Role of PKC, tyrosine kinases, and Rho kinase ... · Role of PKC, tyrosine kinases, and Rho kinase...

Role of PKC, tyrosine kinases, and Rho kinasein �-adrenoreceptor-mediated PASM contraction

DEREK S. DAMRON, NORIAKI KANAYA, YASUYUKI HOMMA,SI-OH KIM, AND PAUL A. MURRAYCenter for Anesthesiology Research, Division of Anesthesiology andCritical Care Medicine, Cleveland Clinic Foundation, Cleveland, Ohio 44195Received 27 August 2001; accepted in final form 8 July 2002

Damron, Derek S., Noriaki Kanaya, YasuyukiHomma, Si-Oh Kim, and Paul A. Murray. Role of PKC,tyrosine kinases, and Rho kinase in �-adrenoreceptor-medi-ated PASM contraction. Am J Physiol Lung Cell Mol Physiol283: L1051–L1064, 2002. First published July 12, 2002;10.1152/ajplung.00345.2001.—Our objectives were to iden-tify the relative contributions of intracellular free Ca2� con-centration ([Ca2�]i) and myofilament Ca2� sensitivity in thepulmonary artery smooth muscle (PASM) contractile re-sponse to the �-adrenoreceptor agonist phenylephrine (PE)and to assess the role of PKC, tyrosine kinases (TK), and Rhokinase (ROK) in that response. Our hypothesis was thatmultiple signaling pathways are involved in the regulation of[Ca2�]i, myofilament Ca2� sensitization, and vasomotor tonein response to �-adrenoreceptor stimulation of PASM. Simul-taneous measurement of [Ca2�]i and isometric tension wasperformed in isolated canine pulmonary arterial stripsloaded with fura 2-AM. PE-induced tension development wasdue to sarcolemmal Ca2� influx, Ca2� release from inositol1,4,5-trisphosphate-dependent sarcoplasmic reticulum Ca2�

stores, and myofilament Ca2� sensitization. Inhibition ofeither PKC or TK partially attenuated the sarcolemmal Ca2�

influx component and the myofilament Ca2� sensitizing ef-fect of PE. Combined inhibition of PKC and TK did not havean additive attenuating effect on PE-induced Ca2� sensitiza-tion. ROK inhibition slightly decreased [Ca2�]i but com-pletely inhibited myofilament Ca2� sensitization. These re-sults indicate that PKC and TK activation positively regulatesarcolemmal Ca2� influx in response to �-adrenoreceptorstimulation in PASM but have relatively minor effects onmyofilament Ca2� sensitivity. ROK is the predominant path-way mediating PE-induced myofilament Ca2� sensitization.

intracellular calcium ion; myofilament calcium ion sensitiv-ity; phenylephrine; vascular smooth muscle; pulmonary ar-tery smooth muscle; protein kinase C

CATECHOLAMINE-INDUCED ACTIVATION of �-adrenoreceptorson vascular smooth muscle cells results in an increasein vasomotor tone. Vasomotor tone is regulated byintracellular free Ca2� concentration ([Ca2�]i) andmyofilament Ca2� sensitivity. The initial increase intone is mediated by an increase in [Ca2�]i, which trig-gers activation of myosin light chain kinase, myosin

light chain phosphorylation, and an increase in tension.Maintenance of vascular smooth muscle contraction ismore complex and likely involves several signaling path-ways. Sensitization of the contractile apparatus to Ca2�

appears to be one important mechanism of pharmaco-mechanical coupling (12). Activation of PKC (4, 5, 11,22), tyrosine kinases (TK) (7, 18, 19, 42), and/or Rhokinase (ROK) (18, 46) have been suggested to playimportant roles in the signal transduction events asso-ciated with vascular smooth muscle contraction. How-ever, the relative roles of PKC, TK, and ROK in regu-lating [Ca2�]i, myofilament Ca2� sensitivity, andtension in response to �-adrenoreceptor stimulationhave not been fully elucidated.

Although the cellular mechanisms involved in �-ad-renoreceptor-mediated vasoconstriction have beenstudied in a variety of systemic vascular smooth mus-cle types (14, 40, 45, 49), comparatively less is knownabout the cellular mechanisms mediating �-adrenore-ceptor activation in the pulmonary circulation. More-over, differential responses to hypoxia in the systemic(vasodilation) vs. the pulmonary (vasoconstriction) cir-culation underscore the difficulty in extrapolating re-sults obtained in systemic vascular smooth muscle tocellular mechanisms that regulate pulmonary vasomo-tor tone. In the pulmonary circulation, the extent towhich PKC, TK, and ROK mediate �-adrenoreceptorpulmonary vasoconstriction is controversial (5, 18, 19,22). In endothelium-intact canine pulmonary arterysmooth muscle (PASM) rings, norepinephrine-inducedcontractions were insensitive to PKC inhibitors butwere abolished by TK inhibition and ROK inhibition(18). In contrast, others demonstrated that the vaso-constrictor response to norepinephrine in the felinepulmonary vascular bed was attenuated by PKC inhib-itors (22) and likely mediated by Ca2�-independentPKC-� isozyme/calmodulin-dependent kinase III (5).Others suggested that the contractile response to nor-epinephrine in isolated rat pulmonary artery is largelymediated by TK activation (42). In none of these stud-ies were [Ca2�]i and tension simultaneously measuredin the same tissue so that the role of PKC, TK, or ROK

Address for reprint requests and other correspondence: P. A.Murray, Center for Anesthesiology Research, FF-40, ClevelandClinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195 (E-mail:[email protected]).

