(Hypertension. 1995;25:365-371.)
© 1995 American Heart Association, Inc.
Articles |
From Serv. Endocrinología, Hospital Ramón y Cajal, Madrid (M.R., J.S.), and the Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias Badajoz (E.G.-M., C.G.-M.), Spain.
| Abstract |
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Key Words: calcium pump Na+,K+-transporting ATPase Ca2+-transporting ATPase sodium-potassium pump inhibitor
| Introduction |
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| Methods |
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HHIF was purified from bovine hypothalamus and hypophysis as previously indicated.3 4 This nonpeptidic, nonlipidic compound is purified to a single, homogeneous peak with a characteristic UV spectrum. This purified material has chemical and chromatographic characteristics different from those of any known cardioglucoside.3 When this homogeneous material is subjected to chromatography in two different chromatographic systems,4 we again obtain a single, symmetrical peak with a spectrum and biological activity identical to those obtained from the purified material, suggesting the purity of the final product. The inhibition by HHIF of the Na+,K+-ATPase activity from porcine kidney outer medulla was measured with a coupled assay,14 15 and 1 U is defined as the amount of HHIF required to inhibit the activity of 8 µg purified Na+,K+-ATPase by 50%.3
Total Ca2+,Mg2+-ATPase activity was measured as previously reported12 with the coupled pyruvate kinaselactate dehydrogenase enzyme system. Buffer was used as the control to obtain 100% activity. The HHIF activity at the concentrations used in this study was found to have no effect on the coupled enzyme system. The composition of the standard assay medium was as follows: 50 mmol/L TES (pH 7.4), 0.1 mol/L KCl, 2 mmol/L MgCl2, 2 mmol/L ATP, 50 µmol/L CaCl2, 2 mmol/L ß-mercaptoethanol, 5 mmol/L sodium azide, 0.42 mmol/L phosphoenol pyruvate, 0.22 mmol/L reduced NADH, 10 IU/mL pyruvate kinase, 28 IU/mL lactate dehydrogenase, and 0.02 g protein/L. Under these experimental conditions, the contribution of the Na+,K+-ATPase activity to the total ATPase activity was 10%. In experiments concerning the Ca2+-activated Mg2+-dependent ATPase activity, the free Ca2+ concentration was fixed in the submicromolar range by use of the buffering ligand EGTA, taking an apparent dissociation constant of the Ca2+-EGTA complex of 10-7.2 at pH 7.4.15 The concentrations of free cations and Ca2+-EGTA, Ca2+-ATP, and Mg2+-ATP in the assay medium were calculated with a program described by Perrin and Sayce17 and developed for multiple equilibrium analysis as indicated by Cuenda et al18 and García-Martín and Gutiérrez-Merino.8 The following Kd values were used8 : Kd(Ca2+-ATP)=1.17x10-4 mol/L and Kd(Mg2+-ATP)=2.46x10-5 mol/L.
45Ca2+ uptake by plasma membrane vesicles was measured by Millipore filtration through HAWP025000 filters as previously described.4 8 The reaction medium contained the following: 50 mmol/L TES (pH 7.4), 0.1 mol/L KCl, 2 mmol/L MgCl2, 1 mmol/L ATP, 50 µmol/L CaCl2 (0.4 µCi/mL; free Ca2+, 42 µmol/L), 2 mmol/L ß-mercaptoethanol, and 0.12 g synaptosomal protein/L. Aliquots of the reaction mixture were pooled at 2 minutes. These aliquots were added to a 3-mL solution of ice-cold LaCl3 (0.4 mmol/L) to stop the reaction then vacuum filtered through Millipore filters. The filters were washed with 6 to 9 mL ice-cold solution containing 50 mmol/L TES (pH 7.4), 0.1 mol/L KCl, 1 mmol/L MgCl2, and 1 mmol/L LaCl3; they were then dissolved with methylglycol and counted with a Beckman scintillation counter with 2-(4'-butylphenyl)-5-(4'-biphenyl)-1,3,4,oxadioazole/toluene (5 g/L) as the scintillation cocktail. Each experimental datum was corrected for the basal uptake, in the absence of ATP with or without HHIF, and for the nonspecific binding measured in the absence of synaptosomes. The free Ca2+ contamination in the buffer arising from salts was measured with Arsenazo III19 and taken into account to fix the total and free Ca2+ concentration.
