AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 279: H1898-H1905, 2000;
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Vol. 279, Issue 4, H1898-H1905, October 2000

Effect of milrinone on left ventricular relaxation and Ca2+ uptake function of cardiac sarcoplasmic reticulum

Masafumi Yano, Michihiro Kohno, Tomoko Ohkusa, Mamoru Mochizuki, Jutaro Yamada, Masateru Kohno, Takayuki Hisaoka, Kaoru Ono, Taketo Tanigawa, Shigeki Kobayashi, and Masunori Matsuzaki

Second Department of Internal Medicine, Yamaguchi University School of Medicine, Ube, Yamaguchi 755-8505, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Milrinone, a phosphodiesterase 3 (PDE3) inhibitor, is known to enhance left ventricular (LV) contractility by an inhibition of the breakdown of cAMP through the mechanism inhibiting PDE3. However, it is unclear whether milrinone also exerts positive lusitropy, like dobutamine. Here, we assessed the effects of milrinone on in vivo LV relaxation, as well as the Ca2+-ATPase activity and the Ca2+ uptake function of the cardiac sarcoplasmic reticulum (SR), compared with the effect of dobutamine on those functions. After dobutamine (3 µg · kg-1 · min-1) was administered, the peak value of the first derivative of LV pressure (+dP/dt) increased by 46%, whereas the time constant (tau ) of LV pressure decay decreased by 6.9%, respectively. After milrinone (10 µg/kg) was administered, the peak +dP/dt increased to a similar extent as dobutamine (46%), whereas tau  decreased much more than dobutamine (19.9%; P < 0.05). In LV crude homogenate, the thapsigargin-sensitive, Ca2+-ATPase activity-cAMP relationships was significantly less increased by milrinone compared with dobutamine (P < 0.05), indicating the higher sensitivity of the SR Ca2+-ATPase activity on cAMP by milrinone than by dobutamine. In the SR vesicles purified from LV muscles, the addition of cAMP increased the SR Ca2+ uptake in a dose-dependent fashion, and the PDE3 inhibitors (milrinone and cGMP) significantly augmented this response (P < 0.05). Hence, milrinone substantially improved LV relaxation in association with an acceleration of the SR Ca2+-ATPase activity and the SR Ca2+ uptake. This acceleration might be due to an inhibition of the membrane-bound PDE3 in the SR, leading to a local elevation of cAMP.

calcium; inotropic agent; ion pumps; ventricular function; phosphodiesterase 3


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A PHOSPHODIESTERASE 3 INHIBITOR milrinone is a potent cardiac bipyridine with inotropic and vasodilator properties (2, 3); therefore, in the clinical setting, it has been frequently used to treat patients with heart failure. Milrinone exerts inotropic action through the mechanism inhibiting the breakdown of cAMP and, hence, elevating the cellular cAMP (7, 25), which in turn activates cAMP-dependent protein kinases with a resultant increase in the transsarcolemmal influx of Ca2+ (21) and the rate of Ca2+ uptake by the sarcoplasmic reticulum (SR) (9). Although several reports (6, 22) have demonstrated that milrinone improves left ventricular (LV) diastolic property as well as systolic function, the mechanism by which milrinone exerts positive lusitropy remains to be elucidated.

At a subcellular level, LV relaxation is closely related to the Ca2+ uptake function by the SR. In cardiac muscle, the SR Ca2+-ATPase activity and the Ca2+ uptake are enhanced when SR membrane-associated phospholamban is phosphorylated by cAMP-dependent protein kinase (31).

Recently, a particulate, cGMP-inhibited phosphodiesterase 3 (PDE3) has been shown to exist in association with the SR vesicles isolated from the mammalian myocardium (14, 16). The potency of the PDE3 inhibitors as inotropic agents in this tissue are considered to correlate with their potency as inhibitors of the SR membrane-bound PDE3 activity (14, 33). Therefore, it is possible that milrinone interacts with the SR-associated PDE3 and, hence, activates cAMP-dependent protein kinase, resulting in an acceleration of the SR Ca2+ uptake and LV relaxation.

