Phosphorylase b kinase and phosphorylase a phosphatase activities in contracting vascular smooth muscle: stimulation by fatty acid

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Bioehimica et Biophysica Acre. 1073 (1991) 550-554 r,~ 1991 Elsevier Science Publishers B.V. 0304-4165/91/$o3.~0

ADONIS 030&41659100132C BBAG EN 23493

Phosphorylase b kinase and phosphorylase a phosphatase activities in contracting vascular smooth muscle: stimulation by fatty acid J o h n T. B a r r e n 1 a n d S t e p h e n J. K o p p 2 Section of Cardiolo~; Department of Medicine, The Rush Heart Institute, Rush-Presbyterian.St. LuAe's Medical Center, R l~sh Medical College, Chicogo, II.. ( U.S.A. ) a rid - Dep artmettt of Physiology, Chicago College of Osteopathic Medicin e. Chicago, IL ( U.S.A. ) (Received 3 July 1990}

Key words: Phosphorylase/~ kinase; Phosphorylase a phosphatase; Muscle contraction; Fatty acid; (Pig vascular smooth muscle;,

The activities of phesphorylase b kinase and phesphorylase a phosphamse were determined during the phases of KCl-lndueed contraction in porcine carotid artery. Phosphorylase b kinase exhibited a biphasie paflem with activity increasing 70% above basal bevels during the early phase of ectlve force generation (4~ s into contraction) fellowed by a decline in activity daring the phase of steady-state tension maintenance. Phosl~horylase a phoslJatase was stimulated simultaneously with phosphorylase b kinase, with activity [nareasing 100% over basal levels at 45 s into contraction, bat remaining elevated at 30 min. Incubation of arteries in 0.5 mM paimitate resulted in a 30% increase in basal activity of phosphorylase b kinase and 117% augmentation of basal phosl',hatase activity, w i ~ no f ~ increase in activity of either enzyme with contraction. The results indicate tbat bolh the kinase and phosphatase are subject to regulation during contractile activation of the muscle, possibly by similar but not identical mechanisms.

Introduction

The pattern of glyeogenolysls upon contraction of arterial smooth muscle is characterized by a rise of activity during the phase of active tension generation followed by reduced activity during the phase of steady-slate tension maintenance [1], This pattern of glyeogenolysis corresponds to the pattern of phosphorytase a activity which also displays an initial rise during the early phase of contraction followed by reduced activity during tension maintenance [2,3]. The pattern of activation of the enzymes regulating phosphorytase, ha':,'aver, has not been determined. The conversion of phosphorylase b to a is catalyzed by the phosphorylation of phosphorylase b by phosphoryiase b kinase. The reverse reaction, i.e., the dephosphorylatiou of phosphorylase a, is mediated by phosphorylase a phosphatase. It is the relative activities of these two enzymes that ultimately determines the activity of phosphorylase a.

Correspondence: J.T. Barren. Seetic,n of Cafdiolog.7, Rush-Presbyterian St. Lukes Medical Center, 1653 W. Congress Parkway. Chicago, IL 6~12, U.S,A,

In (his investigation, the activities of glycogen phosphorylase b kinase and phosphorylase a phosphatase in intact arterial smooth muscle were measured during the different phases of isometric contraction. The effects of incubation of arterial smooth muscle with fatty acids on the activities of the kinase and phosphalas¢ during contractile stimulation were also investigated. These studios were undertaken Because we previously observed that incubation of carotid a~teries in palmitate resulted in augmented contractility, altered glycogen metabolism and enhanced phosphorylase a activity in the early phase of contraction 13]. Materials and Methods

Porcine carotid arteries were obtained from the abattoir, prepared and mounted into muscle chambers as previously described [4] and equilibrated at 37°C in medium consisting of 118 mM NaCI, 4.7 mM KC1, 1.2 mM MgSO4, 1.6 mM CaCI z, 20 mM NaHCO3 and 5.6 mM glucose. The medium was aerated with a gas mixture of 95~ 02/5% CO~, which kept the pH at 7.3-7.4. After 60 rain equilibration, the arteries were incubated for an additional 90 rain in this medium or with medium containing 0.5 mM palmitate. The arterial strips were