Measurement of Fluorescence Polarization
Fluorescence polarization measurements were carried out at
25°C with a spectrofluorometer (HitachiPerkin-Elmer, model 650-40)
and diphenylhexatriene (DPH) as the fluorescence probe, with excitation
and emission wavelengths of 360 and 440 nm, respectively, as previously
described.8
Statistical Analysis
All results are the average of duplicate measurements carried
out with at least three different synaptosomal preparations.
Comparisons between means were analyzed by one-way ANOVA and
Student-Newman-Keuls multiple-range test.
| Results |
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Because HHIF is a moderately hydrophobic compound,3 we studied whether the inhibition of the total Ca2+,Mg2+-ATPase activity by HHIF depends on preincubation time. Synaptosomes were preincubated with different HHIF concentrations in 50 mmol/L TES (pH 7.4) for 30 minutes at 25°C. No difference in the extent of inhibition was detected when the reaction was started by addition of the enzyme to the assay medium containing HHIF or addition of the enzyme preincubated with the inhibitor (data not shown).
We also studied the reversibility of the inhibition produced by HHIF to test experimentally whether this inhibition could reflect enzyme denaturation because denaturation of Ca2+,Mg2+-ATPase is an irreversible process. Synaptosomal plasma membrane vesicles pretreated with HHIF (0.25 g protein/L and 100 U/mL HHIF) were dialyzed against 5 mmol/L TES (pH 7.4), 2 mmol/L ß-mercaptoethanol, 0.3 mol/L sucrose, and 0.5 mmol/L phosphatidylcholine for 4 hours at 4°C. Under these experimental conditions, the Ca2+,Mg2+-ATPase activity of these membranes was inhibited 80% compared with the control. The extent of inhibition after dialysis of synaptosomes was the same; therefore, we can conclude that the inhibition was irreversible and probably related to protein denaturation.
Adsorption of HHIF on Synaptosomal Membranes
Interestingly, the potency of HHIF as inhibitor of the total
Ca2+,Mg2+-ATPase activity
depends on the membrane concentration in the assay medium; higher HHIF
concentrations are needed to reach the same extent of inhibition when
the membrane protein increases (Fig 2). This result
could reflect the change in free inhibitor concentration resulting from
inhibitor-membrane interaction. Adsorption of HHIF to the synaptosomal
membranes has been quantified by measurement of the HHIF concentration
in the supernatant after centrifugation at 100 000g for 2
hours of synaptosomes preincubated for 15 minutes with several HHIF
concentrations. As shown elsewhere,4 HHIF has a
characteristic fluorescence emission spectrum, with a maximum emission
wavelength of 305 nm. We used this fluorescence to measure the HHIF
concentration before addition of synaptosomes and in the supernatant
after centrifugation. Our results show that the apparent partition
coefficient (Kp) for HHIF binding to
synaptosomal membranes is 0.08 (µg protein/mL)-1. The
value of Kp was determined from measurements at
different HHIF and membrane concentrations. The HHIF concentrations
reported in this study are referred to as free HHIF concentrations
corrected by binding of HHIF to plasma membrane.
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Effect of HHIF on Different ATPase Activities of the Plasma
Membrane of Synaptosomes
The results presented above show inhibition by HHIF of the
total ATPase activity, but obviously the target of the inhibitory
effect of HHIF may be any of the different ATPases present in these
membranes. A more accurate study of the inhibitory effect of HHIF on
ATPase activity requires a dissection of the activities more relevant
to the goal of this study, ie,
Ca2+,Mg2+-ATPase and
Mg2+-ATPase. We studied the effects of HHIF on the
dependence of
Ca2+,Mg2+-ATPase and of
the basal Mg2+-ATPase on the concentration of ATP (0
to 250 µmol/L) by adding 1 µmol/L free Ca2+ or 3
mmol/L EGTA, respectively, to the assay medium (Fig 3A and
B). Table 1 lists the relevant kinetic
parameters. Km and
Vmax values are determined from reciprocal plots
of the data obtained by titration of the ATPase activities with HHIF
under different experimental conditions. It can be observed that HHIF
lowers the affinity of both
Ca2+,Mg2+-ATPase and
Mg2+-ATPase activities toward
Mg2+-ATP and that the effects on
Vmax are not reverted by high ATP
concentrations, showing that the inhibition is noncompetitive with the
substrate Mg2+-ATP. However, these data suggest an
antagonistic action between ATP and HHIF. To assess this point further,
we studied the dependence of the
Ca2+,Mg2+-ATPase activity
on the HHIF concentration in the presence of two fixed concentrations
of ATP in the assay medium. The K0.5 of
inhibition by HHIF varied from 1.3 U/mL at 0.1 mmol/L ATP to 2.45 U/mL
at 2 mmol/L ATP (Fig 4).
