phorbol ester, but not ischemic preconditioning, activates protein kinase d in the rat heart

J Mol Cell Cardiol 29, 2273–2283 (1997)

Phorbol Ester, but not IschemicPreconditioning, Activates Protein KinaseD in the Rat HeartGavin Brooks, Martin W. Goss, Enrique Rozengurt1 and Manuel GalinanesCardiovascular Research, The Rayne Institute, St Thomas’ Hospital, and 1Laboratory of GrowthRegulation, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London, UK

(Received 15 October 1996, accepted in revised form 30 April 1997)

G. B, M. W. G, E. R M. G. Phorbol Ester, but not Ischemic Preconditioning,Activates Protein Kinase D in the Rat Heart. Journal of Molecular and Cellular Cardiology (1997) 29, 2273–2283.The signal transduction pathways that mediate the cardioprotective effects of ischemic preconditioning remainunclear. Here we have determined the role of a novel kinase, protein kinase D (PKD), in mediating preconditioningin the rat heart. Isolated rat hearts (n=6/group) were subjected to either: (i) 36 min aerobic perfusion (control);(ii) 20 min aerobic perfusion plus 3 min no-flow ischemia, 3 min reperfusion, 5 min no-flow ischemia, 5 minreperfusion (ischemic preconditioning); (iii) 20 min aerobic perfusion plus 200 nmol/l phorbol 12-myristate 13-acetate (PMA) given as a substitute for ischemic preconditioning. The left ventricle then was excised, homogenizedand PKD immunoprecipitated from the homogenate. Activity of the purified kinase was determined followingincubation with [c32P]-ATP±syntide-2, a substrate for PKD. Significant PKD autophosphorylation and syntide-2phosphorylation occurred in PMA-treated hearts, but not in control or preconditioned hearts. Additional studiesconfirmed that recovery of LVDP was greater and initiation of ischemic contracture and time-to-peak contracturewere less, in ischemic preconditioned hearts compared with controls (P<0.05). Our results suggest that the earlyevents that mediate ischemic preconditioning in the rat heart occur via a PKD-independent mechanism.

1997 Academic Press Limited

K W: Cardioprotection; Ischemia; Phosphorylation; Signal transduction.

in rabbits that adenosine is involved (Liu et al.,Introduction1991), probably via the activation of one or moreprotein kinase C (PKC) isoforms (Liu et al., 1994).The phenomenon of ischemic preconditioning,

where brief non-lethal episodes of ischemia protect Furthermore, a number of investigators have pro-vided evidence that PKC may also mediate theor “precondition” the heart and render the myo-

cardium resistant to a subsequent, more sustained protective effects of preconditioning in rat hearts(Speechly-Dick et al., 1994; Hu and Nattel, 1995;ischemic insult, remains one of the most effective

ways of protecting the heart from ischemia-induced Mitchell et al., 1995). However, despite these re-ports, the involvement of PKC as a general mediatortissue injury. This form of protection has now been

shown to occur in all species tested, including man of preconditioning has been questioned. For ex-ample, other investigators have failed to show PKC(Murry et al., 1986; Lawson and Downey, 1993;

Yellon et al., 1993; Walker et al., 1995; Gho et activation in the rabbit (Simkhovich et al., 1995)and the rat (Galinanes et al., 1996; Moolman etal., 1996). However, there is some uncertainty

regarding the signal transduction mechanism(s) al., 1996). Furthermore, Vogt et al. (1996) andPrzyklenk et al. (1996) recently failed to dem-that mediate this effect. It has been demonstrated

Please address all correspondence to: Dr Gavin Brooks, Cardiovascular Research, The Rayne Institute, St Thomas’ Hospital, LondonSE1 7EH, UK.

0022–2828/97/082273+11 $25.00/0 mc970466 1997 Academic Press Limited

G. Brooks et al.2274

onstrate a role for PKC in the pig or dog, respectively. (75 mmHg) by the Langendorff method at 37°Cwith oxygenated perfusion solution (in mmol/l:Thus, the precise mechanism of preconditioning

remains elusive and has recently become the subject glucose 11.1, NaCl 118.5, KCl 4.8, MgSO4 1.2,KH2PO4 1.2, NaHCO3 25.0, CaCl2 1.4 at pH 7.4of some controversy (Brooks and Hearse, 1996).