The costs of publication of this article were defrayed in part by thepayment of page charges. The article must therefore be herebymarked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

Am J Physiol Lung Cell Mol Physiol 283: L1051–L1064, 2002.First published July 12, 2002; 10.1152/ajplung.00345.2001.

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in [Ca2�]i and myofilament Ca2� sensitivity in re-sponse to �-adrenoreceptor activation could be ade-quately assessed. Only one study assessed the role ofsarcolemmal Ca2� influx, as well as release of Ca2�

from the sarcoplasmic reticulum, in mediating the ac-tions of �-adrenoreceptor activation in PASM (17).However, that study did not directly measure [Ca2�]inor did it assess the role(s) of protein kinase activationin mediating increases in tension in response to �-ad-renoreceptor activation (17). The present study isunique in that our goal was to simultaneously measure[Ca2�]i and tension in PASM strips and to identify theextent to which PKC, TK, and ROK modulate [Ca2�]iand myofilament Ca2� sensitivity in response to �-ad-renoreceptor stimulation with phenylephrine (PE).

MATERIALS AND METHODS

All surgical procedures and experimental protocols wereapproved by the Cleveland Clinic Foundation InstitutionalAnimal Care and Use Committee (Cleveland, OH).

Preparation of pulmonary arterial strips. Heartworm-neg-ative mongrel dogs (20–30 kg) were anesthetized with intra-venous pentobarbital sodium (30 mg/kg) and fentanyl citrate(15 �g/kg) and placed on positive-pressure ventilation. Acatheter was inserted into the right femoral artery, and thedogs were exsanguinated by controlled hemorrhage. A leftlateral thoracotomy was performed through the fifth inter-costal space, and the heart was arrested with electricallyinduced ventricular fibrillation. The heart and lungs wereremoved from the thorax en bloc. Right and left intralobarpulmonary arteries [2- to 4-mm internal diameter (ID)] weredissected free and immersed in cold modified Krebs-Ringerbicarbonate (KRB) solution composed of (in mM) 118.3 NaCl,4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 25 NaHCO3,0.016 Ca-EDTA, and 11.1 glucose. The pulmonary arterieswere cleaned of fat and connective tissue and cut into strips(2 � 8 mm) or rings (2- to 4-mm ID). The endothelium wasdenuded by gently rubbing the intimal surface with a cottonswab. The absence of an intact endothelium was later veri-fied by assessing the vasorelaxant response to 10�6 M ace-tylcholine.

Simultaneous measurement of [Ca2�]i and tension. As de-scribed previously (37), pulmonary arterial strips withoutendothelium were loaded with 5 �M fura 2-AM at roomtemperature (22–24°C). A noncytotoxic detergent, 0.05% cre-mophor EL, was added to solubilize the fura 2-AM in thesolution and act as a vehicle for penetrating the tissue. Afterfura 2 loading, the muscle strips were washed with KRBbuffer for 30–60 min to remove unhydrolyzed fura 2-AM andthen placed between two stainless steel hooks in a tempera-ture-controlled (37°C) cuvette (volume 3 ml), which was con-tinuously perfused (12 ml/min) with KRB solution bubbledwith 95% air-5% CO2 (pH 7.4). One hook was anchored, andthe other was connected to a strain gauge transducer (Grassmodel FTO3; Quincy, MA) to measure isometric force. Theresting tension was adjusted to 4 g, which was determined inpreliminary studies to be optimal for achieving a maximumcontractile response to 20 mM KCl. Fluorescence measure-ments were performed with a dual-wavelength spectroflu-orometer (Deltascan RFK6002; Photon Technology Interna-tional, Lawrenceville, NJ) at excitation wavelengths of 340and 380 nm and an emission wavelength of 510 nm. Fura 2fluorescence signals (340 nm, 380 nm, and 340 nm-to-380 nmratio) were continuously monitored at a sampling frequencyof 2.5 Hz and collected with a software package from Photon