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Effect of HHIF on Ca2+ Modulation of
Ca2+,Mg2+-ATPase
The Ca2+,Mg2+-ATPase
activity is modulated by Ca2+ concentrations.
Submicromolar Ca2+ concentrations stimulate this
activity, whereas submillimolar Ca2+ concentrations
cause inhibition.19 20 We studied the action of HHIF on
calcium modulation of
Ca2+,Mg2+-ATPase activity.
Fig 5 shows the effect of HHIF on the stimulation of the
basal Mg2+-ATPase activity by
Ca2+. The presence of the inhibitor at
concentrations of 0.1 to 2.5 U/mL (producing from 25% to 50%
inhibition of
Ca2+,Mg2+-ATPase activity)
does not change the affinity of the enzyme for Ca2+.
HHIF concentrations that inhibit 50% of the total
Ca2+,Mg2+-ATPase (Fig 1)
produce a 50% inhibition of the stimulation by
Ca2+. This shows that the sensitivity of
Ca2+,Mg2+-ATPase
(Ca2+ pump) to HHIF is similar to other
Mg2+-ATPase activities of the synaptosomal plasma
membrane.
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We also studied the effects of several HHIF concentrations on the inhibition of the total Ca2+,Mg2+-ATPase activity by submillimolar Ca2+ concentrations (Fig 6). In the presence of HHIF, the extent of inhibition of the total ATPase activity by Ca2+ is decreased. The effect of two concentrations of Ca2+ in the medium assay (50 µmol/L and 1 mmol/L) on the dependence of the total ATPase activity on HHIF concentration was determined. We found that the K0.5 of inhibition by HHIF varied from 2.43 U/mL at 50 µmol/L Ca2+ to 4.2 U/mL at 1 mmol/L Ca2+ (Fig 7). This result shows that Ca2+ reduces the sensitivity of the enzyme to HHIF.
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Inhibition of ATP-Dependent Ca2+ Transport by
HHIF
In synaptic membranes, the Ca2+-stimulated ATP
hydrolysis is coupled to ATP-dependent calcium uptake10 ;
therefore, we studied the effect of HHIF on the ATP-dependent
Ca2+ uptake. The Ca2+ uptake by
plasma membrane vesicles was measured by filtration 2 minutes after the
addition of 1 mmol/L ATP. In the absence of HHIF, it was 3.6±0.6 nmol
Ca2+/mg membrane protein, which is in good
agreement with published data.4 9 The ATP-dependent
Ca2+ uptake is inhibited by HHIF with a
K0.5 value of 6.25 U/mL and a Hill coefficient
of 1.42±0.07.4 The K0.5 value
reflects the total HHIF concentration in the assay medium. Because
Ca2+ uptake measurements were carried out at
membrane concentrations of 0.12 g protein/L and the ATPase activity
measurements were done at 0.02 g protein/L, it is necessary to correct
the apparent K0.5 values for HHIF adsorption to
the membrane to compare the data. Using the Kp
of HHIF given above, we obtained a K0.5 value of
inhibition of Ca2+ transport of approximately 0.6
U/mL free HHIF. This concentration is close to the corrected
K0.5 value, 0.9 U/mL, obtained for the
inhibition of the total
Ca2+,Mg2+-ATPase (see
above).
Effect of HHIF on the Order Parameter of the Plasma Membrane of
Synaptosomes
Because HHIF behaves as a lipophilic compound with a high
partition coefficient for synaptosomal membranes (see above), we
considered the possibility that HHIF could produce changes on the
synaptosomal membrane fluidity.21 The order parameter (S)
and the corrected microviscosity (
o) were calculated as
shown by Pottel et al22 from DPH fluorescence polarization
measurements. The values of these parameters in the absence of HHIF are
S=0.67±0.02 and
o=0.97±0.02 (n=11), which are in
agreement with the values reported for these
membranes.8 21 22 Table 2 lists the results
obtained in the presence of different HHIF concentrations. The results
show that there is a small change either on the order parameter or on
the fluidity (microviscosity) of the plasma membrane of synaptosomes in
the presence of HHIF.