In an effort to determine signal transduction when gassed with 95% 02, 5% CO2). All heartswere paced via the right atrium at 400 beats/minpathways other than PKC that may mediate the

early signalling events leading to preconditioning during the pre-ischemic and post-ischemic periodsand during the first 2 min of ischemia. During thein the rat, we have compared the abilities of a

phorbol ester which has been shown to simulate pre-ischemic period of aerobic perfusion, a balloonwas introduced into the left ventricle (LV) andpreconditioning in the rat (Hu and Nattel, 1995),

and ischemic preconditioning to activate the novel inflated to achieve a constant LV end-diastolic pres-sure (LVEDP) of 4 mmHg; this volume was keptenzyme, protein kinase D (PKD). PKD is a serine/

threonine kinase that is distinct from PKC. It was constant for the remainder of the experiment. Theballoon was used to measure LV developed pressurecloned in 1994 from a Swiss 3T3 mouse fibroblast

cDNA library (Valverde et al., 1994) as a gene (LVDP) and LVEDP. Coronary flow (CF) before andafter ischemia was measured by timed collection ofencoding for a protein of 918 amino acids with a

predicted Mr of 102 kDa. The purified protein mi- the coronary effluent.grates with a Mr of 110 kDa by sodium do-decylsulphate-polyacrylamide gel electrophoresis.(SDS-PAGE) and, in common with many of the Experimental time course and study groupsPKC isoforms, PKD binds both phorbol esters anddiacylglycerols with high affinity, and is activated After excision, hearts were aerobically perfused and

subjected to one of the two protocols shown inboth in vitro and in vivo by these agonists (Valverdeet al., 1994; Van Lint et al., 1995; Zugaga et al., Figure 1. In study 1, three groups of hearts (n=6/

group) were studied: group 1 (controls), aerobically1996). Although both enzymes are activated byphorbol esters, PKD is distinct from the PKC family perfused for 36 min; group 2 (ischemic pre-

conditioning), following 20 min aerobic perfusion,of isoforms in a number of respects (see later). Sincepreconditioning has been shown to be mimicked hearts were preconditioned with 3 min ischemia

followed by 3 min reperfusion, then 5 min ischemiaby PKC activators in some species, including therat (Speechly-Dick et al., 1994; Hu and Nattel, followed by 5 min reperfusion; and group 3 (PMA

treated), following 20 min aerobic perfusion, hearts1995; Mitchell et al., 1995), we have determinedwhether PKD could play a role in mediating the received PMA (200 nmol/l) for 3 min followed by

3 min wash-out, then PMA for a further 5 mincardioprotective effects of ischemic preconditioning.Thus, in the present study, we have investigated followed by 5 min wash-out. At the end of each

protocol, hearts were removed from the perfusionwhether PKD is activated during ischemic pre-conditioning in the rat myocardium, and whether rig and the left ventricle was carefully excised and

processed for PKD analysis. In study 2, hearts (n=a stimulatable PKD pathway exists in the rat heartthat can be activated by phorbol esters. 6/group) were subjected to a protocol that was

identical to that for study 1, with the exceptionthat following each protocol they were subjectedto a further 35 min of global ischemia followed by40 min of reperfusion. Preliminary studies in ourMaterials and Methodslaboratory have established the effectiveness of thisischemic preconditioning protocol in the rat v otherAnimals and perfusion proceduresprotocols that utilise 2×3 min or 2×5 min isch-emic episodes. The 1×3 min+ 1×5 min protocolAdult male Wistar rats (230–270 g) were obtained

from Binton and Kingman, Hull, UK. Animals re- provides optimal protection with minimal arrhy-thmogenic effects (Galinanes, unpublished ob-ceived appropriate care in accordance with the UK

Home Office Animals (Scientific Procedures) Act, servations).1986. Rats were anesthetized with pentobarbitol(60 mg/kg, i.p.), the right femoral vein was exposedand heparin (1000 IU/kg) was administered. The Reagentschest was opened and the heart excised and placedin cold (4°C) normal saline. The aorta then was [c-32P]ATP (AA0018; 3000 Ci/mmol), donkey anti-

rabbit Ig horseradish peroxidase-conjugated anti-cannulated rapidly and each heart was perfused

Protein Kinase D and Preconditioning 2275

PMA

Study 2

Group 3 20 min 3 5 35 min 40 min

IschemicpreconditioningGroup 2 20 min 3 5 35 min 40 min

AerobicperfusionGroup 1 36 min 35 min 40 min

PMA

Study 1

Group 3 20 min 5 min3 min 5 min

IschemicpreconditioningGroup 2 20 min 5 min3 min 5 min

AerobicperfusionGroup 1 36 min

3 5

3 5

3 min

3 min

Figure 1 Schematic representation of the perfusion protocols used in this study. PMA was used at a final concentrationof 200 n. n=6 hearts per group. (Φ) Aerobic perfusion/reperfusion period; (Ε) ischemic period; (∆) 200 nmol/lPMA.

body and ECL Western Blotting reagents were pur- of 3H-thymidine over a 48 h-period, according tomethods previously described (Brooks et al., 1993).chased from Amersham International PLC, UK.

