. A specific inhiitor of L-type C 2 þ chnnels, nitrendipine ( mm), highly selective -ARntgonist, isoprolol ( mm) (McGvin & Keting, 22), nd selective inhiitor of PKA, KT572 (2 mm) (Kse et l., 987; Hikl et l., 997; Kiehn et l., 998) were dded to the cell suspension long with Iso. The incution mixture ws rpidly filtered through Whtmn GF/C filters nd wshed three times with 4 ml of wshing solution contining (in mm): NCl 4, HEPES-Tris (ph 7.4), nd EGTA.5. After wshing, the GF/C filters were dded to ml of scintilltion cocktil nd left for 4 min (Universl LSC cocktil for queous smples, Sigm, St Louis, U.S.A.). The rdioctivity ws then mesured in scintilltion counter (LS 65, Beckmn). Iso incresed the 45 C 2 þ uptke in concentrtiondependent mnner nd mm of the drug exerted mximl effect (Figure ). Both sl nd mm Iso-stimulted 45 C 2 þ uptke in solution A were liner from 2 to min (Figure ). Since the liner prt of the curve reflects the rte of 45 C 2 þ uptke, we chose the 5 min point to evlute the rte of 45 C 2 þ uptke. In ll susequent experiments, the sl C 2 þ chnnel ctivity ws defined s the difference etween the sl rtes of 45 C 2 þ uptke in the presence nd sence of mm nitrendipine, specific locker of 45 C 2 þ uptke through C 2 þ chnnels (Luic et l., 994). Iso-stimulted C 2 þ chnnel ctivity ws defined s the difference etween the rtes of 45 C 2 þ uptke in the presence of mm Iso with/ without mm nitrendipine. A similr experimentl protocol ws used for determining C 2 þ chnnels ctivity nd Isostimulted C 2 þ chnnel ctivity in myocytes from shm, Ovx, nd Ovx þ E 2 rts. 45 C 2+ uptke (nmole/ 5 cells/ 5 min) 45 C 2+ uptke (nmole/ 5 cells) Control Time (min) nm nm µm µm Isoprenline Isoprenline (µm) Control Figure Effects of Iso on 45 C 2 þ uptke in ventriculr myocytes from control rts. () Concentrtion-relted effects. () Timedependent chnges. Vlues re men7s.e.m. of four independent experiments. *Po.5 vs control. **Po. vs control. British Journl of Phrmcology vol 44 (7)

4 K.W.L. Km et l Oestrogen, -drenoceptor nd PKA 975 camp ssy camp content ws determined s descried previously (Bin et l., 2). Briefly, 3 5 isolted myocytes were plced in wells to which different concentrtions of forskolin were dded nd incuted for min t 37C. At the end of the tretment, camp ws extrcted nd stored t 2C for susequent ssy. Protein content ws determined y Lowry s method (Lowry et l., 95), nd the intrcellulr camp content ws mesured with n ssy kit (TRK 432; Amershm Interntionl, U.K.) ccording to the mnufcturer s recommended protocol. PKA ctivity PKA ctivity ws mesured with commercilly ville ssy kit from Cliochem s, using the mnufcturer s recommended protocol. Briefly, left ventriculr myocytes were sonicted in ice-cold extrction uffer nd centrifuged t 4, g for 5 min. PKA ctivity ws mesured y the incorportion of 32 P from g 32 P-ATP into Kemptide, the specific synthetic sustrte for PKA. PKA smple (5 ml) ws dded to the rection uffer nd incuted t 3C for min nd the rection ws stopped with 8 M gunidine hydrochloride. The mixture ws trnsferred to centrifugl ultrfiltrtion unit nd wshed three times with the uffer. The smple reservoir of the unit ws then trnsferred to scintilltion vil with ml of scintilltion cocktil (Universl LSC cocktil for queous smples, Sigm). The rdioctivity in the smple reservoir ws mesured in scintilltion counter (LS 65, Beckmn). To study the effects of Iso on PKA ctivity, myocytes were incuted with nm of Iso in the presence/sence of mm isoprolol for min in serum-free DMEM with.5 mm 3- isoutyl-l-methylxnthine (IBMX) followed y soniction in ice-cold extrction uffer nd centrifugtion s descried ove. PKA ctivity ws mesured without exogenously dded camp, using the mnufcturer s recommended protocol. To