551 then contracted for 30 min with 80 mM KCI, and immersed in liquid N z at time O, 4.5 s, and 30 rain into contraction. Frozen muscles were pulverized and the muscle powder weighed_ Extracts for phosphoryla~e b kinase determination were made by homogenizi'ag frozen muscle powder using a Polytron at a setting of 4 for 30 s in a cold solution containing 20 mM B-glycerol phosphate (pH 6.8), 20 mM NaF, 2 mM EDTA, 15 mM mercaptoethanol and 0.01% bovine serum albumin, Extracts for phosphorylase a phosphatase determinatiort were prepared similarly except that NaF was omitted. The ho.~.ogenates were treated with nonte and centrifuged at 3000 × g for 10 rain at 4 ° C . Phosphorylase b klnase was assayed in aliquots of extracts according to procedures described by Krebs et al. [5] except that 0.1 mM CaCI., was included in the reaction mixture, which contained 10 units phosphorylase b per ml. The time of incubation was 30 rmn. Phospho~lase a phosphatase was assayed using 10 units phosphorylase a per ml and was based on the disappearance of phosphorylase a from the reaction mixture according to H u r d et a l [6]. The time of incubation was 90 mia. The appearance or disappearance of phosphorylase a in the reaction mixtures for either the ldnase or phosphatase was m e a s u ~ d using a coupled enzyme system [7]. Enzyme activities are expressed as units of phosphorylase/g per rain; a unit of phosphorylase is v.mol glucose 1 - p h o s p h a t e / m i n and is referenced to the wet weight of the muscle. All enzymes were purchased from Sigma. Results and Discussion

Fig. l A depicts ~ typical, tracing of the pattern of isometric force generation in response to 80 mM KCI. It is compared with the tracing of a carotid artery incubated in medium containing 0.5 mM palmitate. Previous studies have shown that arteries incubated in medium containing palmitate generate significantly more tension than artedes incubated in medium without palmitat¢ [3], The activity of phosphorylase b kinase of arterial muscles frozen at the different phases of muscle contraction upon depolarization with K + is shown in Fig+ lB. Phosphorytase kinases from mammalian striated muscles have the property that they may be phosphorylated by ATP and cAMP.dependent protein kinase. Phosphorylation of ph0sphorylase kinase results in its "activation" which confers a greater affinity of the enzyme for its substrate (phosphorytase b) and imparts operation at a pH in the physio|ogieal range [5]. Although Ca 2+ is not required for the phosphorylation a n d hence 'activation' of the enzyme, the presence of Ca 2+ greatly stimulates the 'activated' kinase [8]. Accordingly, phosphorylase kinase activity in extracts

of porcine carotid artery was assayed at p H 6.8, ia the presence of Ca 2.. Basal activity in resting (time 0)+ unstimulated. muscles incubated in normal modia was 0.134-0.02 u u i t s / g per mln. At 45 s into K+-contraetion, during the phase at which the rate of force generation by the muscle is ma.'dmal, activity at p H 6.8 increased significantly to 0,22 -I- 0.04- t m i t s / g per rain {P < 0.002, n = 6). rials amounted to a change of 0.09 u n i t s / g per min or approx, a 70% increase above basal levels. At the end of 30 rain of sustained isometric contractile force, (the phase of tension maintenance) activated phosphorylase kinase declined to a level of 0.17 +_ 0.01 u n i t s / g per rain. This value is significantly less than the activity at 45 s ( P < 0.02, n = 8), but significantly greater than the activity in resting unstimulated muscles ( P < 0.002. n = 8). Thus, ~he pattern of activated phosphorylase kinase activity iB porcine carotid artery during a K"-stimulated connaction is biphesic and resembles the pattern of phosphJrylase a activity [2,3] with activity rising to peak leve's during the phase of te,'~sion genera*ion, followed by a decline in activity that nevertheless rem~;,¢d _bore control. The pattern of activation of phosphoryiase a phosphatase during K+--contraction is depicted in Fig. 1C. Basal activity (time 0) in unstimulated muscle wa~ 0.06 +__0.005 u n i t s / g per rain. Phosphatase activity increased at 45 s to 0.12+_0.02 u n l t s / g per mira ( P < 0 . 0 0 1 , n = 6) which is a 100~ increase over basal levels, The value at 30 mln was essentially unchanged from that at 45 s (0.12 + 0.91 u n i t s / g per rain). Unlike activated phosphorylase b kinase, therefore, the pattern of activation of p h o ~ h o r y l a s e phosphatase was not biphasie.