In the present study using dogs, we assessed the positive inotropic and positive lusitropic effects of milrinone compared with those of dobutamine, which also elevates the cytosolic level of cAMP through a mechanism of beta -receptor stimulation, and we demonstrated that milrinone substantially improved LV relaxation and was associated with an enhancement of the SR Ca2+ uptake function, probably through the direct inhibition of the SR membrane-bound PDE3.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Milrinone was provided by Yamanouchi Pharmaceutical (Tokyo, Japan). cGMP, rolipram, cAMP, cAMP-dependent protein kinase, and cAMP-dependent protein kinase inhibitor were all purchased from Sigma (St. Louis, MO). One microgram of cAMP-dependent protein kinase has 1.0-2.0 units of phosphorylating activity, where one unit transfers 1.0 picomole of phosphate from [gamma -32P]ATP to hydrolyzed and partially dephosphorylated casein per minute at pH 6.5 at 30°C in the presence of 6.0 µM cAMP (Sigma). One microgram of cAMP-dependent protein kinase inhibitor inhibits 0.75-2.0 phosphorylating units of cAMP-dependent protein kinase (Sigma).

Twelve beagle dogs weighing 10-15 kg were sedated with morphine sulfate (15 mg sc) and cromazine maleate (10 mg sc). They were then anesthetized with isoflurane (2%, 1.5 l/min) and a mixture of nitrous oxide and oxygen (50:50), intubated with a cuffed endotracheal tube, and ventilated at a tidal volume of 22 ml/kg and a respiratory rate of 15 breaths/min. LV pressure was measured by means of a high-fidelity 7-Fr micromanometer-tipped catheter (Millar) inserted from the left carotid artery. Before it was inserted, the catheter was calibrated at 37°C with a mercury manometer. Zero shift of the pressure transducer was checked by simultaneous recording of a fluid-filled transducer, in which the zero reference point was taken at the level of the right atrium. The care of the animals and the protocols used were in accord with guidelines laid down by the Animal Ethics Committee of Yamaguchi University School of Medicine.

Experimental protocol. After the control recording was measured, a stepwise intravenous infusion of dobutamine (1-10 µg · kg-1 · min-1) was started in six dogs. Five to ten minutes were allowed to obtain a steady state at each dose, and hemodynamic measurements were made at the end of each infusion rate. After dobutamine was infused, premilrinone baseline hemodynamic values were established by waiting at least 30 min. After the full recovery of hemodynamics was confirmed, milrinone was intravenously administered by a stepwise cumulative infusion of 1-20 µg/kg, with repeat hemodynamic measurements. Five to ten minutes were allowed to obtain a steady state at each dose, and hemodynamic measurements were made before increasing each infusion rate. The order of drug administration was not randomized due to the long duration of hemodynamic effects by milrinone.

In six different dogs, milrinone (4-12 µg/kg) was administered until the peak value of the first derivative of LV pressure (+dP/dt) increased by about 50%, followed by a stepwise infusion of phenylephrine hydrochloride (0-2 µg · kg-1 · min-1) to elevate LV pressure.

All data were recorded at the end of an expiration on a multichannel recorder digitized at intervals of 2 ms with an online analog-to-digital converter. To obtain data for analysis, we used the average of 10 consecutive cardiac cycles. End diastole was defined by the peak of the R wave on the electrocardiogram. The time of peak value of dP/dt decrease (-dP/dt), obtained from the digital data of the dP/dt signal, was used to estimate end systole. The time constant (tau ) of LV pressure decay during isovolumic relaxation period was calculated as the negative inverse slope of the natural log of the pressure-versus-time relationship, with the assumption of a pressure asymptote of 0 mmHg and with use of data from peak -dP/dt to 10 mmHg above the end-diastolic pressure (34).