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| Discussion |
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Inhibition of Ca2+,Mg2+-ATPase by HHIF does not show competitive characteristics with the substrate Mg2+-ATP or a significant change of the affinity of Ca2+,Mg2+-ATPase for Ca2+. Therefore, in kinetics terms, it resembles the inhibition mechanism of the Ca2+,Mg2+-ATPase of synaptosomal plasma membrane by other compounds such as local anesthetics.8
The modulation of membrane enzymes by the viscosity (or fluidity) of lipid bilayers is a well-known phenomenon,26 leading to the conclusion that increased rigidity of the lipid bilayer produces inhibition of intrinsic membrane enzymes. This has been shown for the sarcoplasmic reticulum Ca2+,Mg2+-ATPase (the prototype of Ca2+ pumps)27 and for the plasma membrane Na+,K+-ATPase.28 29 However, in membranes with relatively high lipid fluidity, as in mammalian cell membranes in which the lipid phase transition is well below body temperature,30 moderate fluidity changes do not appear to play a major regulatory role on membrane enzymes and transporters.31 In this regard, it has been shown with the sarcoplasmic reticulum Ca2+,Mg2+-ATPase reconstituted into a series of phospholipid bilayers of different fluidities that there is no correlation between ATPase activity and fluidity, with the most important factor in activity modulation being the surrounding phospholipid of the lipid bilayer.31 Measurements of the fluorescence polarization of DPH show that the addition of HHIF at concentrations that effectively inhibit ATPase activity to the plasma membrane of synaptosomes leads to a small increase of the order parameter of the lipid bilayer, which should produce a slight activation of Ca2+,Mg2+-ATPase instead of inhibition of this activity.
Together, these results suggest that HHIF modulates the Ca2+ pump of the synaptosomal plasma membrane by direct interaction with hydrophobic sites of this transport site in the same way that local anesthetics modulate the Ca2+ pump of the synaptosomal plasma membrane8 and the sarcoplasmic reticulum.32 33 Particular types of hydrophobic sites of modulatory relevance for membrane proteins are the annular lipid sites. Annular lipids have been shown to modulate the Ca2+ pump of the synaptosomal plasma membrane34 and the sarcoplasmic reticulum.35 36 With this latter system, it has been proved that disruption of the lipid annulus leads to irreversible inactivation of Ca2+,Mg2+-ATPase, which is very rapid under the usual condition of kinetic assays.37 38 39 Because inhibition of Ca2+,Mg2+-ATPase of synaptosomal plasma membrane by HHIF also shows characteristics of irreversible inactivation, it is likely that HHIF disrupts the lipid annulus of this enzyme (Ca2+ pump). It is to be noted that the inhibition of the sarcoplasmic reticulum Ca2+ pump (unpublished observations) by HHIF shares many common kinetic characteristics with that produced by local anesthetics, and this latter inhibition can be rationalized in terms of disruption of the lipid annulus.32
In conclusion, the endogenous factor HHIF inhibits not only the plasma membrane Na+,K+-ATPase but also the Ca2+ pump of synaptic plasma membrane. This dual effect confirms that HHIF seems to have a different mechanism of action than cardiac glycosides. This inhibition could account for its known effect on rat mesangial cell proliferation and contractility.7 Besides the possible direct effect on vascular smooth muscle, it is tempting to speculate on the effect of such an inhibitor on synaptic activity. Owing to the relevance of the plasma membrane Ca2+ pump in the control of cytosolic Ca2+ concentration in neurons,25 an inhibition of this pump by such a circulating inhibitor should produce an increase of cytosolic Ca2+ concentration in the resting state and an increased synaptic activity (eg, an increased basal rate of neurotransmitter release) of terminals innervating vascular smooth muscle. The relevance of this effect on synaptic hyperactivity and increased tone of the vascular smooth muscle is under investigation.
| Acknowledgments |
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| Footnotes |
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Received January 24, 1994; first decision March 2, 1994; accepted November 2, 1994.
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