Murine PKD anti-peptide polyclonal antibody was All cells were kept in an humidified atmospherecontaining 5% CO2, 95% air at 37°C.raised against the synthetic peptide EER-

EMKALSERVSIL (Van Lint et al., 1995). Phorbol12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDB) were obtained from Calbiochem-

Protein preparation and immunoprecipitation analysisNovabiochem (UK) Ltd (Nottingham, UK) and madeup as 5 mg/ml stock solutions in ethanol and stored

Protein samples were prepared from left ventricularin the dark at−20°C. Phorbol esters were diluted totissue as follows: tissue was cut into small pieces20 lmol/l stock solutions in Krebs–Henseleit bufferand as quickly as possible placed into an ice-coldimmediately before use, and all further dilutionslysis buffer containing: 50 mmol/l Tris HCl (pH 7.4),were made directly from these stock solutions in2 mmol/l ethylenediaminetetraacetic acid (EDTA),Krebs–Henseleit buffer. All other chemicals used2 mmol/l ethylene glycol-bis(b-aminoethylether)were of the purest grade available commercially.tetraacetic acid (EGTA), 2 mmol/l dithiothreitol,10 lg/ml leupeptin, 1 lg/ml pepstatin, 10 lg/mlaprotonin, 1 mmol/l AEBSF (Pefabloc) and 1.0%Triton X-100, and homogenized using a polytronCell culturehomogenizer. Samples then were centrifuged atmaximum speed in a benchtop microfuge (Heraeus)Murine Swiss 3T3 fibroblasts and Rat 1 fibroblasts

were cultured routinely, as described previously for 30 min at 4°C. Supernatants (200 ll) were thenimmunoprecipitated with the PKD antibody (1:100(Brooks et al., 1993; Herget et al., 1994). Cells were

maintained in Dulbecco’s Modified Eagle’s Medium dilution) overnight at 4°C on a rotating blood wheel.Twenty ll of protein A sepharose (100 mg/ml in(DMEM) containing 10% fetal calf serum (FCS), and

were rendered quiescent by maintaining confluent lysis buffer) was then incubated with each samplefor 1 h at 4°C on a rotating blood wheel, followedmonolayers in the above medium for 7–8 days.

That cells were quiescent and in the Go phase of by centrifugation at maximum speed in a benchtopmicrofuge for 2 min at 4°C. The pellet was washedthe cell cycle was demonstrated by fluorescence-

activated cell sorting analysis and by incorporation twice with lysis buffer then twice with Kinase Assay


Buffer (30 mmol/l Tris, pH 7.4; 15 mmol/l MgCl2). constituted the cytosolic fraction. The remainingpellet was resuspended by homogenization in 750 llKinase Assay Buffer then was added to each pellet

to obtain a final volume of 30 ll. of ice-cold Buffer A containing 5 mmol/l EDTA,10 mmol/l EGTA and 1% Triton X-100, made upto a volume of 1.5 ml and kept on ice for 15 min.The sample then was centrifuged at 200 000×gIn vitro PKD activation assayfor 1 h at 4°C and the supernatant retained (TritonX-100 soluble membrane fraction). The remainingThe immunoprecipitated PKD samples described

above (in 30 ll) were added to the following re- pellet was resuspended in 1.5 ml Buffer A con-taining 5 mmol/l EDTA, 10 mmol/l EGTA and 1%agents in an Eppendorff tube: 2.5 ll 1 mmol/l ATP,

0.4 ll [c32P]-ATP, 2.1 ll Kinase Assay Buffer and Triton X-100. This fraction constituted the TritonX-100 insoluble membrane fraction. After pre-5 ll 10 mg/ml syntide-2. Each reaction then was

incubated at 30°C for 20 min. Eighty ll of Kinase paration, an equal volume of 2X Sample Bufferwas added to each subcellular fraction. Fifty lg ofAssay Buffer then was added to each reaction tube

followed by centrifugation of samples for 15 s at cytosolic protein was loaded per lane and a volumeequal to the volume loaded for the cytosolic fraction4°C. One hundred ll of the supernatant then was

spotted onto P-81 phosphocellulose filter papers was loaded for both Triton X-100 soluble membraneand Triton X-100 insoluble membrane fractionsand allowed to air dry. Non-incorporated [c32P]-

ATP was removed by washing the filters four times from the same heart. Proteins were separated bySDS-PAGE and immunoblotted as described below.(5 min each) in 5% acetic acid. The amount of

incorporated radioactivity into syntide-2 was thendetermined by adding 3 ml of scintillation cocktailto each filter paper in a scintillation vial and samples

Immunoblot analysiscounted in a b-counter.

Proteins were separated electrophoretically on 8%SDS-polyacrylamide gels and transferred to PVDF

PKD autophosphorylation assaymembranes (Immobilon-P, 0.45 lm, Millipore, UK)using a semi-dry blotting apparatus (LKB Multiphor

Immunoprecipitated protein A sepharose-PKD com-II, Pharmacia, UK) and a transfer buffer containing

plexes were incubated exactly as described above0.037 mol/l Tris base, 0.039 mol/l glycine, 0.04%

for the PKD in vitro kinase assay, except that anSDS and 20% methanol. Membranes were blocked

equal volume of 2X Sample Buffer (0.125 mol/l Trisfor 2 h at room temperature in Tris buffered saline

HCl, pH 6.8, 10% b-mercaptoethanol, 4.6% SDS,(TBS)/0.2% Tween 20 containing 5% non-fat milk

20% glycerol) was added to the reaction at the endfollowed by incubation overnight at 4°C with the

of the kinase assay, and the resultant mix boiledrabbit anti-mouse PKD antibody (1:2500 dilution)

for 10 min followed by storage on ice for 10 min.in TBS/0.2% Tween 20 containing 1% non-fat milk.