Preparation of LV crude homogenates and SR vesicles. The homogenates and the SR vesicles were prepared as described previously (11, 24). LVs were homogenized in a solution containing 30 mmol/l Tris-maleate, 0.3 mol/l sucrose, 5 mg/l leupeptin, and 0.1 mmol/l phenylmethanesulfonyl fluoride (PMSF) at pH 7.0 (solution I). The homogenate was centrifuged at 5,500 g for 10 min, and the resultant supernatant was filtered through four layers of cheesecloth before centrifugation at 12,000 g for 20 min (LV homogenates). The supernatant was then again filtered through cheesecloth and centrifuged at 143,000 g (55,000 rpm; model TLA 100.4, Beckman Optima) for 30 min. The pellet was resuspended in a solution containing 0.6 mol/l KCl, 30 mmol/l Tris-malate, 0.3 mol/l sucrose, 5 mg/l leupeptin, and 0.1 mmol/l PMSF at pH 7.0 (solution II). This suspension was centrifuged at 143,000 g for 45 min. The pellet was resuspended in solution II, homogenized, and centrifuged at 143,000 g as described above. The pellet was suspended in solution I and centrifuged at 143,000 g. The resultant pellet represents the microsomal fraction rich in SR vesicles, and it was suspended in a solution containing 0.1 mol/l KCl, 20 mmol/l Tris-maleate, 0.3 mol/l sucrose, 5 mg/l leupeptin, and 0.1 mmol/l PMSF at pH 7.0 to give a final concentration of 10-20 mg protein/ml. This fraction was rapidly frozen in liquid nitrogen and stored at -80°C. An aliquot was retained for determination of protein concentration by the method of Lowry et al. (18).

Ca2+-ATPase activity and cAMP assays in LV crude homogenates. The Ca2+-ATPase activity in LV crude homogenates was obtained by measuring the amount of Pi released during the reaction after adding ATP. The assay mixture had a total assay volume of 500 µl and contained 150 mmol/l KCl, 20 mmol/l MES (at pH 6.8), 0.3 mmol/l MgCl2, 10 mmol/l NaN3, 10 mmol/l NaF, 6 µM of the ionophore A-23187, 0.32 mmol/l CaCl2, 0.5 mmol/l EGTA (free [Ca2+] = 1 µmol/l), and 0.125 mg crude homogenate. To start the reaction, 1.0 mmol/l ATP was added to the above priming solution in the presence or absence of dobutamine (0-0.3 µmol/l) or milrinone (0-1 µmol/l). The amount of reacted Pi was calculated by converting nanometers (absorbance of 0.1% malachite green) to nanomoles by means of a standard linear line (31, 33). The above procedures were repeated in the presence of 1 µmol/l thapsigargin. The thapsigargin-insensitive portions of the reacted Pi (82.3 ± 7.7% of total Ca2+-ATPase activity) were subtracted from the total reacted Pi. The thapsigargin-sensitive portions of the reacted Pi were then obtained and defined as the SR Ca2+-ATPase activity.

The cAMP content in LV crude homogenate was determined with an enzyme immunoassay kit (Biotrak, cAMP enzyme immunoassay system, Amersham International) according to the kit instructions.

Ca2+ uptake assay in purified SR vesicles. The SR vesicles (0.6 mg/ml) were preincubated in a solution containing 0.15 mol/l KCl, 1 mmol/l MgCl2, 10 mmol/l NaN3, 20 mmol/l MES (at pH 6.8), 5 mmol/l oxalate, 0.2 mmol/l EGTA, 0.09 mmol/l CaCl2 (free [Ca2+] = 0.1 µmol/l), and 2.5 µmol/l fluo 3 as a Ca2+ indicator. ATP (1 mmol/l) was then added to the above priming solution to load the SR with Ca2+.

The SR Ca2+ uptake was measured by the change in the fluorescence intensity of fluo 3, recorded in a cuvette with an excitation wavelength of 480 nm and an emission wavelength of 530 nm using a spectrophotometer (model F2000, Hitachi, Tokyo, Japan). The Ca2+ uptake (nmol/mg) was calculated from the fluorescence intensity of fluo 3 after determining the coefficient of fluo 3 signal divided by the change in [Ca2+] at each [Ca2+] in the range of 0.03-0.3 µmol/l adjusted with the EGTA-Ca2+ buffer (13, 35).

The effect of cAMP-dependent phosphorylation on the SR Ca2+ uptake was determined by addition of cAMP to the SR vesicles in the presence or absence of 5 µg/ml cAMP-dependent protein kinase. PDE3 inhibitors (milrinone and cGMP) were also added in the presence of 0.1 µmol/l cAMP and 5 µg/ml cAMP-dependent protein kinase. To evaluate the effect of phosphodiesterase 4 (PDE4) inhibition on the SR Ca2+ uptake, rolipram was added in the presence of 0.1 µmol/l cAMP and 5 µg/ml cAMP-dependent protein kinase. The cAMP-dependent protein kinase inhibitor (5 µg/ml) was also used to quantify the extent of cAMP-dependent activation of the SR Ca2+ uptake mediated through cAMP-dependent protein kinase.