Technology International. The 340-to-380 fluorescence ratiowas used as an indicator of [Ca2�]i. In control experiments,the baseline fluorescence intensities, as well as the signalratios measured in the absence of dye, were not altered byany of the experimental interventions. After changes in ten-sion and [Ca2�]i in response to 60 mM KCl were measured,the strips were washed with fresh KRB for 30 min. All stripswere pretreated with propranolol (5 �M, 30 min) to inhibitthe �-agonist effect of PE. A high dose of PE (10 �M) wasused throughout the study to achieve a significant increase intension in protocols in which extracellular Ca2� was absent.The response to PE (10 �M) was assessed in the presence (10min) or absence of the PKC inhibitor (48) bisindolylmaleim-ide I (Bis I; 3 �M), the TK inhibitor (26) tyrphostin (Tyr) A47(10 �M), the ROK inhibitor (15) Y-27632 (10 �M), or theinositol1,4,5-trisphosphate(IP3)receptorinhibitor(2)2-amino-ethoxydiphenyl borate (2-APB; 100 �M). Structurally differ-ent inhibitors of PKC (calphostin C; 0.1, 0.3, and 1 �M) andTK (genistein; 100 �M) were also investigated. Structurallysimilar inactive analogs of Bis (Bis V; 3 �M), Tyr A47 (TyrA1; 10 �M) and genistein (daidzein, 100 �M) were alsoassessed. Nonspecific effects of the inhibitors on voltage-gated Ca2� channels and myosin light chain kinase activitywere assessed in strips contracted with KCl. Bis I, Bis V,calphostin C, Tyr A47, Tyr A1, genistein, daidzein, and2-APB had no effect on KCl-induced contraction, whereasY-27632 inhibited the increase in tension by 20 � 9%. Early[Ca2�]i and tension values represent the peak increases ineach parameter in the 2 min after administration of PE. Late[Ca2�]i and tension values represent measurements for eachparameter 15 min after administration of PE. Summarizeddata for the changes in [Ca2�]i and tension after an inter-vention are expressed as the percent change from the previ-ous response measured in that particular protocol.

Materials. Phenylephrine HCl, propranolol HCl, Tyr A47,Tyr A1, genistein, daidzein, Bis I, and Bis V were purchasedfrom Sigma (St. Louis, MO). 2-APB and calphostin C wereobtained from Calbiochem (La Jolla, CA). Y-27632 was ob-tained from Biomol (Plymouth Meeting, PA).

Statistical analysis and data presentation. Results areexpressed as means � SE. The sample size, n, represents thenumber of dogs from which strips or rings were studied.Statistical comparisons used ANOVA and the Bonferroni-Dunn post hoc test. Student’s t-test for paired comparisonswas used when appropriate. Differences were consideredstatistically significant at P 0.05.

RESULTS

Effect of KCl and PE on [Ca2�]i and tension. Figure1A illustrates simultaneous recordings of [Ca2�]i andtension during exposure to 60 mM KCl and 10 �M PEin a PASM strip. Addition of KCl resulted in rapid,early increases in [Ca2�]i and tension, followed by late,sustained increases near peak levels until washout. PEcaused early peak increases in [Ca2�]i and tensionfollowed by late, sustained increases at lower values.PE increased tension to a similar degree as KCl,whereas the increase in [Ca2�]i was only about half ofthat observed with KCl. During washout of PE, tensionremained elevated even when [Ca2�]i had returned tobaseline (Fig. 1A). PE caused a leftward shift in thecontinuous [Ca2�]i-tension relationship (Fig. 1B). Thereproducibility of the PE response was assessed byadministering PE three successive times to PASMstrips. As summarized in Fig. 2, increases in [Ca2�]i

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and tension in response to the second and third appli-cations of PE were similar although somewhat greaterthen the first response to PE. We next investigated theeffects of the various interventions on the early (de-

fined as the peak changes occurring within 2 min of PEapplication) and late (measured 15 min after adminis-tration of PE) changes in [Ca2�]i and tension in re-sponse to PE in PASM strips.

Effect of removing extracellular Ca2�. To test thehypothesis that influx of extracellular Ca2� is requiredfor PE-induced contraction, we measured [Ca2�]i andtension in response to PE in the presence or absence ofextracellular Ca2�. As illustrated in Fig. 3, removal ofextracellular Ca2� decreased the early increase in[Ca2�]i and tension by 32 � 6% and 55 � 5%, respec-tively. In the absence of extracellular Ca2� the latephase of the PE-induced increase in [Ca2�]i was abol-ished, whereas the late phase of tension developmentwas reduced by 65 � 2%.

Effect of IP3 receptor inhibition in absence of extra-cellular Ca2�. Our next goal was to test the hypothesisthat IP3-mediated release of Ca2� from intracellularstores is involved in the PE-induced contractile re-sponse. We pretreated the strips with the IP3 receptorblocker 2-APB before PE stimulation in the absence ofextracellular Ca2� (Fig. 4). Inhibition of IP3 receptorswith 2-APB (100 �M) abolished the early increase in[Ca2�]i in response to PE and reduced the early in-crease in tension by 43 � 5% compared with thatobserved in the absence of extracellular Ca2�. 2-APBhad no effect on the late PE-induced increase in ten-sion.

Effect of PKC inhibition. To test the hypothesis thatPKC activation plays a role in PE-induced increases in[Ca2�]i and tension, PASM strips were pretreated withthe PKC inhibitor Bis I before PE stimulation. In thepresence of extracellular Ca2�, pretreatment with Bis I(3 �M) had no effect on the early increase in [Ca2�]i butreduced the late increase in [Ca2�]i by 60 � 3% (Fig. 5).

