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(Hypertension. 1995;25:365-371.)
© 1995 American Heart Association, Inc.

Articles

Modulation of the Ca²⁺ Pump by the Hypothalamic-Hypophysary Inhibitory Factor

Mercedes Ricote; Elena García-Martín; Jose Sancho; Carlos Gutiérrez-Merino

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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Abstract We previously purified to homogeneity an endogenous sodium pump inhibitor from bovine hypothalamus and hypophysis that is different from digoxin or ouabain and studied the effects of this factor on the total Ca²⁺,Mg²⁺-ATPase activity of plasma membrane of synaptosomes. This factor inhibits the calcium pump and the total Mg²⁺-ATPase activity of these membranes with approximately the same K_0.5 values of inhibition. The potency of this factor as an inhibitor depends on the membrane concentration in the assay medium. The inhibition of the magnesium-dependent ATPase activities of these membranes was of a noncompetitive type with respect to the substrate Mg²⁺-ATP and did not significantly shift the calcium dependence of the Ca²⁺,Mg²⁺-ATPase activity. We suggest that the calcium pump of the synaptosomal plasma membrane is inhibited by this factor through disruption of the lipid annulus; this inhibition could play a role in the control of calcium homeostasis by increasing the cytosolic free calcium concentration.

Key Words: calcium pump • Na⁺,K⁺-transporting ATPase • Ca²⁺-transporting ATPase • sodium-potassium pump • inhibitor

	Introduction

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The etiology of and molecular events underlying hypertension are poorly understood. Many reports support the idea that factors that inhibit the plasma membrane Na⁺,K⁺ -ATPase are involved in the cause of certain forms of hypertension.^{1 2} One of these factors, the hypothalamic-hypophysary inhibitory factor (HHIF),³ is an endogenous compound that is different from digoxin or ouabain. HHIF was initially isolated from bovine hypothalamus and hypophysis and was later found to be present in other bovine tissues and in human plasma, although at lower concentrations.⁴ An accepted hypothesis to account for certain forms of hypertension links a high tone of vascular smooth muscle to the inhibition of Na⁺,K⁺-ATPase that produces a rise of cytosolic Na⁺ concentrations and, through the Na⁺-Ca²⁺ exchanger of the plasma membrane, a sustained increase of cytosolic Ca²⁺ concentration of vascular smooth muscle cells in the resting state.⁵ On the other hand, it is well known that different ion-pumping ATPases, namely Na⁺,K⁺-ATPase and Ca²⁺,Mg²⁺-ATPase, show little specificity toward inhibitors.⁶ A physiological factor with inhibitory properties over the sodium and calcium pumps could have a decisive role in the control of cytosolic Ca²⁺ concentrations in different tissues and explain some of the inconsistencies of the natriuretic hormone hypothesis. HHIF inhibits the sodium pump of human red blood cells and vascular smooth muscle cells^{3 4} as well as purified Ca²⁺,Mg²⁺-ATPase (unpublished observation). We have also observed that HHIF produces an increased proliferation in cultured rat mesangial cells, a model of vascular smooth muscle proliferation.⁷ HHIF also produces a rise in cytosolic Ca²⁺ concentrations and contractility in the same cells (unpublished observations). These observations led us to study the effect of HHIF on the Ca²⁺ pump of the plasma membrane to elucidate whether we are dealing with an inhibitor of both Na⁺ and Ca²⁺ pumps. We carried out this study with synaptosomal plasma membranes for the methodological reason that the plasma membrane Ca²⁺ pump can be measured accurately with these preparations^{8 9 10} ; in addition, the neuronal plasma membrane Ca²⁺ pump has a high homology with that present in other cell lines such as erythrocytes or muscular cells.⁶ In this article, we report that HHIF inhibits the Ca²⁺ pump of synaptosomal plasma membranes with a K_0.5 value approximately twofold higher than the K_0.5 value of inhibition of Na⁺,K⁺-ATPase.

	Methods

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Synaptosomes were prepared from female Wistar rat brains (200 to 250 g) according to the method of Michaelis et al,¹¹ as modified by García-Martín and Gutiérrez-Merino.¹² The whole brains were rapidly removed after cervical dislocation. Rats were kept and used according to government guidelines for the use of experimental animals. Plasma membrane vesicles were prepared from synaptosomes by hypotonic lysis as previously described.¹² Protein concentration was measured by the method of Lowry et al¹³ with bovine serum albumin as standard.