study the effect of camp on PKA ctivity, 5 mm of camp ws dded to the cellulr extrcts. The PKA ctivity ws then mesured ccording to the mnufcturer s recommended protocol. The camp-induced increse in PKA ctivity ws defined s the difference etween the PKA ctivities in the presence nd sence of 5 mm exogenously dded camp. Serum oestrogen level Blood smples were collected from the rts fter decpittion nd serum E 2 levels were mesured using the solid-phse 25 I rdioimmunossy technique (Dignostic Reserch Lortory, U.S.A.) ccording to the mnufcturer s instructions. In ddition to serum oestrogen level, the ody weight/hert weight rtio ws clculted s n idex of oestrogen depletion fter Ovx. Drugs nd chemicls Forskolin, type- collgense, nitrendipine, nti-g s, nti- G,2, nd nti-g 3 ntiodies were from Sigm-Aldrich, St Louis U.S.A. Bisoprolol ws from Tocris Cookson Ltd, Bristol, U.K. 45 C 2 þ (74 MBq, 2.4 mci ml ) ws from Amershm Phrmci, Buckinghmshire, U.K. KT572 ws from Cliochem, Drmstdt, Germny. HRP-conjugted nti-rit secondry ntiody ws from Snt Cruz Ltd, CA, U.S.A. The ECL detection kit ws from Amershm Phrmci. All drugs were dissolved in de-ionized H 2 O or Kres solution except forskolin, which ws dissolved in DMSO. The finl concentrtion of DMSO ws p.%, which itself hd no effect on the hert. Sttisticl nlysis Dt were expressed s men7s.e.m. Two-wy ANOVA with post hoc test ws used to determine differences mong multiple groups. The nonprmetric Kruskl Wllis test ws used to nlyse drug effects. Computer-ssisted nonliner regression nlysis for sigmoidl dose responses ws used to determine EC 5 (GrphPd Prism 3). A difference of Po.5 ws considered sttisticlly significnt. Results Generl fetures of experimentl nimls At 6 weeks fter Ovx, the rts hd significntly reduced serum oestrogen levels ccompnied y significnt increse in ody weight. These chnges were reversed in oestrogen-treted nimls, with the exception of the hert/ody weight rtio, which ws significntly reduced in Ovx rts, ut ws not restored y oestrogen replcement (Tle ). Bsl PKA nd C 2 þ chnnel ctivity, camp ccumultion, nd expression of G-proteins in the hert from control, Ovx, nd Ovx þ E 2 rts Bsl PKA ctivity ws significntly greter in myocytes from Ovx rts thn in those from controls ( vs pmol P i (mg protein) min ), nd the increse ws reversed y oestrogen replcement (Figure 2). The PKA Tle Generl feture of experimentl nimls Body weight (g) Hert weight (g) % Hert/ody weight Serum oestrogen (pg ml ) Femle (8) Ovx (8) * * 774. * Estrogen replcement (9) * * Ech vlue represents the men7s.e.m. The figures in prentheses indicte numers of nimls. * Po.5 vs shm group. British Journl of Phrmcology vol 44 (7)

5 976 K.W.L. Km et l Oestrogen, -drenoceptor nd PKA PKA ctivity (pmole P i /mg protein/min) camp +camp # 45 C 2+ uptke (nmole/ 5 cells/ 5 min) Shm Shm + nitrendipine Bsl ctivity of L-type C 2+ chnnels camp-induced PKA Activity (pmole P i /mg protein/min) 4 Shm Ovx 3 2 Shm Ovx Shm Ovx Figure 2 Enzymtic ctivity of PKA in ventriculr myocytes from control, Ovx, nd Ovx þ E 2 rts. () PKA ctivity. The ctivity of PKA ws mesured with/without dded camp (5 mm) () campinduced ctivity. The camp-induced PKA ctivity ws defined s the difference in PKA ctivity with nd without 5 mm camp. Vlues re expressed s men7s.e.m. of triplicte mesurements from three herts in ech group. *Po.5 vs control group. ctivity ws mrkedly incresed y chllenge with 5 mm camp, in greement with the previous oservtion (Person & Kemp, 99). Interestingly, the camp-induced increse in PKA ctivity ws the sme mong the three groups (Figure 2), so the tretment did not chnge the responsiveness of PKA to camp. The sl C 2 þ chnnel ctivity ws significntly higher in myocytes from Ovx rts thn tht of