Inhibition of guinea pig aortic sarcolemmal Ca2+-Mg2+ ATPase and cAMP phosphodiesterase activity by milrinone

Drug Development Research 19:435-442 (1990)

Inhibition of Guinea Pig Aortic Sarcolemmal Ca2+-Mg2+ ATPase and cAMP Phosphodiesterase Activity by Mi lrinone Bernard O'Connor and Paul J. Silver

Department of Pharmacology, Sterling Research Group, Rensselaer, New York

ABSTRACT

OI'Connor, B. and P.J. Silver: Inhibition of guinea pig aortic sarcolemmal Ca2+ -Mg2+ ATPase and cAMP phosphodiesterase activity by milrinone. Drug Dev. Res. 19:435-442, 1 !390.

Mlilrinone is a positive inotropeivasodilator that inhibits cardiovascular low K, cAMP phos- pliodiesterase (PDE) and not Na+-K+ ATPase activity. To explore other possible mechanisms of action, we quantitated the effects of milrinone on Ca2+-stimulated Mg" ATPase activity in guinea pig aortic smooth muscle plasma membranes. Milrinone inhibited Ca'+- stimulated activity, but not basal activity, in aortic microsomes. Maximum inhibition (70%) occurred at 1 pM, which coincided with the inflection point of a parabolic dose-response curve. In a sarcolemmal-enriched (Fl) aortic preparation, 1 pM milrinone, 0.5 pM CAMP, 1 pIM CI-930 (another low K, cAMP PDE inhibitor), and 100 pM W-7 (a calmodulin antagonist) all inhibited Ca2+-stimulated Mg'' ATPase activity. This F1 preparation contained cAMP PDE activity which was inhibited by 1 pM milrinone (26%) and 1 pM CI-930 (40%) but not by 100 pM W-7. The inhibition of F1 Ca2+-Mg2+ ATPase activity by 1 pM milrinone could be diminished by increasing the concentration of CaCI, in reaction mixtures. In sum, these studies show that milrinone can inhibit vascular sarcolemmal Ca2+-stimulated Mg2+ ATPase activity. However, inhibition may be direct or may be secondary to cAMP PDE inhibition in vascular sarcolemma, since inhibition also occurs with cAMP and another low-K, cAMP PDE inhibitor, CI-930.

Kt?y words: cyclic AMP, cyclic nucleotides, PDE inhibition

Received final version September 14, 1989; accepted October 9, 1989.

Address reprint requests to Dr. Paul .I. Silver, Department of Pharmacology, Sterling Research Group, Rensselaer, NY 12144.

0 1990 Wiley-Liss, Inc.

436 O’Connor and Silver

INTRODUCTION

Milrinone, a positive inotropicivasodilator agent in both preclinical and clinical studies [Alousi et al., 1983; Jaski et al., 19851, is currently used in the acute therapy of congestive heart failure. Previous studies have shown that milrinone does not inhibit cardiac Nat -Kt ATPase activity [Alousi et al., 19831 and is a selective inhibitor of the low K, CAMP phosphodiesterase (PDE; also known as peak I11 PDE) in both cardiac and vascular smooth muscle [Silver et al., 1988a; Weishaar et at., 1986; Harrison et al., 19861. Numerous studies have shown that both cardiotonic and vasodilator activities of milrinone and other peak 111 PDE inhibitors can be ascribed to low-K, cAMP PDE inhibition and subsequent activitation of the cAMP system [Ahn et al., 1986; Endoh et al., 1986; Silver et al., 1988b, 19891.

However, other possible mechanisms of action for milrinone and other low-K, cAMP PDE inhibitors have been proposed. One of these possible additional mechanisms involves direct stimulation of cardiac sarcolemmal ATPase activity by milrinone [Mylotte et al., 19851. However, the possible dissociation from possible effects of milrinone on membrane-bound low-K,,, cAMP PDE activity, or of CAMP-mediated effects, was not made in the Mylotte et al. study. To date, no studies on the effect of milrinone on vascular sarcolemmal Ca2+-ATPdse activity or sarcolemmal PDE activity have yet been reported.