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 kinase–lactate 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 K_d values were used⁸ : K_d(Ca²⁺-ATP)=1.17x10^-4 mol/L and K_d(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 (Hitachi–Perkin-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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HHIF Inhibits Total Ca²⁺,Mg²⁺-ATPase of Synaptosomal Plasma Membrane Vesicles
Fig 1 shows that HHIF inhibits the total Ca²⁺,Mg²⁺-ATPase activity of synaptic plasma membrane vesicles. From a Hill plot of these data, a K_0.5 value of 2.45 U/mL and a Hill coefficient of 0.8±0.1 are obtained. The dependence of the Ca²⁺,Mg²⁺-ATPase activity on the HHIF concentration is not significantly altered by a change of pH of the assay medium from 7.4 to 7 or by a change in temperature from 25° to 37°C.

View larger version (16K): [in this window] [in a new window]   Figure 1. Line graph shows inhibition of the total Ca2+,Mg2+-ATPase activity by hypothalamic-hypophysary inhibitory factor (HHIF). Data shown are for standard assay conditions, pH 7.4 and 25°C (), pH 7 and 25°C (), and pH 7.4 and 37°C (). The reference (100%) is the ATPase activity measured in the absence of HHIF for every experimental condition. Inset shows Hill plot of the inhibition data in the standard conditions (pH 7.4 and 25°C). Each point is the mean activity of duplicate determinations from three experiments. Estimated SD is within 10% of the mean values.

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 (K_p) for HHIF binding to synaptosomal membranes is 0.08 (µg protein/mL)^-1. The value of K_p 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.

View larger version (14K): [in this window] [in a new window]   Figure 2. Line graph shows dependence of the inhibitory effect of hypothalamic-hypophysary inhibitory factor (HHIF) on Ca2+,Mg2+-ATPase activity on membrane concentration. Protein concentrations were 10 (), 20 (), or 40 () µg/mL. Each point is the mean activity of duplicate determinations from three experiments. Estimated SD is <10%.

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. K_m and V_max 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 V_max 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 K_0.5 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).

View larger version (24K): [in this window] [in a new window]   Figure 3. Line graphs show effect of hypothalamic-hypophysary inhibitory factor (HHIF) on the dependence on Mg2+-ATP (0 to 250 µmol/L) of the Mg2+-ATPase (A) and Ca2+,Mg2+-ATPase activities (B). All ATPase assays were carried out under standard conditions (pH 7.4 and 25°C) as described in "Methods." Special conditions of the reaction medium are as follows: A, The reaction medium containing 250 µmol/L free Mg2+ and EGTA-buffered medium was supplemented with HHIF at the following concentrations (U/mL): 0 (), 0.5 (), and 2.5 (). B, The same medium was used plus 1 µmol/L free Ca2+ and the following HHIF concentrations (U/mL): 0 (), 0.5 (), 1.5 (), and 2.5 (). Each point is the mean activity of duplicate determinations from three experiments. Estimated SD is <10%.

View this table: [in this window] [in a new window]   Table 1. Effects of HHIF on the Basic Kinetic Parameters of the Mg2+-ATPase and Ca2+,Mg2+-ATPase Activities of Synaptosomal Plasma Membranes

View larger version (13K): [in this window] [in a new window]   Figure 4. Line graph shows inhibition of the total ATPase activity by hypothalamic-hypophysary inhibitory factor (HHIF) in the presence of 2 () or 0.1 () mmol/L ATP. Reference (100%) corresponds to the ATPase activity measured in the absence of HHIF and in the presence of 2 mmol/L ATP. Other experimental conditions are indicated in "Methods." Results represent the means of three experiments in duplicate. Estimated SD is <10%.

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.

View larger version (17K): [in this window] [in a new window]   Figure 5. Line graph shows Ca2+ dependence of the basal Mg2+-ATPase activity measured in the presence of hypothalamic-hypophysary inhibitory factor (HHIF) (U/mL): 0 (); 0.1 (); 0.5 (); and 2.5 (). The y axis represents the ATPase acitivity relative to the ATPase activity obtained in the presence of 50 µmol/L Ca2+ and the absence of HHIF. Each point is the mean activity±SD of duplicate determinations from three experiments.

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 K_0.5 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.

