(Hypertension. 1995;25:365-371.)
© 1995 American Heart Association, Inc.
Articles |
Modulation of the Ca2+ Pump by the Hypothalamic-Hypophysary Inhibitory Factor
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.
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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 K0.5 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
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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 cells3 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 preparations8 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 K0.5 value approximately twofold higher than the K0.5 value of inhibition of Na+,K+-ATPase.
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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 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 (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.
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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 K0.5 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.
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), 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 (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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), 20
(
) 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. 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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). Each point is the mean activity of
duplicate determinations from three experiments. Estimated SD is
<10%.
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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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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 documented14 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 K0.5 values of inhibition. HHIF is a lipophilic compound that partitions between synaptosomal membranes and water. Once corrected for binding to the membranes, the K0.5 value of inhibition of the Ca2+ pump is 0.9 U/mL, which is approximately twofold higher than the K0.5 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.
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Acknowledgments |
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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.
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Footnotes |
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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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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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1. Hamlyn JM, Ringel R, Schaeffer J, Levinson PD, Hamilton BP, Kowarski AA, Blaustein MP. A circulating inhibitor of (Na+,K+)ATPase associated with essential hypertension. Nature. 1982; 300:650-652.
2. de Wardener HE, MacGregor GA. The relation of a circulating sodium transport inhibitor (the natriuretic hormone?) to hypertension. Medicine. 1983;62:310-326. [Medline] [Order article via Infotrieve]
3. Illescas M, Ricote M, Mendez E, G-Robles R, Sancho J. Complete purification of two identical Na+-pump inhibitors isolated from bovine hypothalamus and hypophysis. FEBS Lett. 1990;261:436-440. [Medline] [Order article via Infotrieve]
4. Sancho JM, García-Martín E, García-Robles R, Santirso R, Villa E, Gutiérrez-Merino C, Ricote M. Properties of the purified hypothalamic pituitary Na/K-ATPase inhibitor. J Cardiovasc Pharmacol. 1993;22(suppl 2):S32-S34.
5.
Blaustein MP. Sodium ions, calcium ions, blood pressure
regulation and hypertension: a reassessment and a hypothesis. Am
J Physiol. 1977;232:C165-C173.
6. Pedersen PL, Carafoli E. Ion motive ATPases, I: ubiquity, properties, and significance to cell function. Trends Biochem Sci. 1987; 12:146-150.
7. Montero A, Rodriguez-Barbero A, Ricote M, Sancho J, López-Novoa JM. Effect of ouabain and hypothalamic, hypophysary inhibitory factor on rat mesangial cell proliferation. J Cardiovasc Pharmacol. 1993;22(suppl 2):S35-S37.
8. García-Martín E, Gutiérrez-Merino C. Modulation of the Ca2+,Mg2+-ATPase activity of synaptosomal plasma membrane by the local anesthetics dibucaine and lidocaine. J Neurochem. 1990;54:1238-1246.[Medline] [Order article via Infotrieve]
9. García-Martín E, González-Cabanillas S, Gutiérrez-Merino C. Modulation of calcium fluxes across synaptosomal plasma membrane by local anesthetics. J Neurochem. 1990;55:370-378. [Medline] [Order article via Infotrieve]
10.
Gill DL, Grollman EF, Kohn LD. Calcium transport mechanisms in
membrane vesicles from guinea pig brain synaptosomes. J Biol
Chem. 1981;256:184-192.
11.
Michaelis EK, Michaelis ML, Chang HH, Kitos TE. High affinity
Ca2+-stimulated Mg2+-dependent
ATPase in rat brain synaptosomes, synaptic membranes and microsomes.
J Biol Chem. 1983;258:6101-6108.
12. García-Martín E, Gutiérrez-Merino C. Local anesthetics inhibit the Ca2+,Mg2+-ATPase activity of rat brain synaptosomes. J Neurochem. 1986;47:668-672.[Medline] [Order article via Infotrieve]
13. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193:265-275.
14.
Carilli CT, Berne M, Cantley LCJ, Haupert GT Jr. Hypothalamic
factor inhibits the (Na+/K+)ATPase from
the extracellular surface. J Biol Chem. 1985;260:1027-1031.
15. Josephson L, Cantley LCJ. Isolation of a potent (Na+-K+)ATPase inhibitor from striated muscle. Biochemistry. 1977;16:4572-4578. [Medline] [Order article via Infotrieve]
16.
Harafuji H, Ogawa Y. Re-examination of the apparent binding
constant of ethylene glycol bis (ß-aminoethyl
ether)-N,N,N',N',-tetraacetic acid with calcium around neutral pH.
J Biochem. 1980;87:1305-1312.
17. Perrin DD, Sayce IG. Computer calculation of equilibrium concentrations in mixtures of metal ions and complexing species. Talanta. 1967;14:833-842.
18. Cuenda A, Henao F, Gutiérrez-Merino C. Distance between functional sites of the Ca2++Mg2+-ATPase from sarcoplasmic reticulum using Co2+ as a spectroscopic ruler. Eur J Biochem. 1990; 194:663-670.