Accordingly, the purpose of the present study was to determine if milrinone could modulate membrane-bound Ca*+-stimulated Mg2+ ATPase activity in microsomal and sarcolemmal-enriched fractions prepared from guinea pig aortic smooth muscle. Milrinone has previously been shown to be a low-K, cAMP PDE inhibitor and vasorelaxant in guinea pig aortic smooth muscle [Silver et al., 1988b3. Comparisons with CAMP, another low-K, cAMP PDE inhibitor (CI-930), a calmodulin antagonist (W-7), and a guanylate cyclase activator (sodium nitroprusside) were also made in the present study. Moreover, the effects of added CaC& on milrinone-mediated inhibition of Ca’+ -stimulated Mg’+ ATPase activity and the presence of membrane-associated low-K, cAMP PDE activity were also quantitated.

METHODS Preparation of Membrane Fractions

The descending aorta from the aortic arch to the right renal artery was excised from 12 to 14 stunned male guinea pigs (500-700 g body weight). In all studies where a sarcolemmal enriched (FI) microsomal preparation was used, the animals were anesthetized with 40 mg pentobarbital per kg (i.p.) prior to stunning. The vessels were immediately placed in ice-cold isotonic saline until all were collected (up to 30 min). All subsequent procedures were performed at 0-4°C. The aortae were rinsed, blotted, weighed, and transferred to 19 volumes of ice-cold buffer composed of 250 mM sucrose, 20 mM MOPS, and 1 mM dithiothreitol (DTT), pH 7.0 [modified from Sharmd and Bhalla, 19861. The tissues were homogenized with three 15 sec bursts of an Ultra-Turrax Tissumizer at half full power. The homogenate was centrifuged at 1,OOOg for 15 min to remove cellular debris. Mitochondria were removed by a second centrifugation at 10,OOOg for 15 min. The supernatant fraction was centrifuged at 100,OOOg for 60 min to collect microsomes. Microsomes were resuspended in 0 .6 ml of fresh homogenizing buffer and stored overnight at 4°C.

Sarcolenimal enrichment of microsomal preparations was accomplished by discontinuous sucrose gradient centrifugation according to the procedure of Matlib 119841. This method was slightly modified by the use of 2 ml of 3076, 35%, and 50% sucrose (WiW) prepared with 20 mM MOPS, pH 7.0, containing 1 mM DTT. The crude heterogeneous microsomal pellet obtained from differential centrifugation ( 1 mg protein) was resuspended in 0.6 ml ice-cold homogenizing buffer (8.5% sucrose w / W ) and injected at the interface of the 30% sucrose layer and of 10 ml of supernatant sucrose-free buffer. The gradient was centrifuged in a swinging bucket rotor (Beckman SW 28) at 108,000g for 9.5 min. The distinct bands at the

Milrinone Inhibits Ca2+ ATPase Activity 437

F;.5%-30% sucrose and 30%-35% sucrose interfaces, designated FI and F2 fractions respectively, were collected, as were the 35%-50% sucrose interface (F3), which contained no visible band. The sucrose concentrations of these collected fractions were measured with a I3ausch and Lomb Abbe 3L refractometer and diluted to 8.5% sucrose with sucrose-free buffer before centrifugation at 100,OOOg for 60 min to collect the pellets. The pellets were resuspended in 0.4 ml homogenizing buffer and protein concentrations were determined by using bovine serum albumin prepared in homogenizing buffer as a standard [Bradford, 19761; 5-nucleotidase activity was used as a marker for plasma membranes following sucrose gradient separation of the microsomal fractions as described by Matlib [ 19841. Inorganic phosphate was measured by the method of Rockstein and Herron (19511.