The samples then were centrifuged briefly and theThe membrane then was washed twice (10 min

supernatants loaded onto 8% SDS-PAGE gels andeach) with TBS/0.2% Tween 20 prior to incubation

proteins separated according to their molecularwith donkey anti-rabbit horseradish peroxidase-

weights. Resolved gels were dried under vacuumconjugated antibody (30 min at room temperature,

and phosphorylated proteins visualized by auto-1:2000 dilution) in TBS/0.2% Tween 20 containing

radiography at −70°C with intensifying screens.1% non-fat milk. Filters were washed four times inTBS/0.2% Tween 20 (5 min each) followed by one5 min wash in TBS and incubated for 1 min

Subcellular fractionation of PKD from rat heartwith ECL Western Blotting Reagents (AmershamInternational) prior to exposure to film. The res-

Fractionation of LV samples into cytosolic and mem-ultant autoradiographs were quantified using den-

brane fractions was achieved by homogenizationsitometric scanning (LKB Densitometer).

of LV tissue in 10 ml ice-cold extraction buffer(Buffer A) containing 50 mmol/l Tris HCl (pH 7.5),1 mmol/l DTT, 10 lg/ml leupeptin, 1 lg/ml pep-statin, 10 lg/ml aprotonin and 1 mmol/l AEBSF. Protein determinationHomogenates were aliquoted into 1.5 ml fractionsand centrifuged at 200 000×g for 1 h at 4°C. The Protein concentrations in homogenized tissue and/

or cell preparations were determined according tosupernatant from each fraction was retained and


(P<0.05). In addition, the magnitude of peak con-tracture was greater in the preconditioned group(89.0±4 mmHg) than in control hearts(66.0±4 mmHg). However, compared to controlvalues, PMA did not significantly affect either para-meter (10.5±0.9 min, 16.0±0.5 min and68.0±5 mmHg respectively, P=..).

Post-ischemic recovery of cardiac function35

100

01

Duration of ischemia (min)

Lef

t ve

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pres

sure

(m

mH

g)

15

80

60

40

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3 5 9 11 19 21 25 297 13 17 23 27 31 33

Figure 3(a) shows that post-ischemic recovery ofFigure 2 Graph to show the profiles of contracture LVDP after 40 min of reperfusion was only 19±5%during 35 min of ischemia. (Β) controls; (Χ) ischemic in control hearts, whereas the recovery with isch-preconditioning; (Ε) PMA (see text for details). Each

emic preconditioning was 69±4% (P<0.05). Nopoint represents the mean±... of six hearts.significant protective effect was observed with PMAwhere hearts recovered only 14±3% of the pre-ischemic values. Post-ischemic LVEDP [Fig. 3(b)]the method of Bradford (1976) using bovine serumwas 69±4 mmHg in control hearts, whereas thisalbumin as a standard.value was significantly lower in ischemic pre-conditioned hearts (24±4 mmHg, P<0.05). Again,PMA failed to improve recovery compared withStatistical analysiscontrol values (60.0±4 mmHg, P=..). As ob-served with LVDP, the post-ischemic recovery of CFResults were expressed as mean±standard error of[Figure 3(c)] was significantly improved by ischemicthe mean (...) and analysed using ANOVA andpreconditioning (76±3% v 54±3% in controlthe Bonferonni t-test. Values were considered stat-hearts; P<0.05). In contrast, PMA resulted in aistically significant at P<0.05.significantly poor recovery of CF (18±2%, P<0.05v control and ischemic preconditioned hearts).

Results

All hearts that were entered into the study were PKD expression in the rat heartincluded in the analysis. The mean pre-ischemicvalues for LVDP (range 118±7 to 127±7 mmHg) In order to determine that the mouse PKD antibodyand CF (range 12.3±0.6 to 13.3±0.6 ml/min) for cross-reacted with rat tissue, we carried out im-the two studies were similar in all groups. munoblot analyses with mouse Swiss 3T3 fibroblast

and Rat 1 fibroblast cell lysates. As shown in Figure4, PKD protein was expressed in both cell lines

Effects of PMA on cardiac function with a Mr of >110 kDa, although higher levels ofexpression were detected in Rat 1 cells. The presence

The infusion of PMA had a rapid and profound of a second immunoreactive band with a lower Mr

effect on cardiac function such that LVDP was of>100 kDa also was detected at consistently highdecreased by 53±3% and CF was reduced by levels in Rat 1 cells and weakly in Swiss 3T352±2% of the 20 min aerobic control values. fibroblasts, although the nature of this band was

not investigated further in the present study. Thespecificity of antibody binding was demonstratedby competition with immunizing peptide (Fig. 4,Effects of PMA and ischemic preconditioning on

ischemic contracture lane 3). Also shown in Figure 4, is the expressionof PKD protein in the left ventricular tissue lysate