controls (.7.3 vs nmol ( 5 cells) min ) nd the effect ws reversed y oestrogen replcement (Figure 3). Blockde of PKA with selective inhiitor, KT572 t 2 mm, concentrtion shown in our l to exert the mximl inhiitory effect, completely olished the increse in sl C 2 þ chnnel ctivity in myocytes from Ovx rts without ffecting the ctivity in myocytes from the other two groups. This indictes tht higher sl ctivity of PKA in myocytes from Ovx rts is responsile for the incresed sl C 2 þ chnnel ctivity. There ws no difference in the sl camp ccumultion mong ll three groups (Figure 4). Nor ws there ny difference in their response to forskolin over the rnge from 7 to 4 M (Figure 4). So, the greter sl PKA ctivity in the herts of Ovx rts ws not due to higher sl cellulr camp concentrtion. Figure 5 shows the sum of 45 nd 52 kd peptides detected y ntiody to G s. Bnds migrting t 4 nd 4 kd detected y ntiodies to G i 2 nd G i3 re shown in Figure 5 nd c, Bsl ctivity of L-type C 2+ chnnels (nmole/ 5 cells/ min) c Bsl ctivity of L-type C 2+ chnnels (%) Shm Ovx Shm Ovx Shm Ovx respectively. The mounts of G s,g i 2, nd G i3 proteins did not significntly differ mong the three groups, so the higher sl PKA ctivity in the hert of Ovx rts ws not due to chnges in expression of G-proteins. Effect of -AR stimultion with Iso on PKA nd C 2 þ chnnel ctivity, E[C 2 þ ], nd contrctile functions in the herts from control, Ovx, nd Ovx þ E 2 rts +KT572 Figure 3 C 2 þ chnnel ctivity in ventriculr myocytes of control, Ovx, nd Ovx þ E 2 rts effect of PKA lockde. () 45 C 2 þ uptke y ventriculr myocytes from control rts. Bsl C 2 þ chnnel ctivity ws defined s the difference etween sl 45 C 2 þ uptke in the sence nd presence of mm nitrendipine, selective L-type C 2 þ chnnel locker. A similr experimentl protocol ws used for determining C 2 þ chnnel ctivity in myocytes from the other groups of rts. () Bsl C 2 þ chnnel ctivity. (c) Effects of KT572 (2 mm), selective inhiitor of PKA, on sl C 2 þ chnnel ctivity (ctivity in myocytes from control rts expressed s %). Vlues re men7s.e.m. of four independent experiments. *Po.5 vs control nd Ovx þ E 2 groups. To test the hypothesis tht the femle sex hormone suppresses crdic responses to -ARstimultion with Iso y suppressing PKA, we first determined the PKA ctivity following -AR stimultion in ll three groups of rts. Iso significntly British Journl of Phrmcology vol 44 (7)

6 K.W.L. Km et l Oestrogen, -drenoceptor nd PKA 977 camp (pmole /mg protein) camp (pmole /mg protein) 5 Shm Ovx Shm Ovx Control Forskolin concentrtion (M) Figure 4 Bsl nd forskolin-induced camp content in ventriculr myocytes of control, Ovx, nd Ovx þ E 2 rts. () Bsl camp content nd () concentrtion-relted effect of forskolin. Cells were chllenged with nm to. mm forskolin for min nd camp ws extrcted nd mesured. All mesurements were mde in the sence of phosphodiesterse inhiitor. Vlues represent men7 s.e.m. of triplicte determintions from 3 herts in ech group. *Po.5 vs corresponding vlue in the control group. incresed the PKA ctivity in ll groups nd the increse ws significntly greter in the herts of Ovx thn in those of shm nd Ovx þ E 2 rts (Figure 6). In the presence of mm isoprolol, specific -ARntgonist, the PKA ctivity ws reduced y 8%, confirming receptor-medited ction (Figure 6). This incresed PKA ctivity my e responsile for the greter crdic response to -ARstimultion in Ovx rts. The Iso-stimulted PKA ctivity in Ovx rts ws.7 pmol P i (mg protein) min higher thn in the control rts (Figure 6c), much greter increse thn tht in sl PKA ctivity (.67 pmol P i (mg protein ) min ) shown in Figure 2, so the significntly higher Iso-stimulted PKA ctivity in Ovx rts cnnot e ccounted for y the greter sl PKA ctivity. Iso-stimulted