Ca2+-Mg2+ ATPase Assay

Ca2+ -Mg2+ ATPase activity of microsomal fractions was determined by modifications of previously published procedures [Grover and Samson, 1986; Furukawa and Nakamura, 1984; Takeo and Sakanashi, 19851. Reaction mixtures contained 20 mM TRIS maleate, pH '7.0, 100 mM KCI, 5 mM sodium azide. either EGTA (1 mM) or CaCI, (0.1 mM), 2 p M tcalmodulin, 100 pgiml microsomal protein, and either test agents or vehicles. Following a 5 min preincubation interval at 30°C, reactions were initiated by the addition of Mg2+-ATP (pH=7.0) to produce a final concentration of 5 mM. After the addition of the substrate, 150 pl aliquots of the incubate were transferred to an equal volume of ice-cold 10% trichlo- roacetic acid at 0, 2, and 4 min after initiation of the reaction. These acidified aliquots were centrifuged at 2 , 0 0 0 ~ for 15 min (4°C) and kept in an ice bath until assayed for inorganic phosphate (Pi). The supernatant fractions from the ATPase reactions were assayed in duplicate for Pi by the procedure of Rockstein and Herron [I9511 by using monobasic potassium phosphate in water as a standard. Absorbance was measured at 720 nm.

Cyclic Nucleotide PDE Assay

PDE activity of the FI microsomal fraction was determined after substituting 1 p M [3H]-cAMP for ATP in the ATPase activity assay. Only the incubate composition containing CaCI, was used. The incubation was performed at 30°C for 10 rnin after the addition of substrate. Heating the reactants for 60 sec at 100°C terminated the reaction. Subsequent treatment of the reaction mixture with 5'-nucleotidase and Dowex 1-X8 column isolation of [3H]adenosine were performed according to the procedure of Thompson et al. [1979] as previously described [Silver et al., 1988al.

Materials

Milrinone (prepared at Sterling Research Group, Rensselaer) and CAMP (Sigma Chem- ical Co. A-4137; lot no. 112F-9002) were prepared in 1 mM KOH as 800 p M and 80 pM stock solutions, respectively. Sodium nitroprusside dihydrate (Sigma Chemical Co. S-0501; lot no. 64F-0224), C1-930 hydrochloride (Warner-Lambert PD-l12548-2), and W-7 hydrochloride (Sigma Chemical Co. A-3281; lot no. 35F-5204) were prepared in distilled deionized water as 8 nM, 80 p M , and 800 pM stock solutions, respectively.

RESULTS

The effects of milrinone on Ca2'-stimulated Mg2+ ATPase activities of guinea pig aortic smooth muscle microsomes are shown in Figure 1 . There was no significant change in basal activity relative to vehicle over a concentration range of 0.01-100 p M milrinone (range of 1.63 t 0.09 to 1.66 2 0.10 pnio1 Pi/mg proteinlmin). However, milrinone inhibited the net Ca2+ stimulation of Mg2+ ATPase activity in a biphasic concentration response. A peak significant (P < 0.05) reduction occurred at 1 p M milrinone.

Smooth muscle microsomal membrane preparations contain primarily sarcolemmal and


-I

I 0.01 0.10 1 10 100

B :: IMILRINONEI /1 M

0 s Fig. 1. Concentration-related inhibition of vascular niicrosomal Ca2 i- -stimulated Mg2+-ATPase activity by milrinone. Microsomes from guinea pig aortic smooth muscle were prepared as described in the text. The effect of different concentrations of milrinone (closed circles) and vehicle (125 p,M KOH; open circle) on Ca2+-stimulated MgZf ATPase activity is shown. There were no effects of either vehicle or milrinone on basal Mg2+ ATPase activity. Values are the mean 2 S.E. for seven or eight aortic microsomal preparations. * indicates statistical significance (P<0.05) with the Dunnett’s test for multiple comparisons.