As shown in Figure 2, the initiation of contracture obtained from an adult rat heart. Two immuno-reactive bands were detected in rat LV tissue thatand the time-to-peak contracture were shortened

significantly by ischemic preconditioning from migrated at exactly the same Mr as the two bandsobserved in Rat 1 cells, and both were competed11.7±0.8 min and 15.8±0.5 min in control hearts

to 4.8±0.3 min and 9.2±0.8 min, respectively out when the immunoprecipitation reaction was


1 2 3 4Mr(kDa)

97.4

PKD

Figure 4 Autoradiograph of an immunoblot showingexpression of PKD protein in Swiss 3T3 fibroblasts (lane1), Rat 1 fibroblasts (lane 2), Rat 1 fibroblasts wherethe PKD antibody has been incubated with immunizingpeptide (lane 3), and adult rat heart (lane 4). Cell andtissue lysates were prepared as described in Materials andMethods and 50 lg protein was loaded per lane onto 8%SDS/PAGE gels. Resolved proteins were transferred tonitrocellulose and probed with the anti-PKD antibody.Detection of immunoreactive antigens was with donkeyanti-rabbit Ig, conjugated to horse-radish peroxidase andthe enhanced chemiluminescence system from Amer-sham International Inc. as described in Materials andMethods.

carried out in the presence of immunizing peptide(lane 3).

PKD activation by ischemic preconditioning and PMA

Having established that PKD is expressed in the ratmyocardium, we next investigated the ability of theenzyme to be activated in vivo by phorbol esters inRat 1 cells and by phorbol esters and ischemicpreconditioning in the rat myocardium by twomethods: autophosphorylation and phos-phorylation of syntide-2, a substrate of PKD usedalso by calmodulin-dependent protein kinases(Bruder et al., 1992; Lorca et al., 1993; Mochizukiet al., 1993). Unlike PKC, PKD can be im-munoprecipitated as an active kinase, since it hasbeen proposed that activation of PKD results in acovalent modification of the enzyme (Van Lint etal., 1995). We have utilized this property in Figure5(a), which shows an autoradiograph of im-munoprecipitated PKD obtained from Swiss 3T3fibroblasts that have been pretreated for 10 mineither with vehicle, 200 nmol/l PMA or 400 nmol/

80

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(%

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Control

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*

l PDB (lanes 1–3), and Rat 1 fibroblasts that havebeen pretreated in a similar manner (lanes 4–6).Figure 3 Post-ischemic recoveries of (a) left ventricular

developed pressure (LVDP); (b) left ventricular end-dia- The figure clearly shows that phorbol esters inducestolic pressure (LVEDP); and (c) coronary flow (CF) in a rapid (within 10 min) activation and subsequentcontrol, ischemic preconditioned and PMA-treated hearts autophosphorylation of PKD in both mouse and(see Fig. 1 and text for further details) subjected to 35

rat cells, with a 110 kDa phosphoprotein beingmin of global ischemia and 40 min reperfusion. Columnsobserved. Although PKD has been shown to berepresent the mean±... ∗P<0.05 when compared

with controls. activated in intact cells by PKC (Zugaza et al.,1996), the phosphorylation of PKD observed in ourstudies occurs as a result of autophosphorylation


Con

trol

3T3

Mr(kDa)

97.4

PKDP

MA

PD

B

Con

trol

Rat 1

PM

A

PD

B

(a)

1 2 3 4Mr(kDa)

97.4

PKD

(b)

5 6 7

Figure 5 (a) Autoradiograph of PKD autophosphorylation in Swiss 3T3 fibroblasts and Rat 1 fibroblasts. (b)Autoradiograph of PKD autophosphorylation in left ventricular tissue obtained from hearts that were either: aerobicallyperfused (lanes 1,2), ischemic preconditioned (lanes 3,4) or PMA-treated (lanes 5,6). Lane 7 shows competition forantibody binding by immunizing peptide when included in an immunoprecipitation reaction with a PMA-treated heartsample. Immunoprecipitated PKD from the above sources was incubated with [c32P]-ATP as described in Materials andMethods and the phosphorylation reaction resolved by 8% SDS/PAGE. The result shown is representative of at least sixseparate experiments.

of the enzyme since (i) PKD was purified by im- phosphoprotein. Figure 5(a) demonstrates also thatthere is a higher level of PKD autophosphorylationmunoprecipitation before the addition of [c32P]-ATP

to the in vitro kinase assay (i.e. no contaminating in Rat 1 cells than in mouse Swiss 3T3 fibroblasts.Figure 5(b) shows an autoradiograph of im-PKC was present); (ii) PKC does not retain its

activity following immunoprecipitation (see above), munoprecipitated PKD obtained from control (lanes1 and 2), preconditioned (lanes 3 and 4) and PMA-and even if another kinase that is highly homo-

logous to PKD in the region recognized by the treated (lanes 5 and 6) hearts that have undergonean autophosphorylation reaction in the presence ofantibody were co-immunoprecipitated by the PKD