Statistics. Data are presented as means ± SE or SD. Changes within the same group were analyzed by one-way analysis of variance (ANOVA) for repeated measures and subsequent Fisher's protected least-significant difference. Differences between two groups were analyzed by two-way ANOVA and subsequent paired Student's t-test. Statistical significance was defined by P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hemodynamic data before and after administration of milrinone or dobutamine. Hemodynamics are summarized in Table 1. After dobutamine (1-10 µg · kg-1 · min-1) was administered, the heart rate tended to increase, and peak LV pressure gradually increased. LV end-diastolic pressure tended to increase. The peak +dP/dt of LV pressure significantly increased, and tau  was shortened. After milrinone was administered, LV peak systolic pressure did not change, and LV end-diastolic pressure tended to decrease. Although the peak +dP/dt increased to a lesser extent than dobutamine, tau  was shortened even more than dobutamine. Figure 1 shows the relationship between percent change in tau  and that in +dP/dt after milrinone or dobutamine administration. At a similar percent increase in +dP/dt, tau  was shortened significantly more by milrinone than by dobutamine.

                              
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  		Table 1.   Effect of dobutamine or milrinone on hemodynamics



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  		Fig. 1.   Relationship between the percent change in the time constant (tau ) of left ventricular (LV) pressure decay and that in peak value of the first derivative of LV pressure over time (+dP/dt) during the infusion of milrinone (; 3, 6, 10, or 20 µg/kg) or dobutamine (open circle ; 1, 2, 3, or 4 µg · kg-1 · min-1). At the similar percent increase in peak +dP/dt, tau  was shortened significantly more with dobutamine. Data are means ± SE. *P < 0.05 vs. baseline; #P < 0.05 vs. dobutamine.

Table 2 summarizes the hemodynamic data after the addition of milrinone and phenylephrine hydrochloride. tau , which was shortened by preinfusion of milrinone, was not prolonged after phenylephrine hydrochloride, although the peak LV pressure increased by about 25%. Figure 2 shows a representative example of dP/dt-pressure loops of LV during the administration of milrinone or milrinone plus phenylephrine hydrochloride. The slope of the linear relation between dP/dt and pressure during the isovolumic relaxation period that indicates LV relaxation function (5, 10) became steeper with milrinone, and it was not significantly influenced by a small rise in LV pressure by phenylephrine hydrochloride.

                              
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  		Table 2.   Afterloading effect of phenylephrine hydrochloride on hemodynamics



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  		Fig. 2.   dP/dt-pressure loops of LV at baseline (solid line) and after the administration of milrinone (dashed line) or milrinone plus phenylephrine hydrochloride (dotted line). After milrinone was administered, the slope of the linear relation between +dP/dt and pressure during isovolumic relaxation period became steep compared with dobutamine, and it was not influenced by a small rise of LV pressure after phenylephrine hydrochloride infusion.

Effects of dobutamine and milrinone on SR Ca2+-ATPase activity and cAMP level in LV crude homogenates. Figure 3 shows the relationship between the SR Ca2+-ATPase activity and the cAMP level after the addition of milrinone or dobutamine in LV crude homogenates. At a given increase in the SR Ca2+-ATPase activity, the cAMP level was significantly less increased by milrinone than by dobutamine, indicating the higher sensitivity of Ca2+-ATPase activity on cAMP in the case of milrinone.


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  		Fig. 3.   Relationship between Ca2+-ATPase activity and cAMP level by addition of milrinone (; 0, 0.3, or 1 µM) or dobutamine (open circle ; 0, 0.1, or 0.3 µM) in LV crude homogenate. Data represent means ± SE of 5 experiments from 3 different preparations. Note that at a given similar increase in Ca2+-ATPase activity, cAMP level was significantly less increased by milrinone than by dobutamine. *P < 0.05 vs. dobutamine.