sarcoplasmic reticular membranes. In order to more fully examine Ca2+ activation of ATPase activity, a sarcolemmal-enriched (F 1) fraction was prepared via discontinuous sucrose gradient centrifugation of the aortic smooth muscle microsomal preparation. The specific activity of Ca*+-stimulated Mg2+ ATPase activity was increased in the F1 fraction, from 105 2 15 nmol Piimg proteinimin in microsomes to 500 t 60 nmol Piimg proteinimin in the F1 fraction. The F1 fraction also exhibited a roughly 2 X enhancement of 5’-nucleotidase activity, a sarcolem- ma1 enzyme marker assay, when compared with the starting microsomal preparation (Table 1). However, while an enhancement was evident, the increase was not 7-8 x as previously reported for other smooth muscle preparations [Takeo and Sakanashi, 19851. The reason for this difference is not clear but may be related to different smooth muscle sources.

The effects of 1 y M milrinone, as well as 0.5 y M CAMP, 1 y M (3-930, 100 pM W-7, and 1 nM sodium nitroprusside, on Ca2+ stimulation of Mg2+ ATPase activity in these Fl fractions are shown in Figure 2. There were no significant effects of any treatments on basal Mg2+ ATPase activity. However, milrinone, CAMP, the low-K, cAMP PDE inhibitor CI- 930, and the reference calmodulin antagonist W-7 all inhibited the Ca2’ -induced activation in ATPase activity.

To examine if this inhibition was direct or possibly secondary to inhibition of the low-K, cAMP PDE isozyme, the presence of cAMP PDE activity in the F1 fraction and the effects of 1 pM milrinone, 1 p M CI-930, and 100 p M W-7 on this PDE activity were determined (Fig. 2). Both milrinone and CI-930 inhibited PDE activity while W-7 had no significant effect.

To further characterize inhibition of Ca2 + -Mg2+ ATPase activity by milrinone, the effects of milrinone at differing CaCl, concentrations were quantitated. Inhibition of Ca2+ -

Milrinone Inhibits Ca2+ ATPase Activity

TABLE 1. 5‘-Nucleotidase Activity in the Crude Heterogeneous Microsomal and Sucrose Gradient Fractions (Fl-3) From Guinea Pig Aortic Smooth Muscle

Membrane fraction (pmol Piimg proteidmin)”

Crude microsomes 0.246 ? 0.011 (5 ) F1 0.578 t 0.059 ( 5 ) P<0.05 F2 0.160 2 0.009 ( 5 ) F3 0.160 t 0.064 (4)

aResults are the mean 2 S.E. for the number of aortic preparations shown in parenthe- ses. Statistical analysis (P<0.05) was conducted with the Dunnett’s test comparing gradient fractions to crude microsomes.

5‘-nucleotidase activity

> 5 700 c

A

T

KOH MIL CAMP HflCI-930 SNP W-7

CONCENTRATION 1 0.5 1 0.001 100 (UM)

* T

MIL CI-930 W-7

1 1 1oc

439

Fig. 2. Effects of agents on (A) Ca*+-stimulated Mg’+ ATPase activity or (B) CAMP phosphodi- (:sterase activity in sarcolemmal-enriched FI fractions. Sarcolemmal-enriched FI fractions were prepared as described in the text. A: Effects of the distilled water vehicle, 125 pM KOH vehicle, 1 pM milrinone (MIL), 1 pM C1-930, 0.5 pM CAMP, 100 pM W-7, or 1 nM sodium nitropmsside (SNP) on Ca*+- stimulated ATPase activity are shown. Values are the mean t S.E. for five preparations: * indicates statistical significance (P<0.05, Dunnett’s test) for each agent relative to the respective vehicle control (KOH; MIL, CAMP: H,O; CI-930, SNP, W-7). B: Effects of 1 p M milrinone (MIL), 1 p M CI-930, or 100 pM W-7 on CAMP ( I pM) stimulated PDE activity in sarcolemmal-enriched aortic fractions. Inhibition of PDE activity was quantitated in reaction mixtures which were identical to those used in part A. Values are the mean 2 S.E. for three preparations. * indicates statistical significance (P<0.05) by the Dunnett’s test.

stimulated Mgz+ ATPase activity in the F1 fraction by 1 pM milrinone was diminished by increasing CaZf concentrations (0.1 mM CaCI,, 75 ? 5% inhibition; 1.0 mM CaCI,, 58 t 12% inhibition; 3.0 mM CaCI,, 40 2 7% inhibition).