Fig. 1. A: changes in intracellular freeCa2� concentration ([Ca2�]i) (indicatedby 340 nm-to-380 nm ratio) and ten-sion during contractions induced by 60mM KCl and 10 �M phenylephrine(PE) in a canine pulmonary arterysmooth muscle (PASM) strip. Extracel-lular Ca2� concentration was 2.5 mM.Vertical bar represents the period afterwashout (w/o) of PE when tension re-mains increased above baseline whereas[Ca2�]i has returned to baseline. B:continuous changes in the [Ca2�]i-tension relationship in response to KCland PE. Data presented in A are plot-ted as a phase-plane loop in B.

Fig. 2. Summarized data depicting the reproducibility of early (mea-sured within 2 min) and late (measured at 15 min) increases in[Ca2�]i (A) and tension (B) in response to 3 consecutive applicationsof PE. *Significantly different from first PE (PE-1) response (P 0.05); n 6.

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Bis I reduced both the early (20 � 5%) and late (33 �4%) increases in tension in response to PE. The inac-tive analog of Bis I (Bis V) had no effect on PE-inducedincreases in [Ca2�]i or tension. To further confirm arole for PKC in PE-induced contraction, concentration-response curves for KCl and PE were obtained inPASM rings in the presence or absence of anotherspecific PKC inhibitor, calphostin C. Because of thelight-sensitive nature of calphostin C, [Ca2�]i could notbe measured in these studies. Pretreatment with cal-phostin C (1 �M) for 60 min had no effect on restingtension or KCl-induced contraction (Fig. 6). However,PE-induced contraction was inhibited by calphostin C,resulting in a 43 � 5% decrease in maximal tension.

Effect of PKC inhibition in absence of extracellularCa2�. We next tested the hypothesis that PKC activa-tion is involved in regulating the PE-induced increasein tension observed in the absence of extracellular

Ca2�. We pretreated strips with Bis I in the absence ofextracellular Ca2� before stimulation with PE. In theabsence of extracellular Ca2�, Bis I (3 �M) had noeffect on the early increase in [Ca2�]i induced by PEbut reduced the early increase in tension by 25 � 6%(Fig. 7). Bis I had no significant effect on the latePE-induced increase in tension.

Effect of PKC inhibition in absence of extracellularCa2� and IP3-mediated SR Ca2� release. We tested thehypothesis that the increase in tension observed in theabsence of any increase in [Ca2�]i is mediated by activa-tion of PKC. PASM strips were pretreated with Bis I inthe absence of extracellular Ca2� and in the presence of2-APB before stimulation with PE. Under these condi-tions, PE-induced increases in tension were not associ-ated with concomitant increases in [Ca2�]i. As summa-rized in Fig. 8A, Bis I reduced both the PE-induced early(20 � 5%) and late (18 � 6%) increases in tension.

Fig. 3. A: Changes in [Ca2�]i and tensioninduced by 10 �M PE in the presence (2.5mM; Control) or absence of extracellular Ca2�

in a PASM strip. Note that the time scales for[Ca2�]i and tension are different. The originaltracings for [Ca2�]i are designed to highlightthe early response to PE. B: summarizeddata. *Significantly different from responsemeasured in the presence of extracellularCa2� (P 0.05); n 8.



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Effect of TK inhibition. To test the hypothesis thatactivation of TK plays a role in PE-induced increases in[Ca2�]i and tension, we pretreated PASM strips withthe TK inhibitor Tyr A47 before PE stimulation. Pre-treatment with Tyr A47 (10 �M) had no effect on theearly increase in [Ca2�]i but, surprisingly, enhancedthe late increase in [Ca2�]i by 79 � 20% (Fig. 9). TyrA47 had no effect on the early increase in tension butenhanced the late increase in tension by 20 � 8%. Theinactive analog of Tyr A47 (Tyr A1; 10 �M) did notalter PE-induced increases in [Ca2�]i or tension. Incontrast to Tyr A47, pretreatment with genistein (100�M) reduced the early increase in [Ca2�]i and tensionby 55 � 6% and 38 � 12%, respectively. Genistein alsoreduced the late increases in [Ca2�]i and tension by60 � 8% and 20 � 8%, respectively. The inactive analogof genistein (daidzein; 100 �M) had no effect on PE-induced increases in [Ca2�]i or tension.

Effect of TK inhibition in absence of extracellularCa2�. We tested the hypothesis that activation of TK isinvolved in regulating the PE-induced increase in ten-sion observed in the absence of extracellular Ca2�.PASM strips were pretreated with Tyr A47 in theabsence of extracellular Ca2� before stimulation withPE. Similar to Bis I, Tyr A47 (10 �M) had no effect onthe early increase in [Ca2�]i in response to PE butreduced the early increase in tension by 22 � 6% (Fig.10). Tyr A47 had no effect on late changes in tension.Pretreatment with genistein (100 �M) reduced theearly PE-induced increase in [Ca2�]i and tension by68 � 12% and 60 � 16%, respectively. Genisteinreduced the late phase of PE-induced contraction by55 � 15%.