C 2 þ chnnel ctivity ws concentrtiondependent nd ntgonized y mm isoprolol (Figure 7). This ntgonist shifted the EC 5 for Iso from 42.5 nm to 2. mm, indicting -AR-medited effect on 45 C 2 þ uptke through C 2 þ chnnels. The Iso-stimulted C 2 þ chnnel ctivity in the hert of Ovx rts ws much greter thn those of the other two groups (Figure 7c). In ddition, the increment ws.22 nmol ( 5 cells) min over tht of the control, vlue much greter thn the difference (.4 nmol ( 5 cells) min ) in sl C 2 þ chnnel ctivity etween Ovx nd control Aritrry densitometric unit Aritrry densitometric unit c Aritrry densitometric unit Shm Ovx Shm Ovx Shm Ovx Shm Ovx Shm Ovx Shm Ovx 52kD 45kD 4kD 4kD Figure 5 Western lotting nlysis of G s nd G i 3 in the memrne frction of left ventricles. () G s protein, () G i 2 protein, nd (c) G i3 protein. Upper pnels show representtive Western lots for the respective proteins. Ech lne represents the smple from one hert. Lower pnels show quntified dt. Vlues represent men7s.e.m. of six independent experiments. rts (Figure 3), showing tht -ARstimultion elicits n increse in C 2 þ chnnel ctivity tht cnnot e ccounted for y greter sl ctivity. To determine whether the differences in the effects of -AR ctivtion on C 2 þ chnnel ctivity were PKA-dependent, we mesured the effect of KT572, which significntly reduced the Iso-stimulted 45 C 2 þ influx vi C 2 þ chnnels (Figure 8). The degree of inhiition ws significntly greter in myocytes from Ovx rts thn in those from the other two groups, indicting greter influence of PKA on C 2 þ chnnels (Figure 8). Iso ( nm mm) concentrtion dependently incresed the E[C 2 þ ] i in crdic myocytes from ll groups of rts (Figure 9). The mximum responses of myocytes from control, Ovx, nd Ovx þ E were 96726, 22573, nd 87736%, respectively. The corresponding EC 5 vlues were British Journl of Phrmcology vol 44 (7)

7 978 K.W.L. Km et l Oestrogen, -drenoceptor nd PKA PKA ctivity (pmole P i /mg protein/min) PKA ctivity (pmole P i /mg protein/min) 4.5 -Iso Iso shm shm Ovx Ovx -Iso +Iso+isoprolol # 45 C 2+ uptke (nmole/ 5 cells/ 5 min) 2 45 C 2+ uptke % Shm Shm + Iso Shm + Iso +nitrendipine Control Bisoprolol Isoprenline-stimulted ctivity of L-type C 2+ chnnels Isoprenline (log [M]) Isoprenline-induced PKA ctivity (pmole P i /mg protein/min). 3 2 shm shm Ovx Ovx shm Ovx shm Ovx µm isoprolol Figure 6 Effect of Iso on enzymtic ctivity of PKA in ventriculr myocytes from control, Ovx, nd Ovx þ E rts. () Iso-stimulted PKA ctivity, () Iso-stimulted PKA ctivity in the presence of isoprolol ( mm), nd (c) Iso-stimulted PKA ctivity. The Isostimulted PKA ctivity is defined s the difference in PKA ctivity with/without Iso preincution in the sence/presence of isoprolol, respectively. Vlues re expressed s men7s.e.m. of triplicte mesurements from three herts in ech group. *Po.5 vs corresponding vlues without Iso. # Po.5 vs corresponding vlues with Iso , 57.6, nd nm. Over concentrtion rnge from nm to mm Iso, the percentge increse in E[C 2 þ ] i ws significntly higher in myocytes from Ovx rts thn those from the other two groups nd there ws no significnt difference etween those from control nd Ovx þ E 2 rts. So, -ARstimultion in Ovx rts cuses significntly higher C 2 þ relese during E C coupling, which my in turn increse contrction. As in the cse of E[C 2 þ ] i, which is directly correlted to contrction (Yu et l., 999), nm of Iso significntly incresed hert rte, LVDP, nd 7dp/dt mx in ll three groups nd the increse over seline ws significntly greter in Ovx c Isoprenline-stimulted L-type C 2+ chnnel ctivity (nmole/ 5 cells/ min) thn those in shm nd Ovx þ E 2 rts (Tle 2), indicting significnt higher excittion nd contrction, nd fster relxtion in the herts