19. Thomas MV. Metallochronic indicators. In: McCormack JG, Cobbold PH, eds. Cellular Calcium: A Practical Approach. New York, NY: Oxford University Press; 1991:115-122.
20. Sorensen RG, Mahler HR. Calcium-stimulated adenosine triphosphatases in synaptic membranes. J Neurochem. 1981;37:1407-1418. [Medline] [Order article via Infotrieve]
21. Hitzemann RJ, Harris RA, Loh HH. Synaptic membrane fluidity and function. In: Shinitzky M, ed. Physiology of Membrane Fluidity. Boca Raton, Fla: CRC Press; 1984:109-126.
22. Pottel H, Van Der Meer W, Herreman W. Correlation between the order parameter and the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and evaluation of membrane fluidity. Biochim Biophys Acta. 1983;730:181-186.
23. Blaustein MP, Hamlyn JM. Role of a natriuretic factor in essential hypertension: an hypothesis. Ann Intern Med. 1983;98:785-792.
24. Rasgado-Flores H, Santiago EM, Blaustein MP. Kinetics and stoichiometry of coupled Na+ efflux and Ca2+ influx (Na+/Ca2+ exchange) in barnacle muscle cells. J Gen Physiol. 1989;93: 1219-1241.
25. Akerman KEO, Nicholls DG. Ca2+ transport and the regulation of transmitter release in isolated nerve endings. Trends Biochem Sci. 1983;8:63-64.
26. Sandermann H. Regulation of membrane enzymes by lipids. Biochim Biophys Acta. 1978;515:209-237. [Medline] [Order article via Infotrieve]
27. Warren GB, Toon PA, Birdsall NJM, Lee AG, Metcalfe JC. Reversible lipid titrations of the activity of pure adenosine-triphosphatase-lipid complexes. Biochemistry. 1974;13:5501-5507. [Medline] [Order article via Infotrieve]
28.
Chong PL-G, Fortes PAG, Jameson DM. Mechanisms of inhibition
of (Na+,K+)ATPase by hydrostatic
pressure studied with fluorescent probes. J Biol Chem. 1985;260:14484-14490.
29. Yeagle PL. Cholesterol modulation of the (Na++K+)ATPase hydrolyzing activity in the human erythrocyte. Biochim Biophys Acta. 1983;727:39-44.[Medline] [Order article via Infotrieve]
30. Melchor DL, Steim JM. Thermotropic transitions in biomembranes. Annu Rev Biophys Bioeng. 1976;5:205-238. [Medline] [Order article via Infotrieve]
31.
East JM, Jones OT, Simmonds AC, Lee AG. Membrane fluidity is
not an important physiological regulator of the
(Ca2+-Mg2+)-dependent ATPase of
sarcoplasmic reticulum. J Biol Chem. 1984;259:8070-8071.
32. Gutiérrez-Merino C, Molina A, Escudero B, Diez A, Laynez J. Interaction of the local anesthetics dibucaine and tetracaine with sarcoplasmic reticulum membranes: differential scanning calorimetry and fluorescence studies. Biochemistry. 1989;28:3398-3406. [Medline] [Order article via Infotrieve]
33. García-Martín E, Escudero B, Fernández-Salguero P, Gonález-Cabanillas S, Gutiérrez-Merino C. Modulation of (Ca2++Mg2+)-ATPases and Ca2+ fluxes through the plasma membrane of synaptosomes and sarcoplasmic reticulum by the local anesthetics. Biochem Soc Trans. 1989;17:960-962. [Medline] [Order article via Infotrieve]
34. Niggli V, Adunyah ES, Penniston JT, Carafoli E. Purified (Ca2+-Mg2+)-ATPase of the erythrocyte membrane: reconstitution and effect of calmodulin and phospholipids. J Biol Chem. 1981; 256:395-401.
35. East JM, Lee AG. Lipid selectivity of the calcium and magnesium ion dependent adenosinetriphosphatase, studied with fluorescence quenching by a brominated phospholipid. Biochemistry. 1982;21:4144-4151. [Medline] [Order article via Infotrieve]
36. Caffrey M, Feigenson GW. Fluorescence quenching in model membranes, 3: relationship between calcium adenosinetriphosphatase enzyme activity and the affinity of the protein for phosphatidylcholines with different acyl chain characteristics. Biochemistry. 1981;20:1949-1961. [Medline] [Order article via Infotrieve]
37.
Lundt S, Orlowski S, de Foresta B, Champeil P, Le Maire M,
Moller JV. Detergent structure and associated lipid as determinants in
the stabilization of solubilized Ca2+-ATPase from
sarcoplasmic reticulum. J Biol Chem. 1989;264:4907-4915.
38. Hardwicke PMD, Green NM. The effect of delipidation on the adenosine triphosphatase of sarcoplasmic reticulum: electron microscopy and physical properties. Eur J Biochem. 1974; 42:183-193.
39. Warren GB, Monslay D, Metcalfe JC, Birdsall NJM. Cholesterol is excluded from the phospolipid annulus surrounding an active calcium transport protein. Nature. 1975;255:684-687.[Medline] [Order article via Infotrieve]
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