Effect of TK inhibition in absence of extracellularCa2� and IP3-mediated SR Ca2� release. To test thehypothesis that the increase in tension observed in the

Fig. 4. A: Changes in [Ca2�]i and tensioninduced by 10 �M PE in the presence orabsence (Ca2� free) of 2-aminoethoxydiphe-nyl borate (2-APB; 100 �M) in a PASM strip.Extracellular Ca2� concentration was 0 mM.B: summarized data. *Significantly differentfrom response measured in the absence ofextracellular Ca2� (P 0.05); n 7.



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absence of any increase in [Ca2�]i is mediated by acti-vation of TK, we pretreated strips with Tyr A47 in theabsence of extracellular Ca2� and in the presence of2-APB before stimulation with PE. As noted above,under these conditions PE-induced increases in ten-sion were not associated with concomitant increases in[Ca2�]i. As summarized in Fig. 8B, Tyr A47 reducedthe early (34 � 3%) and late (30 � 5%) increases intension in response to PE.

Effect of combined TK and PKC inhibition in absenceof extracellular Ca2� and IP3-mediated SR Ca2� re-lease. We also tested the hypothesis that the effects ofPKC and TK on myofilament Ca2� sensitization wereadditive (i.e., the signaling pathways are in parallel).PASM strips were pretreated with both Bis I and TyrA47 in the absence of extracellular Ca2� and in thepresence of 2-APB before stimulation with PE. As sum-marized in Fig. 8C, the effect of combined PKC and TK

inhibition on PE-induced contraction was no greaterthan that observed with Bis I or Tyr A47 alone.

Effect of ROK inhibition. To test the hypothesis thatROK plays a role in PE-induced increases in [Ca2�]iand tension, PASM strips were pretreated with theROK inhibitor Y-27632 before PE stimulation. In thepresence of extracellular Ca2�, pretreatment withY-27632 (10 �M) reduced the early (21 � 7%) but notthe late increase in [Ca2�]i in response to PE (Fig. 11).However, pretreatment with Y-27632 reduced both theearly and late PE-induced increases in tension by 52 �7% and 54 � 8%, respectively.

Effect of ROK inhibition in absence of extracellularCa2� and IP3-mediated SR Ca2� release. Finally, totest the hypothesis that the increase in tension ob-served in the absence of any increase in [Ca2�]i ismediated by activation of ROK, we pretreated stripswith Y-27632 in the absence of extracellular Ca2� and

Fig. 5. A: changes in [Ca2�]i and tension in-duced by 10 �M PE in the presence or ab-sence (Control) of bisindolylmaleimide (Bis) I(3 �M) in a PASM strip. Extracellular Ca2�

concentration was 2.5 mM. B: summarizeddata. *Significantly different from responsemeasured before PKC inhibition (P 0.05);n 6.



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in the presence of 2-APB before stimulation with PE.Under these conditions, inhibition of ROK withY-27632 (10 �M) abolished PE-induced contraction(Fig. 8D).

DISCUSSION

To our knowledge, this is the first study to simulta-neously measure PE-induced changes in [Ca2�]i andtension in PASM in the presence and absence of extra-cellular Ca2� before and after inhibition of PKC, TK,ROK, and IP3 receptors. Our results indicate the fol-lowing. Transsarcolemmal Ca2� influx and myofila-ment Ca2� sensitization contribute to both the earlyand late increases in tension. IP3 receptor-mediatedrelease of Ca2� from the sarcoplasmic reticulum con-tributes to the increase in early tension but not the latephase of tension. PKC and TK activation positivelyregulate both PE-induced Ca2� influx and myofilamentCa2� sensitization. The effects of PKC and TK onmyofilament Ca2� sensitivity were comparatively mi-

nor and not additive. Finally, ROK is the predominantpathway mediating PE-induced myofilament Ca2� sen-sitization. However, it should be noted that the relativeroles of these pathways may vary depending on thestimulus intensity and/or degree of resting tension. Inaddition, cross talk (30, 39) likely occurs between thesepathways and may account for some of the differencesobserved in other studies (18, 42).

[Ca2�]i-tension relationship. Our first goal was todetermine the relationship between [Ca2�]i and ten-sion in response to membrane depolarization with KCl(electromechanical coupling), in which no phospholipid-derived second messengers are liberated, compared withthat observed with �-adrenoreceptor stimulation withPE (pharmacomechanical coupling). KCl essentiallycaused monotonic increases in [Ca2�]i and tension. Incontrast, PE caused a transient early increase in[Ca2�]i and tension, followed by a late phase of tensionand [Ca2�]i that remained elevated above baseline butbelow peak values. Because the PE-induced increase intension was similar to that of KCl, whereas the in-crease in [Ca2�]i was only about half of that observedwith KCl, our results suggest that PE increases myo-filament Ca2� sensitivity. A PE-induced increase inmyofilament Ca2� sensitivity is also suggested by thecontinued contraction during the washout period when[Ca2�]i had returned to baseline and by the leftwardshift in the continuous [Ca2�]i-tension relationshipcompared with KCl. A PE-induced leftward shift in the[Ca2�]i-tension relationship was also observed in rab-bit PASM (12). We then investigated the roles of trans-sarcolemmal Ca2� influx and IP3-mediated Ca2� re-lease in mediating the PE-induced increases in [Ca2�]iand tension.