from Ovx rts. Discussion Shm Ovx Figure 7 Effects of Iso on C 2 þ chnnel ctivity in ventriculr myocytes from control, Ovx, nd Ovx þ E 2 rts. () Effects of Iso on 45 C 2 þ uptke in ventriculr myocytes from control rts. The ctivity of the Iso-stimulted L-type C 2 þ chnnel ws defined s the difference etween Iso-stimulted 45 C 2 þ uptke in the sence nd presence of mm nitrendipine. A similr experimentl protocol ws used for determining C 2 þ chnnel ctivity in myocytes from the other groups of rts. () Concentrtion-relted effect of Iso on 45 C 2 þ uptke in the presence nd sence of mm isoprolol, selective -ARntgonist. (c) Effects of Iso on C 2 þ chnnel ctivity in ventriculr myocytes from control, Ovx, nd Ovx þ E 2 rts. Vlues re men7s.e.m. of four independent experiments. *Po.5 vs control nd Ovx þ E 2 groups. There is compelling evidence for oestrogen-medited modultion of G-protein coupled receptors (GPCR) in the reproductive system (Roerts et l., 977; Mggi et l., 988; Pinto et l., 997). A previous study from our lortory showed tht 7estrdiol confers crdioprotection y inhiiting -ARexpression nd the ccumultion of camp fter -ARctivtion (Km et l., 24). In the present study, we further demonstrted tht -ARstimultion with Iso led to significntly greter increses in PKA ctivity, C 2 þ uptke 45 through C 2 þ chnnels E[C 2 þ ] i, hert rte nd contrctility British Journl of Phrmcology vol 44 (7)

8 K.W.L. Km et l Oestrogen, -drenoceptor nd PKA Isoprenline-stimulted L-type C 2+ chnnel ctivity(%) Fluorescence rtio (34/38 nm) Iso Iso Iso Shm Ovx Inhiition of isoprenline-stimulted L-type C 2+ chnnel ctivity y KT572 (%) Shm Ovx Shm Ovx +KT572 in the herts of Ovx rts thn in those from controls, nd these responses were restored to norml y oestrogen replcement. The most importnt finding of the present study ws tht the sl PKA ctivity ws much higher in the herts of Ovx rts thn in controls, n effect reversed y oestrogen replcement. The incresed sl PKA ctivity is responsile for greter C 2 þ chnnel ctivity since lockde with its selective inhiitor, KT572, completely olished the effect. The higher PKA ctivity is not due to ltered expression of G- proteins, or camp ccumultion, or responsiveness of PKA to camp, s we found no chnge in the expression of G-proteins, or camp ccumultion, or camp-induced PKA ctivity in the herts from shm, Ovx, nd Ovx þ E 2 rts. Following -AR stimultion with Iso, PKA ctivity incresed to significntly greter extent in the herts from Ovx rts thn in the herts of the other two groups of rts, nd ws ccompnied y significntly greter increse in C 2 þ chnnel ctivity, indicting cusl reltionship. This greter increse in PKA ctivity cnnot e ccounted for y higher sl ctivity. Rther it is due to n incresed influence from the -AR, which is upregulted in the hert of Ovx rts, s reflected y Shm Ovx Figure 8 Iso-stimulted L-type C 2 þ chnnel ctivity in ventriculr myocytes from control, Ovx, nd Ovx þ E 2 rts effect of PKA lockde with KT572. () Effects of Iso on C 2 þ chnnel ctivity in the sence (left) nd presence (right) of KT572 (2 mm) in ventriculr myocytes from control, Ovx, nd Ovx þ E 2 rts. The vlue in the myocytes from control rts ws expressed s %. () Inhiition of Iso-stimulted C 2 þ chnnel ctivity y KT572 (defined s the difference in the presence nd sence of KT572). Vlues re men7s.e.m. of four independent experiments. *Po.5 vs control nd Ovx þ E 2 groups. Increse of electriclly induced [C 2+ ] i trnsient (percentge of mximl effect) 5 Ovx Shm 5 * Isoprenline (M) Figure 9 Concentrtion-relted effects of Iso on E[C 2 þ ] i in ventriculr myocytes from control, Ovx, nd Ovx þ E 2 rts. () Representtive trces showing the effect of nm Iso nd () concentrtion response curve. Y-xis represents percentge