antibody, it would be very unlikely to be both active [c32P]-ATP followed by separation of 32P-labelledproteins by SDS-PAGE. Perfusion of hearts withand activate PKD; and (iii) recent studies have

shown that a kinase-defective PKD mutant, in PMA resulted in a rapid and significant auto-phosphorylation of PKD observed as a 110 kDawhich the Lys 618 residue within the ATP binding

site was substituted by a methionine residue, when phosphoprotein. This response was inhibited whenthe immunoprecipitation reaction was carried outtransfected into COS-7 cells, was not activated by

phorbol ester treatment, whereas wild-type PKD in the presence of the immunizing peptide (lane 7),confirming the specificity of the antibody for PKD.was activated (Zugaza et al., 1996). Taken together,

these results demonstrate that phorbol ester-in- In contrast, hearts that had undergone ischemicpreconditioning (Group 2) or aerobic perfusionduced protein kinase activity measured in PKD

immunoprecipitates is due to activation of PKD (Group 1) failed to induce autophosphorylation ofthe slower migrating 110 kDa PKD protein.and not to activation of a co-immunoprecipitating

kinase. We also determined the ability of PKD to phos-phorylate the substrate syntide-2 in cells of ratAn additional phosphoprotein that migrates with

a lower molecular weight also is observed in all origin by incubating Rat 1 fibroblasts with PMAand PDB for 10 min followed by im-samples from Rat 1 cells, in an analogous manner

to the immunoblot studies (Fig. 4), although it munoprecipitation with the PKD antibody. The im-munoprecipitated PKD was incubated with [c32P]-would appear that only the upper band, which

comigrates with PKD in Swiss 3T3 cells, is phos- ATP and syntide-2. Both PMA and PDB induced asignificant level of syntide-2 phosphorylationphorylated significantly upon treatment with phor-

bol esters compared with control cells. This was (140.0±9.7% and 136.4±13.4% of controlvalues, respectively, P<0.05) in Rat 1 cells. Theconfirmed by densitometric scanning of all bands,

which demonstrated that the faster migrating band response was more pronounced in Swiss 3T3 fibro-blasts with PMA and PDB producing 278.0±34.6%did not increase in intensity between treatment

groups (data not shown). Densitometric analysis of and 244.8±29.1% of control values, respectively(P<0.05). The reason for this difference betweenthe autoradiograph shown in Figure 5(a) dem-

onstrated that, in Swiss 3T3 fibroblasts, PMA and Swiss 3T3 fibroblasts and Rat 1 fibroblasts probablyreflects high basal phosphorylation levels in Rat 1PDB induced a 4.7-fold and 5.5-fold increase in the

degree of PKD autophosphorylation, respectively, cells, similar to the autophosphorylation resultsshown in Figure 5(a). Figure 6 shows that in ratwhereas, in Rat 1 cells, these same phorbol esters

induced a two-fold and 1.8-fold increase in phos- hearts, PMA was able to activate PKD, as measuredby the ability of immunoprecipitated PKD fromphorylation, respectively, of the upper 110 kDa


C M PMr(kDa)

97.4

PKD

PMAIschemic

preconditioningControl

C M P C M P

Figure 7 Subcellular distribution of PKD in rat leftventricular tissue obtained from hearts that were either:aerobically perfused controls, ischemic preconditioned orPMA-treated. Subcellular fractionation and separation ofproteins by SDS-PAGE were carried out exactly as de-scribed in the Methods section. C, cytosolic fraction; M,Triton X-100 soluble membrane fraction; P, Triton X-100 insoluble membrane fraction.

Subcellular distribution of PKD in rat heart

Downey and colleagues have previously proposeda two-phase process to explain many of the ob-servations associated with PKC and preconditioning(Liu et al., 1994). They suggest that during thepreconditioning ischemic period, PKC is trans-located to the sarcolemma in the absence of ac-tivation and phosphorylation of the enzyme andsubstrate proteins. Activation of the enzyme, asmeasured by autophosphorylation and substratephosphorylation, then occurs during the longerperiod of ischemia. In order to determine whetherthe Downey hypothesis could also be applied to PKD,we measured the ability of ischemic preconditioningand PMA to induce translocation of PKD in the ratheart. Figure 7 shows that in the rat heart, the

200

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majority of the PKD is associated with the TritonFigure 6 Phosphorylation of syntide-2 by im- X-100 insoluble membrane fraction (lanes markedmunoprecipitated PKD obtained from rat left ventricular P); however, a portion of the enzyme is found intissue obtained from hearts that were either: aerobically

the cytosolic fraction (lane marked C) when theperfused controls, ischemic preconditioned or PMA-autoradiograph is overexposed (as in Fig. 7), withtreated. The results shown in Figure 6(a) represent means

and standard deviations obtained from three separate no detectable levels of protein in Triton X-100immunoprecipitation reactions carried out on each heart soluble fractions (lane marked M) in control hearts.with two hearts per group. The results shown in Figure A similar subcellular distribution pattern of ex-6(b) represent the means and standard deviations from