Ca2� influx and Ca2� release. Removal of extracel-lular Ca2� abolished the late increase in [Ca2�]i inresponse to PE, indicating that it is entirely depen-dent on transsarcolemmal Ca2� influx. The earlyincrease in [Ca2�]i was also modestly reduced, likelyreflecting a small depletion in the Ca2� content of thesarcoplasmic reticulum in response to removal ofextracellular Ca2�. Both the early and late increasesin tension were reduced when extracellular Ca2� wasremoved, indicating that transsarcolemmal Ca2� in-flux plays a major role in PE-induced contraction inPASM. For reasons that remain unclear, our resultsappear to be in direct contrast to a recent study thatreported that removal of extracellular Ca2� or ad-ministration of voltage-gated Ca2� channel blockershad no effect on tension development in response to�-adrenoreceptor stimulation in endothelium-intactPASM (18). However, in a separate study, PE-in-duced contractions were found to be reduced by�75% in a nominally Ca2�-free buffer and involved anisoldipine-insensitive, SK&F-96365-sensitive Ca2�

influx pathway (17). In that same study, depletion ofIP3-sensitive Ca2� stores, but not caffeine-sensitiveCa2� stores, nearly abolished the PE response. Thisstudy by Jabr and coworkers (17) suggests that anisoldipine-insensitive Ca2� entry pathway is nor-mally involved in filling IP3-sensitive Ca2� stores

Fig. 6. Summarized data depicting the effect of calphostin C (Cal;0.1, 0.3, 1.0 �M) on KCl (A) and PE (B) dose-response relationship inpulmonary arterial rings. Extracellular Ca2� concentration was 2.5mM. *Significantly different from response measured in the absenceof calphostin C (P 0.05); n 10 in each group.



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and that IP3-sensitive and caffeine-sensitive Ca2�

stores are functionally separate and independententities in PASM. In our study, Ca2� influx could bemediated via activation of receptor-operated Ca2�

channels (9), by activation of voltage-gated Ca2�

channels in response to PE-induced membrane depo-larization (35), or by capacitative Ca2� entry in re-sponse to depletion of Ca2� stores in the sarcoplas-mic reticulum (8). In the absence of extracellularCa2�, IP3 receptor inhibition abolished the earlyincrease in [Ca2�]i, thereby indicating that Ca2�

release from IP3-sensitive Ca2� stores in the sarco-plasmic reticulum was the mechanism responsiblefor the early increase in [Ca2�]i in response to PE.Our data are consistent with the findings of Jabr etal. (17) that both sarcolemmal Ca2� influx and Ca2�

release from intracellular stores (IP3 sensitive) areinvolved in PE-induced contraction.

Myofilament Ca2� sensitization. After removal of ex-tracellular Ca2� and inhibition of IP3 receptor-medi-

ated Ca2� release, PE increased tension without aconcomitant increase in [Ca2�]i. The most likely expla-nation for this result is a PE-induced increase in myo-filament Ca2� sensitivity. An alternative explanationcould be that there is a Ca2�-independent mechanisminvolved, rather than an increase in myofilament Ca2�

sensitivity. However, these two possibilities are notmutually exclusive and may exist in parallel or belinked to each other (i.e., Ca2�-independent pathwaytriggering an increase in myofilament Ca2� sensi-tivity). This could be achieved through activation ofPKC, TK, ROK, or a yet unidentified pathway. Thus wenext investigated the roles of PKC, TK, and ROK inmediating this effect, as well as PE-induced changes inCa2� influx.

PKC. PKC activation is concomitant with IP3 pro-duction in response to agonist stimulation of phospho-lipase C (50). Alternatively, agonist activation of phos-pholipase D (1, 34) or phospholipase A2 (10, 38) canresult in PKC activation in the absence of IP3 produc-

Fig. 7. A: changes in [Ca2�]i and tension in-duced by 10 �M PE in the presence or ab-sence (Ca2� free) of Bis I (3 �M) in a PASMstrip. Extracellular Ca2� concentration was 0mM. B: summarized data. *Significantly dif-ferent from response measured before PKCinhibition in the absence of extracellularCa2� (P 0.05); n 7.