of mximl effect. The effect of mm Iso stimultion on E[C 2 þ ] i in shm rts ws tken s. Vlues re men7s.e.m. N ¼ 5 24 cells from three different rts in ech group. *Po.5 vs corresponding vlue in the control group. greter camp ccumultion in response to -ARstimultion (Km et l., 24). So the oestrogenic environment downregultes the -ARnd suppresses PKA ctivity. The suppressed PKA ctivity in turn reduces C 2 þ chnnel ctivity nd ssocited crdic functions. In crdiomyocytes, C 2 þ flux through the C 2 þ chnnel triggers C 2 þ relese from srcoplsmic reticulum, leding to contrction, which is modified y sympthetic stimultion. The C 2 þ influx is potentited y PKA-ctlysed phosphoryltion of the C nd 2 suunits (Go et l., 997; Kpiloff, 22). Since the sl PKA ctivity in the herts of Ovx rts ws significntly higher thn in the other two groups, the sl conductnce of C 2 þ chnnels would e expected to e higher. Indeed, our experiments demonstrted 64% elevtion. This increment ws function of PKA ctivity, since KT572, t concentrtion known to mximlly inhiit it, restored the chnnel ctivity to norml in myocytes from Ovx rts. Previous studies reported tht oth the expression nd ctivity of C 2 þ chnnels re significntly incresed in ER knockout mice (Johnson et l., 997), nd tht oestrogen replcement suppresses oth the expression nd ctivity of crdic C 2 þ chnnels in the Ovx rit (Ptterson et l., 998). We recently lso oserved tht the numer of L-type C 2 þ chnnels in left ventriculr myocytes from Ovx rts ws much higher thn in those from control nd Ovx þ E 2 rts (Km & Wong, unpulished dt). Bsed on these oservtions, we elieve * * British Journl of Phrmcology vol 44 (7)

9 98 K.W.L. Km et l Oestrogen, -drenoceptor nd PKA Tle 2 Crdic vriles in control, Ovx, nd groups Vriles Bseline Isoprenline ( 8 M) Fold-increse LVDP (mmhg) Con Ovx 872* * * LVEDP (mmhg) Con Ovx dp/dt mx (mmhg/s) Con Ovx * * 37.3* dp/dt mx (mmhg/s) Con Ovx * 84927* * HR (ets/min) Con Ovx *.647.6* Vlues re expressed s men7s.e.m. (n ¼ 8 in ech group). +dp/dt mx, the velocity of contrction; dp/dt mx, the velocity of relxtion, LVDP, left ventriculr developed pressure; LVEDP, left ventriculr end distolic pressure; HR, hert rte. *Po.5 vs Con. tht oth higher levels of PKA nd incresed density of L-type C 2 þ chnnels re responsile for the enhnced sl C 2 þ chnnel ctivity in the herts of Ovx rts. In contrst to sl C 2 þ trnsport, lockde of PKA with KT572, t concentrtion tht completely olished the effect of PKA on L-type C 2 þ chnnel ctivity, ws unle to reduce the chnnel ctivity in Iso-treted myocytes from Ovx rts to tht of myocytes from control nd Ovx þ E 2 rts. This result would not e expected if -ARnd C 2 þ chnnel coupling occurred only through PKA-dependent mechnism. There is evidence to support mechnism independent of PKA. A recent study on CHW firolsts stly trnsfected with crdic L-type C 2 þ chnnel nd 2 suunits long with either -or 2 -ARreveled PKA-independent preferentil coupling of the -ARto C 2 þ chnnels (Ytni et l., 999). -ARmy directly increse the C 2 þ chnnel ctivity in myocytes from Ovx rts without involving PKA. Furthermore, evidence supports direct interction of G s nd C 2 þ chnnels with -ARstimultion (Ytni & Brown, 989). We lso found tht oth the sl nd forskolin-stimulted camp ccumultion ws the sme in ll the experimentl groups, so the oestrogen does not ffect the ccumultion of camp or its response to ctivtion of AC. Since no phosphodiesterse inhiitor ws used in the camp ssy, the cellulr camp content represents lnce etween camp formtion vi AC nd rekdown vi PDE. The possiility tht AC nd PDE ctivities re ltered y the oestrogenic environment cnnot e excluded. We found in the present study tht 8 M Iso incresed LVDP nd þ dp/dt mx y 2.5- nd 