DISCUSSION

Milrinone exhibits the multiphasic properties of stimulating contraction and the rate of relaxation in cardiac musculature, and relaxing vascular smooth muscle. As previously dis-


cussed, these activities are consistent with inhibition of the low-K, cAMP PDE and activation of the cAMP system [Silver et al., 1988b, 19891. However, it is possible that other mechanisms may also contribute to efficacy. A previous study with rabbit myocardial membranes revealed that milrinone stimulated the Ca2+-Mg2 ATPase activity with a parabolic dose- response curve [Mylotte et al., 19851. This activity is not consistent with positive inotropic activity but is consistent with a cardiac muscle relaxant drug, since stimulation of Ca2+ ATPase activity would be expected to result in a greater rate of Ca2+ removal from the myocyte. It is also possible that the biphasic positive inotropic response to milrinone previously demonstrated in cardiac muscle [Farah et al., 19871 might be a net effect of cAMP PDE inhibition and Ca2+ -Mg2 + ATPase stimulation at submicromolar, therapeutic concentrations of milrinone, while the more pronounced increases in contractile force at higher, micromolar concentrations of milrinone may represent the effect of cAMP PDE inhibition [Silver et al., 19891.

Although the study by Mylotte et al. [I9851 demonstrated an effect of milrinone in cardiac muscle, no studies which we are aware of have examined the effects of milrinone on vascular Ca’+-Mg’+ ATPase activity. In the present study with guinea pig aortic smooth muscle membranes, we found that milrinone inhibits non-nuclear, non-mitochondria1 sarcolemmal-enriched membrane Ca2+-Mg2+ ATPase activity. This is the opposite of the stimulatory effect reported in cardiac sarcolemma [Mylotte et al., 191151 although a similar parabolic concentration-response was apparent. In comparing both studies, not only was the direction of the dose-response curve different (i.e., stimulation of cardiac vs. inhibition of vascular), the potency range was also different. Maximum stimulation of cardiac Ca2 + -Mg2+ ATPase activity occurred at 0.1 p M , while maximum inhibition of vascular Ca2+-ATPase activity occurs at 1.0 p M . This further emphasizes the difference of the effects of milrinone on cardiac and vascular Ca2’ -Mg2+ ATPase activity.

Since milrinone is a low-K, cAMP PDE inhibitor, we also sought to determine if inhibition of vascular Ca2+ -stimulated Mg2+ -ATPase activity might be possibly secondary to inhibition of this PDE. If this assumption was true, the following criteria should be met: 1) cAMP should also inhibit this ATPase activity; 2) a structurally dissimilar agent from milrinone, which shares the common activity of low-K,, cAMP PDE inhibition, should also inhibit Ca2+-stimulated Mg2+ ATPase activity; and 3) low-K,,, cAMP PDE, which is inhibited by ATPase-inhibitory concentrations of milrinone and CI-930, should be present under the ATPase assay conditions in this membranous preparation.

All three criteria were met in the current study. That is, both cAMP and (21-930 also inhibited Ca’+-Mg’+ ATPase activity. The concentration of cAMP (0.5 pM) used in this study is one which activates CAMP-dependent enzymes (i.e., PDE and protein kinase) and is thought to be physiologically relevant. CI-930, which also inhibited Ca2+-Mg2+ ATPase activity, is a pyridazinone low-K,, cAMP PDE inhibitor that is slightly (2-3 x ) more potent than milrinone [Silver et al., 1988al. The previous study by Mylotte et al. [1985] suggested that the bipyridine structure of inilrinone was related to thyroxine and that this may have been responsible for stimulating Ca2+-Mg2+ ATPase activity. However, they did not evaluate low-K,, cAMP PDE inhibition in their cardiac membranes. In the current study, both low-K,, cAMP PDE inhibitors affected Ca2+-Mg2+ ATPase activity and shared the property of common mechanism, and not structure.