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tion. PKC has been reported to play a role in �-adre-noreceptor-mediated pulmonary vasoconstriction inisolated cat and rat lung (5, 22). In contrast, a recentreport indicated that PKC is not involved in �-adreno-receptor-mediated Ca2� influx or contraction in endo-thelium-intact canine pulmonary artery (18). To re-solve this controversy, we assessed the extent to whichPKC mediates increases in [Ca2�]i and tension simul-taneously in response to PE. PKC inhibition with Bis Iattenuated the PE-induced late increases in [Ca2�]iand tension, which suggests that a component of thelate increase in tension is mediated by PKC-dependentsarcolemmal Ca2� influx. Bis I had no effect on KCl-induced increases in [Ca2�]i or tension, indicating thatits effect was not due to some nonspecific action onmyosin light chain kinase. Moreover, calphostin C, astructurally different PKC inhibitor, also attenuatedPE-induced, but not KCl-induced, contraction. Thesedata are consistent with other studies that demon-strated that PKC activation by phorbol esters en-hances L-type channel-mediated Ca2� influx in somesmooth muscle cells (28) as well as �-adrenoreceptor-stimulated Ca2� influx through voltage-gated Ca2�

channels in rat portal vein and tail artery (25). Ourresults clearly indicate that PKC activation is involvedin �-adrenoreceptor-mediated contraction of caninePASM.

After removal of extracellular Ca2� and pretreat-ment with 2-APB, the PE-induced increase in tension,without a concomitant increase in [Ca2�]i, likely re-flects an increase in myofilament Ca2� sensitivity. Un-der these conditions, Bis I attenuated the PE-inducedincrease in tension, albeit only by 20% (Fig. 7A). Theseresults indicate that PKC activation plays a relativelyminor role in the Ca2�-sensitizing effect of PE. Poten-

tial mechanisms could include PKC-dependent activa-tion of Na�/H� exchange and intracellular alkaliniza-tion causing myofilament Ca2� sensitization (23) orPKC-dependent inhibition of myosin light chain phos-phatase via phosphorylation of CPI-17, an inhibitor ofthe catalytic subunit on the phosphatase (27, 46). Al-ternatively, PKC-dependent activation of the thin fila-ment accessory proteins caldesmon or calponin may beinvolved (3, 13, 33). However, the fact that PE in-creased tension in the absence of an increase in [Ca2�]iand after PKC inhibition indicates that additional sig-naling pathways are involved.

TK. Because TK activation has also been implicatedin the contractile response to �-adrenoreceptor activa-tion in the pulmonary circulation (8, 18, 42), we as-sessed the extent to which inhibition of TK regulates[Ca2�]i and tension in response to PE. Much to oursurprise, pretreatment with Tyr A47 appeared to en-hance the late increase in [Ca2�]i and tension in re-sponse to PE when extracellular Ca2� was present(Fig. 9). Tyr A47 was shown to exert no effect onarginine vasopressin-induced increases in Ca2� inA7r5 aortic smooth muscle cells (21). In contrast, otherstudies demonstrated that inhibition of TK with vari-ous Tyr derivatives results in decreased Ca2� entry invascular smooth muscle cells (21, 43, 51, 52). It shouldbe noted that repetitive exposure to PE alone wasassociated with slightly potentiated late increases in[Ca2�]i and tension (Fig. 2). This effect may explain, atleast in part, the late PE-induced increases in [Ca2�]iand tension after Tyr A47. Alternatively, the effects ofTyr A47 could potentially be explained by the findingthat some TK inhibitors, including Tyr A47, actuallyenhance tyrosine phosphorylation (6, 21). To furtherinvestigate this unexpected finding, we assessed the

Fig. 8. Changes in tension induced by 10 �M PEin the presence or absence of Bis I (A), tyrphostin(Tyr) A47 (B), combined Bis I and Tyr A47 (C), orY-27632 (D) in PASM strips pretreated with2-APB (100 �M). Extracellular Ca2� concentra-tion was 0 mM. *Significantly different from re-sponse measured before administration of thevarious inhibitors in the absence of extracellularCa2� and after inositol 1,4,5-trisphosphate re-ceptor inhibition (P 0.05); n 5 in each group.



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effects of a more widely used and structurally differentTK inhibitor, genistein. In contrast to Tyr A47,genistein attenuated both the early and late increasesin [Ca2�]i and tension in response to PE. This effectwas observed in the presence and absence of extracel-lular Ca2�. These results suggest that a component ofPE-induced contraction is due to Ca2� release andtranssarcolemmal Ca2� influx. Our results withgenistein are consistent with our previous finding (8)that TK inhibition caused a rightward shift in thePE-induced concentration-response relationship inpulmonary arterial rings and provide additional sup-port for the idea that TK plays a role in regulating�-adrenoreceptor-mediated increases in [Ca2�]i andtension in PASM strips.

We also observed that TK inhibition in the absenceof sarcolemmal Ca2� influx and after IP3 receptor

block attenuated PE-induced contraction (Fig. 8B).Under these conditions, the PE-induced increase intension likely involves a TK-dependent increase inmyofilament Ca2� sensitivity. However, the impor-tance of TK activation in the myofilament Ca2�-sensitizing effect of PE appears to be relatively mi-nor (similar to PKC), again suggesting that anothersignaling pathway may play a greater role in PE-induced Ca2� sensitization.