Effects of milrinone, cGMP, and rolipram on SR Ca2+ uptake in purified SR vesicles. Figure 4, A-C, shows the dose-dependent effect of cAMP on the SR Ca2+ uptake in the absence or presence of milrinone, cGMP, or rolipram. The addition of cAMP increased the SR Ca2+ uptake in a dose-dependent fashion, and the half-maximum effect was obtained at ~0.3 µmol/l cAMP. Both 10 µmol/l milrinone and 30 µmol/l cGMP shifted the curves upward and to the left, indicating the stimulation of the SR Ca2+ uptake by milrinone and cGMP. In contrast, rolipram had virtually no effect on the cAMP dependence of the SR Ca2+ uptake. Figure 5, A-C, shows the dose-dependent effect of milrinone, cGMP, or rolipram on the SR Ca2+ uptake in the presence of 0.1 µmol/l cAMP and 5 µg/ml cAMP-dependent protein kinase. Both milrinone and cGMP stimulated the SR Ca2+ uptake in a dose-dependent fashion. The half-maximum stimulating effect was elicited at ~0.5 µmol/l by milrinone and 2 µmol/l by cGMP. However, rolipram again had no dose-dependent effect on the SR Ca2+ uptake.


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  		Fig. 4.   Dose-dependent effect of cAMP on the sarcoplasmic reticulum (SR) Ca2+ uptake in the absence (open circle ) or presence () of 10 µmol/l milrinone (A), 30 µmol/l cGMP (B; black-triangle) or 10 µmol/l rolipram (C; ). Data represent means ± SD of 5 experiments from 3 different preparations. The cAMP-dependent protein kinase (5 µg/ml) was added in the priming solution during the Ca2+ uptake. Both milrinone and cGMP shifted the curve upward and to the left, indicating the stimulation of cAMP-induced Ca2+ uptake by milrinone or cGMP. In contrast, rolipram had no effect on the cAMP-induced activation of Ca2+ uptake. *P < 0.05 vs. baseline (cAMP = 0 µmol/l); #P < 0.05 vs. without milrinone or cGMP.



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  		Fig. 5.   Dose-dependent effect of milrinone (A; ), cGMP (B; black-triangle), or rolipram (C; ) on the SR Ca2+ uptake in the presence of 0.1 µmol/l cAMP and 5 µg/ml cAMP-dependent protein kinase. Data represent means ± SD of 5 experiments from 3 different preparations. Both milrinone and cGMP stimulated the SR Ca2+ uptake in a dose-dependent fashion, whereas rolipram had no effect on the Ca2+ uptake. *P <=  0.05 vs. baseline (0 µmol/l milrinone or cGMP).

Figure 6 shows the effect of cAMP, cAMP-dependent protein kinase, or cAMP-dependent protein kinase inhibitor on the SR Ca2+ uptake. cAMP or cAMP-dependent protein kinase alone did not enhance the SR Ca2+ uptake. Only when cAMP was added together with the cAMP-dependent protein kinase was the SR Ca2+ uptake significantly enhanced. The cAMP-dependent protein kinase inhibitor partially inhibited the baseline Ca2+ uptake (without cAMP and protein kinase) and completely inhibited the increase in the SR Ca2+ uptake by the addition of cAMP plus protein kinase up to the level below the baseline Ca2+ uptake. Both milrinone and cGMP also did not increase Ca2+ uptake without cAMP (Fig. 4) or cAMP-dependent protein kinase (data not shown). Both milrinone and cGMP increased Ca2+ uptake only when both the protein kinase and cAMP were added together with the SR vesicles. The protein kinase inhibitor completely inhibited the augmentation of the SR Ca2+ uptake after the addition of milrinone or cGMP to the level below baseline (data not shown).


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  		Fig. 6.   Effects of cAMP, cAMP-dependent protein kinase A (PKA), and cAMP-dependent protein kinase inhibitor (PI) on the SR Ca2+ uptake. Data represent means ± SD of 5 experiments from 3 different preparations. When cAMP was added together with the cAMP-dependent protein kinase, the SR Ca2+ uptake was significantly elevated. The cAMP-dependent protein kinase inhibitor inhibited the augmentation of Ca2+ uptake by cAMP plus cAMP-dependent protein kinase to the level below baseline of Ca2+ uptake (no cAMP and no protein kinase).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The major findings of this study are as follows. First, the PDE3 inhibitor milrinone accelerated LV relaxation much more than dobutamine, as evidenced by the decrease in tau  of LV pressure decay. Second, milrinone increased the SR Ca2+-ATPase activity in LV crude homogenates and the SR Ca2+ uptake in dose-dependent fashion and also stimulated the response of cAMP-induced augmentation of the SR Ca2+ uptake. Third, cGMP also increased the SR Ca2+ uptake in a dose-dependent fashion, whereas the PDE4 inhibitor rolipram had no effect on the cAMP-dependent augmentation of the SR Ca2+ uptake.