If our supposition that cAMP is the mediator of the inhibitory effect of milrinone on the Ca*+-stimulated Mg2+-ATPase activity was correct, it was paramount to demonstrate that the sarcolemmal preparation in this study, under the conditions of Ca2+ -stimulated Mg2+ ATPase activity, simultaneously expresses cAMP PDE activity. As shown in Figure 2, this PDE activity was confirmed and was shown to be inhibited by both 1 p M milrinone and I p M CI-930. Although a prior study [Silver et al., 1988al demonstrated the existence of the low-K,,, cAMP PDE in guinea pig aortic smooth muscle, the present data have shown that this low-K, cAMP PDE is present in a sarcolemmal-enriched fraction of this vascular smooth muscle.

Milrinone Inhibits CaZ+ ATPase Activity 441

The relationship among milrinone, (3-930, and cAMP may indicate that cAMP plays a role in the inhibition of the Ca2+ -stimulated Mg2 + -ATPase since both milrinone and CI-930 as PDE isozyme inhibitors would be expected to elevate the level of CAMP. Previous studies have implicated calmodulin as a regulator of this Ca2+-Mg2+ ATPase [Furukawa and Naka- mura, 19841. Inhibition of ATPase activity with the absence of low-K,, cAMP PDE inhibition by the calmodulin antagonist W-7 in this study is consistent with previous inhibition by calmodulin antagonists in this system. Another vasorelaxant, sodium nitroprusside (which is thought to relax vascular smooth muscle through cCMP), was inactive at a vasorelaxant concentration (1 nM).

A nexus between cAMP and Ca2+ in the relaxation of smooth muscle has previously been reported by Andersson and Nilsson [1972]. Their studies showed that cAMP promoted the binding of Ca2+ in a smooth muscle microsomal fraction and that this enhanced binding :resulted in a reduction of free myoplasmic concentration of this cation. Thus, in the present :study, it is tempting to speculate that the inhibition of Ca2+-stimulated Mg2+ ATPase activity Iby milrinone may possibly be due to the sequestration of free calcium by cAMP rather than by !direct interaction of cAMP with the ATPase complex. A reduction of Ca2+ available to the .ATPase would decrease CaZf-stimulated ATPase activity. If this hypothesis was correct, then .it should be possible to attenuate this inhibition by increasing the calcium concentration in the assay mixture. This assumption was tested and the results show that increased calcium levels do indeed attenuate the inhibition of Ca2+ -stimulated Mg2+ -ATPase activity by milrinone. However, in the absence of Ca2+-binding studies, these results may also be consistent with attenuation by Ca2+ of direct inhibition of CaZt -stimulated ATPase activity.

In summary, these data show that milrinone does inhibit Ca2+-stimulated Mg2+ ATPase activity in guinea pig aortic smooth muscle membranes. This inhibition is the opposite of a previously reported stimulatory effect on cardiac Ca2+-Mg2+ ATPase activity [Mylotte et al., 19851 and thus may reflect different mechanisms of Ca2+-Mg2+ ATPase modulation by milrinone. In vascular smooth muscle, inhibition may occur via direct inhibition of Ca2+- Tag2 ATPase activity or as a consequence of inhibition of low-K,, CAMP-PDE in these membranes. The contribution of this inhibition to vasorelaxation is not clear but may possibly be associated with an increase in Ca2+ binding to aortic membranes. Further studies are needed to define the role, if any, that this potential mechanism plays in the vasorelaxant efficacy of milrinone and other low-K, cAMP PDE inhibitors.

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Inhibition of guinea pig aortic sarcolemmal Ca2+-Mg2+ ATPase and cAMP phosphodiesterase activity by milrinone