Combined TK and PKC inhibition. Controversysurrounding the relative roles of PKC and TK inmediating the overall Ca2�-sensitizing effect of PE inthe pulmonary vasculature (5, 18) led us to assessthe extent to which combined inhibition of PKC andTK attenuated PE-induced Ca2� sensitization. Theeffect of combined inhibition of PKC and TK on thePE-induced increase in tension in the absence of

Fig. 9. A: changes in [Ca2�]i and tension in-duced by 10 �M PE in the presence or ab-sence (Control) of Tyr A47 (10 �M) in a PASMstrip. Extracellular Ca2� concentration was2.5 mM. B: summarized data. *Significantlydifferent from response measured beforetyrosine kinase (TK) inhibition (P 0.05);n 6.



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extracellular Ca2� and in the presence of 2-APB wasnot additive, suggesting that PKC and TK were act-ing via a final common pathway. Inhibition of myosinlight chain phosphatase is a possible candidate (46).

ROK. Recent evidence in a variety of smooth mus-cle preparations (16, 36), including PASM (18, 46),suggests that activation of ROK by the active GT-Pase RhoA may be a key mediator of Ca2� sensitiza-tion in response to G protein-coupled receptor acti-vation. ROK phosphorylates the regulatory subunitof myosin light chain phosphatase and inhibits itscatalytic activity, thus resulting in increased myosinlight chain phosphorylation, Ca2� sensitization, andincreased tension (46). In our study, inhibition ofROK with Y-27632 attenuated KCl-induced tensionby 20%, with no concomitant effect on [Ca2�]i, indi-cating that Y-27632 may have some nonspecific in-hibitory effect on myosin light chain kinase activity.When extracellular Ca2� was present and intracel-lular Ca2� stores were preserved, inhibition of ROK

resulted in a slight reduction in the PE-inducedincrease in early [Ca2�]i but no effect on late [Ca2�]i.However, both early and late increases in tension inresponse to PE were attenuated. These data suggestthat ROK positively regulates IP3-dependent SRCa2� release while having no significant effect onCa2� influx (16). The reduction in both PE-inducedearly and late tension is likely explained by a modestreduction in Ca2� availability but primarily by adecrease in the sensitizing component of the latecontraction. ROK inhibition abolished the contractileresponse to PE in the absence of extracellular Ca2�

and after IP3 receptor block, indicating that ROKactivation is the predominant pathway mediatingPE-induced Ca2� sensitization (Fig. 8D). Activationof ROK by PE may be due to the GTPase RhoA (46)or the release of arachidonic acid in response to PE(37, 46).

Relative roles of signaling pathways. As noted above,the inhibitory effects of PKC and TK inhibition (alone

Fig. 10. A: changes in [Ca2�]i and tensioninduced by 10 �M PE in the presence orabsence (Ca2� free) of Tyr A47 (10 �M) in aPASM strip. Extracellular Ca2� concentra-tion was 0 mM. B: summarized data. *Signif-icantly different from response measured be-fore TK inhibition in the absence ofextracellular Ca2� (P 0.05); n 6.



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and in combination) on PE-induced contraction in theabsence of changes in [Ca2�]i were relatively modestand not additive, whereas ROK inhibition abolishedPE-induced contraction under these conditions. ROKinhibition also blocked about half of the PE-inducedcontraction when Ca2� was available. These resultssuggest that PKC and TK play a relatively minor rolein mediating PE-induced late tension and that they actvia a final common pathway. TK-dependent activationof ROK (18, 41) or PKC-dependent activation of ROK(32, 44) may explain these findings. PKC activationmay serve dual roles in the PE response by positivelyregulating the L-type Ca2� current (47, 53) and en-hancing myofilament Ca2� sensitivity, possibly via in-teractions with CPI-17 (27). TK activation also appearsto positively regulate sarcolemmal Ca2� influx inPASM, which is consistent with studies in other vas-

cular beds (24, 29). TK positively regulates myofila-ment Ca2� sensitivity, perhaps via direct phosphoryla-tion of tyrosine residues on myosin light chainphosphatase (30, 39, 41). RhoA activation of ROK couldexert a direct effect on myosin light chain phosphataseactivity to enhance myofilament Ca2� sensitivity (46).Phosphorylation of calponin by ROK (20), PKC (33), orTK (31) is another potentially important pathway. Fur-ther studies will be required to fully elucidate theseinteractions.

In summary, our results suggest that PE activatesmultiple signaling pathways that mediate �-adrenore-ceptor contraction in PASM via modulation of both[Ca2�]i and myofilament Ca2� sensitization. PKC andTK activation positively regulate sarcolemmal Ca2�

influx and also play a relatively minor role in theregulation of myofilament Ca2� sensitization via a fi-

Fig. 11. A: changes in [Ca2�]i and tensioninduced by 10 �M PE in the presence orabsence (Control) of Y-27632 (10 �M) in aPASM strip. Extracellular Ca2� concentra-tion was 2.5 mM. B: summarized data. *Sig-nificantly different from response measuredbefore Rho kinase inhibition (P 0.05); n 5.



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nal common pathway. ROK activation is the primarymechanism for regulating myofilament Ca2� sensitiza-tion in response to PE in PASM.

This study was supported by National Heart, Lung, and BloodInstitute Grants HL-38291 and HL-40361.

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