View larger version (15K): [in this window] [in a new window]   Figure 6. Line graph shows effects of hypothalamic-hypophysary inhibitory factor (HHIF) on the Ca2+ dependence of the total Ca2+,Mg2+-ATPase activity. HHIF concentrations are (U/mL) 0 (), 0.75 (), and 2.5 (). The y axis represents the ATPase activity obtained in the presence of a Ca2+ concentration of 50 µmol/L and in the absence of HHIF. Each point is the mean activity±SD of duplicate determinations from three experiments.

View larger version (13K): [in this window] [in a new window]   Figure 7. Line graph shows inhibition of the total ATPase activity by hypothalamic-hypophysary inhibitory factor (HHIF) in the presence of 50 µmol/L () or 1 mmol/L () Ca2+. Other experimental conditions are given in "Methods." The y axis represents the ATPase activity measured without HHIF and in the presence of 50 µmol/L Ca2+. Results represent the mean of three experiments in duplicate with SD within 10%.

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 K_0.5 value of 6.25 U/mL and a Hill coefficient of 1.42±0.07.⁴ The K_0.5 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 K_0.5 values for HHIF adsorption to the membrane to compare the data. Using the K_p of HHIF given above, we obtained a K_0.5 value of inhibition of Ca²⁺ transport of approximately 0.6 U/mL free HHIF. This concentration is close to the corrected K_0.5 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.

View this table: [in this window] [in a new window]   Table 2. Effects of HHIF on the Fluidity of Synaptosomal Membranes

	Discussion

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An increased tone of the vascular smooth muscle is common in certain forms of hypertension. This could arise from at least two different physiological perturbations: an increased concentration of cytosolic Ca²⁺ in the resting state of smooth muscle cells and/or hyperactivity of synaptic terminals of muscle innervation. The occurrence of a perturbation of cytosolic Ca²⁺ concentrations in vascular smooth muscle cells in essential hypertension has been well documented^{14 23 24} and is probably related to inhibition of the Na⁺,K⁺-ATPase that in turn should increase the resting concentration of cytosolic Ca²⁺ through the Na⁺-Ca²⁺ exchanger.⁵ Because of the weak selectivity of inhibitors of P-ATPases,⁶ an inhibitor of Na⁺,K⁺-ATPase could also inhibit the plasma membrane Ca²⁺,Mg²⁺-ATPase (Ca²⁺ pump), and this also would result in an increase in cytosolic Ca²⁺. Inhibition of the plasma membrane Na⁺,K⁺-ATPase should in turn potentiate the strength of muscle contraction, as is usually assumed. HHIF is an inhibitor of the Na⁺ pump, present in bovine tissues and human plasma, that is different from any known glycoside and has been shown to inhibit Ca²⁺,Mg²⁺-ATPase.^{3 4} This inhibitor also induces an increased proliferation in cultured rat mesangial cells, a known model of vascular smooth muscle proliferation,⁷ and a rise in cytosolic Ca²⁺ concentrations and contractility in the same cells (unpublished observations). The study of the inhibition of the plasma membrane Ca²⁺ pump by HHIF seems to be relevant to the understanding of the possible role this inhibitor has in the control of cytosolic Ca²⁺.²⁵ We decided to study this effect on synaptosomal membranes because the plasma membrane Ca²⁺ pump can be measured accurately in these preparations and because of the high analogy of this Ca²⁺ pump with the one present in other cellular lines. The results reported here show that the endogenous factor (HHIF) inhibits the Ca²⁺ pump and the total Mg²⁺-ATPase activity of synaptosomal plasma membrane with approximately the same K_0.5 values of inhibition. HHIF is a lipophilic compound that partitions between synaptosomal membranes and water. Once corrected for binding to the membranes, the K_0.5 value of inhibition of the Ca²⁺ pump is 0.9 U/mL, which is approximately twofold higher than the K_0.5 value of inhibition of the Na⁺,K⁺-ATPase of synaptosomal plasma membrane by HHIF (results not shown).

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

 
This work was supported in part by financial aid from the CACYT, FIS, and DGCYT PB91/0311 and by a fellowship (Dr Ricote) from FIS and the Rich Foundation. We are indebted to Jesus Colilla for excellent technical assistance.

	Footnotes

 
Reprint requests to J.M. Sancho, MD, Department of Endocrinology, Hospital Ramón y Cajal, Carr Colmenar Km 9, 1 Madrid, 28034, Spain.

Received January 24, 1994; first decision March 2, 1994; accepted November 2, 1994.

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