Effects of fatty acid We pre~ously reported that incubation of porcine carotid artery sOdps in medium containing 0.5 mM palmitate enhanced phosphorylase a activity upon contractile stimulation. It was of interest to d e l e t e whether this effect was mediated by alterations in the activities of either phosphorylasc b kinase or phosphorylase a phosphatasc, or both, Incubation with patmitat¢ resulted in a significant elevation (30~) of basal activity of activated phosphorylas¢ b kinase to a level of 0.17 d: 0.01 t m i t / g per rain (vs. control, P < 0+05, n = 6), as seen in Fig. lB. Contractile stimulation by KCI resulted in augmentation of activated kinase activity (0.20 _+_0.03 u n i t s / g per rain) but the increase was not significantly different from the resting unstimulated state. Similarly, phosphorylase kinase activity at 30 rain during the phase of te~nsion maintenance was also not significantly different from basal activity. The effect of incubation of carotid arteries with patmitate, therefore, was to enhance basal activity. It is possible that a significant additional increase in activity at 45 s into contraction was not evident because

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Fig, I, (A) Typical isometric tensior~ recordings of carotid strips incubated in the abs~u¢~ (sofid line) and pre.sence (dashed line) of 0.5 mM palmitatc attd then cantracled with g0 ram gCt. (B) Ph0sphorylase b kinas¢ activities at di[femnt times ~alo contraction of arteries thcuba1¢,.~ in the presence and absence of palmitatc, (C) Phosphorylase a phosphatas~ activities at different tim~ in contraction of arler.es incubated in the presence and absence of palmitate, Open bars, ec,nlrol (absexJc~of palmltate): hatched bars, presence of palmizate~ Values mpresertt mean ± S,E.: n ~ 6-8 ar|erlos from different animals. See text for P value~. basal acti,Aty was already near-maximally stimulated. Alternatively, fatty acid incubation m a y have resulted in a blunting of the peak response.

Phosphorylase a ph0sphatas¢ activity or carotid arteries incubated with palmitate is shown i n Fig. 1C. Basal phosphatas¢ activity was markedly augmented