Different hemodynamic effects between milrinone and dobutamine. Milrinone, a PDE3 inhibitor, is known to enhance LV contractility by an inhibition of the breakdown of cAMP through the mechanism inhibiting PDE3 (2, 3, 7, 25). However, it remains to be elucidated whether milrinone also exerts positive lusitropy, like dobutamine. Although both dobutamine and milrinone elevate the cytosolic level of cAMP, the present results show the clear difference in positive lusitropic action between dobutamine and milrinone. tau  was decreased to a greater extent by milrinone than by dobutamine when compared at a dose by which a similar increase of +dP/dt was elicited. Therefore, in the case of milrinone, an additional mechanism for the improvement of LV relaxation should be considered.

Inhibition of SR membrane-bound PDE3 by milrinone or cGMP. Low Michaelis-Menten constant cGMP-inhibited PDE3 activity has been identified in both cytosolic and SR-enriched microsomal fractions of the mammalian myocardium (14, 16). There are two reported genes for the PDE3 family, PDE3A and PDE3B, and four genes for PDE4 (PDE4A-PDE4D) (4). Some molecular probes have recently been used to define which phosphodiesterase genes are in the canine heart. PDE4D and PDE3A mRNAs (28) and several PDE3A proteins are present in canine ventricles (30). It is not known whether PDE3B is also present. An antibody to human platelet PDE3A cross reacts on Western blots with canine ventricular proteins in both the cytoplasm and SR-enriched fractions (30). However, Liu and Maurice (17) suggested that the microsomal forms of PDE3 are PDE3B (125-135 kDa) in cardiovascular tissues, whereas PDE3A represents the alternatively spiced cytosolic forms (e.g., 80-120 kDa).

Evidence has accumulated to suggest that certain cardiotonic agents (milrinone, imazodan, and amrinone) inhibit SR membrane-bound PDE3 (27, 32, 33) and exert their contractile effects through subtle alterations in the metabolism of cAMP (15, 16). With regard to this, functional compartmentalization of cAMP and protein kinases has previously been proposed for cardiac muscle (1, 12), and, hence, intracellular Ca2+ mobilization might be affected by cAMP located in the particulate compartment of canine cardiac myocytes (12).

The present findings showed that SR Ca2+ uptake was accelerated by cAMP in the presence of cAMP-dependent protein kinase, and both milrinone and cGMP raised the sensitivity of cAMP on the SR Ca2+ uptake. The half-maximum stimulating effect of milrinone on the SR Ca2+ uptake was elicited at 0.3-0.5 µmol/l, which is very similar to the concentration range of milrinone by which half-maximum phosphodiesterase inhibition is elicited. Moreover, in crude LV homogenates, milrinone hypersensitized the SR Ca2+-ATPase activity on cAMP compared with dobutamine. Taken together, it is strongly suggested that milrinone specifically binds the SR membrane-bound PDE3, and inhibition of PDE3 could lead to localized increases in cAMP with a resultant activation in cAMP-dependent protein kinase, followed by an increase in the SR Ca2+ uptake through phosphorylation of phospholamban.

However, milrinone has been reported to have no effect on the phosphorylation of phospholamban in the SR vesicles isolated from guinea pig hearts (26). The reduced inotropic response to the PDE3 inhibitors in guinea pig myocardium has been attributed to an absence of SR-associated activity in these species (32, 33). More recently, Smith et al. (29) demonstrated that, like dogs, humans, and rabbits, guinea pig ventricular SR vesicles contain a 135-kDa PDE3 that is a substrate itself for cAMP-dependent protein kinase. These authors argued that the lack of in vivo inotropic effects of PDE3 inhibitors in rodents cannot be explained by the absence of PDE3 in the SR fraction, as was previously suggested by Weishaar et al. (33). Endogenous levels of endogenously phosphorylated phospholamban, phosphatase, and/or cAMP-dependent protein kinase may differ between guinea pig and dog SR vesicles.