3-fold, respectively in the herts from Ovx rts, which were much greter thn the corresponding increses in the norml femle rt herts (.3- nd 2-fold for LVDP nd þ dp/dt mx, respectively). The oservtion indictes significntly greter increse in contrctility following -ARstimultion in the herts from Ovx rts. In ddition to incresed PKA ctivity shown in the present study, it lso up-egultes the -ARin the herts of Ovx rts (Km et l., 24) nd increse in the C 2 þ sensitivity of myofilments (Wttnpermpool, 998) re lso responsile for incresed contrctility. -ARstimultion ctivtes two signlling pthwys. The first is the G s /AC/cAMP/PKA pthwy, which leds to n incresed influx of C 2 þ vi the C 2 þ chnnel nd to n incresed contrctility (Cmpell & Struss, 995). The second is through PKA-independent CMKII-medited poptotic signlling pthwy (Zhu et l., 23). During myocrdil ischemi, n incresed sympthetic dischrge ctivtes oth pthwys, leding to incresed crdic contrctility nd incresed poptosis. In ddition to poptosis, incresed contrction t time of insufficient oxygen my contriute to myocrdil infrct. In previous study, we showed tht oestrogen confers crdioprotection ginst myocrdil ischemi nd -ARstimultion with Iso in vitro (Km et l., 24). In the present study, we found tht oestrogen decresed the Iso-stimulted contrction. This suggested tht it my confer crdioprotection y reducing crdic contrction. In conclusion, the present study provides the first evidence tht oestrogen inhiits PKA ctivity, thus reducing C 2 þ influx vi the C 2 þ chnnel, which is responsile for reduction in E[C 2 þ ] i nd crdic contrction. However, oestrogen does not lter the expression of G s nd G i proteins, or camp ccumultion, or the responsiveness of PKA to camp. Upon -ARstimultion, the reduction in PKA ctivity in the presence of oestrogen is even greter thn in the sence of the hormone, nd is ccompnied y greter reductions in C 2 þ chnnel ctivity nd crdic responses. So, in norml femle rts, crdic responses to -ARstimultion re suppressed due to decresed sl PKA ctlytic ctivity in ddition to the downregultion of -ARshown previously (Km et l., 24). We thnk Dr I.C. Bruce for dvice on the writing of the mnuscript, Dr C.-M. Co for dvice on the mesurement of crdic vriles, nd Mr C.P. Mok for technicl ssistnce. This study ws supported y grnt to T.M.W. from the Reserch Grnt Council of Hong Kong (HKU732/M). K.W.L.K. ws supported y Postgrdute Studentship from the University of Hong Kong nd n Edwrd Youde Memoril Fund Scholrship. References BERS, D.M. (22). Crdic excittion contrction coupling. Nture, 45, BEUCKELMANN, D.J. & WIER, W.G. (988). Mechnism of relese of clcium from srcoplsmic reticulum of guine-pig crdic cells. J. 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10 K.W.L. Km et l Oestrogen, -drenoceptor nd PKA 98 CAMPBELL, D.L. & STRAUSS, H.C. (995). Regultion of clcium chnnels in the hert. Adv. Second Messenger Phosphoprotein Res., 3, CIRIC, O. & SUSIC, D. (98). Effect of isoprenline on lood pressure nd hert rte in different phses of the oestrous cycle. Endokrinologie, 76, CONRATH, C.E. & OPTHOF, T. (23). Bet3-drenoceptors in the hert. Crdiovsc. Res., 56, GAO, T., YATANI, A., DELL ACQUA, M.L., SAKO, H., GREEN, S.A., DASCAL, N., SCOTT, J.D. & HOSEY, M.M. (997). campdependent regultion of crdic L-type C 2+ chnnels requires memrne trgeting of PKA nd phosphoryltion of chnnel suunits. Neuron, 9, HAIKALA, H., KAHEINEN, P., LEVIJOKI, J. & LINDEN, I.B. (997). The role of camp- nd cgmp-dependent protein kinses in the crdic ctions of the new clcium sensitizer, levosimendn. Crdiovsc. Res., 34, JOHNSON, B.D., ZHENG, W., KORACH, K.S., SCHEUER, T., CATTERALL, W.A. & RUBANYI, G.M. (997). 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