pression was observed in quiescent Rat 1 fibroblastseight separate experiments and show the percentage(data not shown). Whereas treatment of heartssyntide-2 phosphorylation in ischemic preconditioned

and PMA-treated hearts compared to aerobically perfused with PMA leads to a translocation of cytosolic PKDcontrol hearts. ∗P<0.05 v control and ischemic pre- to the Triton X-100 soluble membrane fraction,conditioning group. ischemic preconditioning failed to induce such a

response with levels not being significantly differentto those found in control hearts (Fig. 7). In-terestingly, PMA treatment also resulted in a loss ofPMA-treated hearts to induce phosphorylation of

syntide-2. In contrast, PKD immunoprecipitated immunoreactive PKD in the Triton X-100 insolublefraction without a concomitant rise in PKD levelsfrom control hearts or from hearts that had under-

gone preconditioning was unable to induce phos- in any of the other fractions. The reason for thisfinding remains unknown, but may represent aphorylation of this substrate (Fig. 6), suggesting

that ischemic preconditioning is unable to activate conformational change in the PKD molecule orcytoskeletal elements, that comprise the Triton X-PKD during the first (3 min) and second (5 min)

ischemic episodes. 100 insoluble fraction, that affect the binding of


the PKD antibody. Indeed, the effects of PMA on Despite the distinct differences between PKD andPKC isoforms, some investigators have maintainedcontracture were significant (Fig. 2) suggesting that

such conformational changes could be responsible that PKD is indeed an isoform of PKC and that itrepresents the murine homologue of the humanfor this result.PKCl isoform (Johannes et al., 1994). Despite a92% homology to PKD (extending to 98% in thecatalytic domain), initial reports suggested thatDiscussionPKCl was distinct from PKD (Johannes et al., 1994,1995). However, more recent studies have shownThe early signal transduction mechanisms that

mediate the cardioprotective effects of ischemic pre- that PKCl is distinct from the known PKC isoformfamilies (Gschwendt et al., 1996) and, therefore,conditioning remain obscure, although phorbol

ester-mediated pathways have been suggested to PKD and PKCl probably represent the same genein different species.play a pivotal role (Liu et al., 1991; Hu and Nattel,

1995; Mitchell et al., 1995; Gho et al., 1996). Until In this study we report, for the first time, theeffects of ischemic preconditioning on PKD ac-recently, phorbol esters were known only to directly

activate members of the cPKC and nPKC subfamilies tivation. Ischemic preconditioning failed to activatePKD using two separate measures of enzyme ac-of PKC isoforms. However, in 1994 a novel kinase

that was distinct from PKC, named PKD, was cloned tivation viz. autophosphorylation and phos-phorylation of syntide-2. Furthermore, ischemicfrom a mouse cDNA library that bound, and was

activated by, phorbol esters and diacylglycerols in preconditioning failed to induce a significant trans-location of PKD from the cytosolic to Triton X-100a calcium-independent manner (Valverde et al.,

1994). PKD has been shown to be distinct from soluble fraction in rat heart. Thus, the hypothesisproposed by Downey and colleagues (Cohen andthe PKC family of isoforms in a number of ways.

For example, (i) the length of the sequence sep- Downey, 1993; Liu et al., 1994), that translocationof PKC occurs during the preconditioning ischemia,arating the Zn-finger cysteine-rich motifs is 95

amino acids long in PKD, but either 28 amino acids whereas enzyme activation and phosphorylation ofsubstrate proteins occurs during the subsequentor 35 amino acids in the cPKC family or nPKC

family, respectively; (ii) the amino acid residues long ischemic period, does not apply to PKD. Takentogether, our results provide strong evidence thatAla-146 and Ala-154 in the consensus sequence

of the cysteine-rich region in PKD differ from those activation of the PKD signal transduction pathwaydoes not serve as an early requirement for thein PKCs; (iii) PKD does not contain sequences with

homology to the typical PKC pseudosubstrate motif cardioprotective effect of preconditioning. Fur-thermore, the fact that we were unable to mimicthat is found upstream of the cysteine-rich region,

although it is found in all PKC isoforms from all preconditioning with PMA (i.e. absence of ac-celeration of ischemic contracture and failure tospecies; (iv) the motif in the kinase domain that

guides peptide substrates into the correct ori- improve post-ischemic recovery of cardiac func-tion), despite activating PKD, in the rat heart sug-entation such that catalysis can occur is YRDLKLDN

in all PKCs, but in PKD it is different in every gests that a phorbol ester–mediated signallingpathway is not essential for preconditioning in ourvariable residue (HCDLKPEN); (v) comparisons of

the regulatory and catalytic domains of PKD with system. In contrast to these results, Hu and Nattel(1995) recently showed that PMA, at a dose ofother kinases clearly establish PKD as a novel type

of protein kinase, distinct from PKC (Valverde et al., 100 nmol/l, was able to simulate preconditioningin the rat heart. At the present time, we are unable1994; Van Lint et al., 1995), and it has been

demonstrated that the substrate specificities of these to provide an obvious reason for the lack of pro-tection with PMA treatment in our hands, excepttwo kinases are very different. Thus, whereas all

members of the PKC family can catalyse the phos- to report the finding as a reproducible phenomenon.However, we do not discount the possibility thatphorylation of a peptide based on the pseud-

osubstrate domain of PKCe, PKD cannot catalyse the dose of PMA used had toxic effects on the heart,such that any beneficial effect due to pre-phosphorylation of this substrate (Van Lint et al.,