In the present study, because exogenous cAMP (with or without milrinone and cGMP) did not increase the Ca2+ uptake in the absence of added cAMP-dependent protein kinase, endogenous phosphorylation of phospholamban might not blunt an additional effect of cAMP-dependent protein kinase in dog SR vesicles.

Effect of PDE4 inhibition on SR Ca2+ uptake. The SR microsomes largely contain cGMP-inhibited PDE3, whereas the cGMP-insensitive PDE4 is present in the sarcolemmal fraction (20). However, depending on the purity of the microsomal preparation, the sarcolemmal fraction containing the cGMP-insensitive PDE4 may be contaminated. In this regard, we evaluated the effect of the PDE4 inhibitor rolipram on the SR Ca2+ uptake. As a result, rolipram had no effect on the cAMP-dependent activation of the SR Ca2+ uptake, unlike milrinone or cGMP, suggesting that the inhibition of particulate PDE3 indeed mediates cAMP-dependent activation of the SR Ca2+-ATPase.

Afterload reduction by milrinone and LV relaxation Milrinone is known to exert a vasodilating effect as well as positive inotropic and lusitropic effects. Therefore, afterload reduction by this drug may induce acceleration of LV relaxation. However, when LV pressure was increased by about 25% (mean pressure 30 mmHg) by adding phenylephrine hydrochloride together with milrinone, tau  was not significantly influenced. With regard to this, we (36) previously showed that tau  was not significantly influenced by a small increase (~20-30 mmHg) in peak LV pressure unless systolic loading sequence is dramatically changed, i.e., by early or late systolic loading. In the present study, because the time to peak systolic pressure did not significantly change (data not shown) after administration of milrinone, it seems unlikely that vasodilation by milrinone could account for the changes in tau . In our study, LV end-diastolic pressure decreased in association with the shortening of tau . Because LV preload itself does not influence the isovolumic relaxation rate (8), the shortening of tau  after milrinone administration might be provided by the direct effect of this drug, which in turn may partly contribute to the decreasing tendency of LV end-diastolic pressure.

Direct effect of milrinone on LV relaxation. In the clinical setting, both dobutamine and milrinone have been shown to lead to a significant improvement in the hemodynamic state of patients with acute heart failure. However, much of the research in heart failure has been directed toward the assessment of LV systolic function. Although it is difficult to establish whether the improved diastolic function is due to a direct action on the myocardium or an indirect action due to improved conditions of load, there are some evidences indicating a direct improvement of LV relaxation by milrinone. In patients with heart failure, Monrad et al. (23) reported that milrinone significantly improved LV diastolic function, as evidenced by increases in LV peak filling rate and chamber distensibility, with little change in LV systolic pressure and only a small fall (10%) in mean aortic pressure. Ludmer et al. (19) administered milrinone by an intracoronary infusion technique for separation of the direct myocardial and vasodilator actions. They observed the decrease in LV filling pressures at very low infusion rate, with no change in mean aortic pressure or systemic vascular resistance. Consistent with these findings, we observed that after administration of milrinone, the isovolumic LV relaxation was improved in association with the decreasing tendency in LV end-diastolic pressure. In addition, we demonstrated that milrinone accelerates SR Ca2+ uptake, probably through an inhibition of SR membrane-bound, not cytosolic, PDE3.

In conclusion, milrinone substantially improved LV relaxation in association with an acceleration of Ca2+ uptake by SR. This acceleration might be due to an inhibition of membrane-bound PDE3 in SR, which might induce a local elevation of cAMP.


    ACKNOWLEDGEMENTS

This work was supported by a Grant-In-Aid for scientific research from the Ministry of Education in Japan (Grant C-11670684) and by a Health Sciences Research Grant for Comprehensive Research on Aging and Health from the Ministry of Health and Welfare, Japan.


    FOOTNOTES

Address for reprint requests and other correspondence: M. Yano, Second Dept. of Internal Medicine, Yamaguchi Univ. School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-0067, Japan (E-mail: yanoma{at}po.cc.yamaguchi-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 November 1999; accepted in final form 9 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 279(4):H1898-H1905
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