553 (117%) when compared to that in control muscles (0.13 _+ 0.01 u n i t s / g per rain vs. 0.06 u n i t s / g per rain, P < 0.001, n = 6). U p o n challenge with K +. there was no further increase in activity either at 45 s or az 30 min into contraction. The activities at these time points were not different from those of controls. The major effect of fatty acid on phosphatasc activity, therefore, was to enhance basal activity in resting muscles. The in rive activities of phosphorylase b kinasc and phosphorylase a phosphatase at different times into contraction would depend on the available substrate (i.e., the level of unphosphorylated and phosphorylaled phosphorylase, respectively) and the intracelhilar concentration of Ca 2+, which stimulates activated phosphorylase kinase [8]. These enzymes from tissue extracts were assayed with fixed levels of substrate (phosphorylase) and Ca z ~ in the reaction mixture. Therefore, the measured values for the kinasc and phosphatase must only be considered a qualitative reflection of the state of the activities of these enzymes in rive. Nevertheless, it may be assumed that the net formation of phosphorylase a is the sum of the opposing actions of the Idnase and phosphatase. For intact skeletal muscle, evidence has been presented by Danforth et al. i15] suggesting that the actual regulatory control over formation of phosphorylase a is exerted predominantly by the phosphorylase kinase system, with minimal regulation by the phosphatus¢. In the present study, phosphorylase phosphatas¢ activity was found to increase with contraction. This suggests that phosphorylase phosphatase m intact vascular smooth muscle, like other enzymes involved in glycogen metabolism, is subject to physiological control, and together with phosphorylase kinase play complex regulatory roles in glycogen metabolism upon contractile activation of the muscle. The biochemical systems involved in the regulation of phosphorylase b kinase axe not defined. While an early report indicated that cyclic A M P enhanced activity of a crude preparation of phosphorylase kinase from smooth msucle [10], subsequent studies have indicated that this enzyme cannot be activated by phosphorylaLion with cyclic AMP-dependent protein kinase [11,12]. it is possible that this enzyme is subject to phosphorylation by other protein Idnases present in smooth muscle that are independent of cyclic AMP a n d / o r Ca 2+ [13]. However, it is likely that Ca 2+ plays a central role in the activity of the kinase. In addition to stimulating the enzyme [8] Ca 2+ may also be involved in its 'activation' during contraction because the biphaste pattern paraliris the transient rise and then gradual decfne the concentration of C a z+ in the sarcoplasm observed in porcine carotid arteries challenged by K ~" [9]..q, similar biphasic pattern is observed in the phosphorylation of the 20 kDa light chain of myosin, which is also a Ca 2 +-dependent process [14]. The results also indicate that both phosphorylase

kinase and phosphatasc are activated simultaneously during the contractile process, at least in the phase of initial force generation. The processes activating these two enxymes diverge, ho~, vet, in the phase of steadystate tension maintenance since phosphorylase kinase displayed reduced activi.y at 30 rain. while phosphatase activity was maintained at 30 rain. This observation suggests that the mechanisms of regulation of the kimtse a n d phosphatase are not identical. However, the observation that fatty acid augmented the basal activities of both enzymes does suggest that there are regulatory features common to both enzyme systems. H o w treatment with palmitate results in enhanced activity of the kinase and phosphatase is unknown. Fatty acid may enhance entry of Ca'-* into the cell [16], alter cyclic nucleotide metabolism [17], or possibly alter the function of G T P binding proteins [18]. Whether these factors are involved requires investigation. Phosphorylase p h o s p h a t a s c also catalyzes the dephosphorylation of glycogen synthetase [191, promoting the synthesis of glycogen. Tbas is opposed by the action of phosphorylase a. Although palmitate eu'Lances both activities, it is likely that the synthetase reaction prevails since a net decrease in the level of glycogen was not observed during K ~-contraction in carotid arteries incubated with palmitate (see. Ref. 3, Fig. 2). By contrast, there was marked glycogenolysis in control arteries contracted with KC] [3]. Measurements of glycogen turnover are needed to test this hypothesis. It is important to note that fatty acid is the primary energy fuel utilized in resting unstimulated vascular smooth muscle [20], while glycogen may be the primary substrate utilized during contraction [1]. The effects of fatty acid on the enzymes regulating glycogen metabolism may represent a means by which one substrate influences utilization of another substrate.

Acknowledgments This work was supported by a grant from the Amoco Foundation. We thank Professor Michael Bfirfiny for helpful suggestions and Ms. Lillian Linares for typing the manuscript. References I Lynch, R.M. and Paul, ILL (1983)Science222. 1344-1345. 2 Galvas, P.E.. Kuenner, C., Paul, R.J. and DiSMvo.J. (1985) Prcc, •~ . Exp, a:~,oi. M,~I, t78, 254- 260. 3 BarT~,n, J.T~ and Kopp, S.J. (1989) Bic~hlm. Bioph~s. A c t s I012,

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