1995). However, PKD efficiently catalyses the phos- conditioning was obscured. However, similar con-centrations of phorbol esters have previously beenphorylation of syntide-2 (Van Lint et al., 1995); and

(vi) PKD can be immunoprecipitated as an active shown to produce no ultrastructural damage, evenwhen l doses of these compounds were perfusedkinase, whereas this is not possible with PKC iso-

forms, since they lose their active conformation for up to 30 min (Watson and Karmazyn, 1991).Furthermore, we have shown in pilot studies thatupon immunoprecipitation with specific antibodies.


a lower dose of 40 nmol/l PMA produced identical translation of PKD resulting in two proteins ofdifferent molecular weight. Consistent with thishemodynamic effects on the heart as 200 nmol/l

PMA (data not shown). However, it remains a possibility, is that the amino acid sequence showsthe presence of additional methionine initiationpossibility that, if the concentrations of phorbol

ester used in this study did lead to toxic hemo- codons, and these are present at positions thatwould not be inconsistent with our data showingdynamic effects, thereby masking any protective

effect, direct activation of PKD with a lower, non- the presence of two PKD proteins that migrate withdifferent sizes in SDS-PAGE; and (iii) alternativetoxic dose could still trigger protection. However,

whether such low doses of phorbol ester would be splicing of the PKD gene, which could result intranscription and subsequent translation of twosufficient to activate PKD in the heart would need

to be determined. Despite the fact that PMA does proteins of distinct size. In support of the lattertwo possibilities, both bands were competed outnot mimic preconditioning in our model, it does

serve as an ideal positive control for PKD activation. when the immunoprecipitations were carried outin the presence of immunizing peptide, and theyInterestingly, our results with PMA are in ac-

cordance with the hypothesis proposed by Downey both still could be detected when the stringency ofwashing was increased (data not shown), suggestingand colleagues, since we show that early activation

and phosphorylation is not correlated with pre- that both proteins are recognized specifically bythe PKD antibody. However, we cannot discountconditioning. Furthermore, the fact that PMA ac-

tivated PKD in the time frame studied indicates that the possibility that a protein completely unrelatedto PKD, but which has the same amino acidischemic preconditioning is unlikely to mediate its

effects through this enzyme in the rat. However, it sequence as the synthetic peptide used to raisethe antibody, is not reacting with the polyclonalmight be pertinent to investigate the potential role

of PKD in species other than the rat, e.g. the dog antibody. A determination of the exact nature ofthe additional band requires further experimentaland the pig, where the role of PKC in mediating

the protective effects of preconditioning remains examination and studies are currently underway toclone and sequence this additional “PKD isozyme”controversial (Brooks and Hearse, 1996).

Despite the fact that PKD does not appear to be from a rat cDNA library.In summary, we have demonstrated by moreactivated in the early stages of preconditioning, we

have demonstrated for the first time the existence than one method that PKD is activated in therat heart by phorbol esters, but not by ischemicof an active PKD pathway in intact rat heart, and

have shown that this can be activated in a phorbol preconditioning. In addition, we have shown thatphorbol esters do not provide the protection againstester-dependent manner. It has recently been dem-

onstrated that PKD is regulated by a dual mech- ischemia-induced cardiac dysfunction observedwith ischemic preconditioning. These results sug-anism in intact cells, such that phorbol esters,

diacylglycerol or growth factors can stimulate PKD gest that, in the rat model, a phorbol ester-mediatedpathway is not involved in the cardioprotectivedirectly and/or induce PKD phosphorylation/ac-

tivation via nPKCs (Zugaza et al., 1996). Thus, it effect afforded by ischemic preconditioning. How-ever, we have demonstrated for the first time theis likely that this dual mechanism of regulation is

functional in rat heart. existence of an active PKD pathway in rat heartand have shown that this can be activated in aIt was interesting to note that in Rat 1 cells

and in rat myocardial tissue the PKD antibody phorbol ester-dependent manner.recognized two distinct phosphoproteins at 110 kDaand >100 kDa. The reason why two immuno-

Acknowledgementsreactive proteins were detected by immunoblottingand in autophosphorylation analyses in rat tissues

We wish to thank the Leopold Muller Foundation,compared with a single 110 kDa band in mouseThe British Heart Foundation and STRUTH forSwiss 3T3 fibroblasts is unclear at the present time,financial support, Dr Metin Avkiran for helpfulalthough a number of possibilities exist, including:discussions and Professor David J. Hearse for his(i) the second lower molecular weight band couldsupport and encouragement.represent a proteolytic fragment of full length PKD

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