(Hypertension. 1997;29:1337-1343.)
© 1997 American Heart Association, Inc.
Articles |
Hypothalamic Hypophyseal Inhibitory Factor (HHIF) Increases Intrasynaptosomal Free Calcium Concentration
From the Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, Badajoz (E.G.-M., C.G.-M.), and Servicio de Endocrinología, Hospital "Ramón y Cajal," Madrid (M.R., J.S.), Spain.
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Abstract |
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Abstract We have isolated from bovine hypothalamic and pituitary tissues a sodium pump inhibitor that is structurally different from ouabain. By mass spectrometric analysis, this purified factor revealed a single unique molecular ion with an accurate mass of 412.277 and a mass spectra different from that of ouabain. It has been previously shown that this factor inhibits the Ca2+,Mg2+-ATPase of the plasma membrane of synaptosomes. Because Ca2+ plays a major role in cellular excitability, we carried out a systematic study of the effects of this inhibitor on the Ca2+ transport processes across the plasma membrane of synaptosomes: We measured ATP-dependent calcium uptake, Na+-Ca2+ exchange, and passive permeability using 45Ca2+ and Millipore filtration, chlortetracycline fluorescence, and light-scattering, respectively. This factor inhibits the Na+,K+-ATPase activity of the synaptosomal plasma membrane vesicles in the same range of concentrations that produced an increase of intrasynaptosomal free calcium, with nearly the same K0.5 value. In addition, in this concentration range, this factor stimulated 10- to 11-fold the passive flux of Ca2+ and 2.5- to 3-fold the Ca2+ influx via the Na+-Ca2+ exchange in these membranes with respect to control values. Measurements of fluorescence anisotropy showed that in this concentration range, the inhibitor did not significantly change the order parameter (fluidity) of these membranes. These results suggest that besides its known inhibition of the sodium pump, this factor could play a role in the control of Ca2+ homeostasis by direct modulation of transport systems implicated in the control of intracellular calcium.
Key Words: Na+,K+-exchanging ATPase • ion exchange • Ca2+-transporting ATPase • synaptosomes
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Introduction |
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A controversial hypothesis to explain the etiology of essential hypertension is based on the presence of a humoral factor able to inhibit Na+,K+-ATPase activity. Inhibition of active sodium transport may lead to a rise in intracellular free calcium concentrations and thus to an increase of vascular tone and blood pressure.1 2 We have recently purified an endogenous factor—the hypothalamic hypophyseal inhibitory factor (HHIF)—that is different from digoxin or ouabain.3 HHIF inhibits the sodium pump of human red blood cells4 and produces a rise in cytosolic Ca2+ concentration and contractility in cultured rat mesangial cells.5 In addition, we have previously shown that HHIF inhibits the Ca2+,Mg2+-ATPase activity and active calcium transport of the plasma membrane of synaptosomes.6 Synaptosomal plasma membranes were chosen for methodological reasons and because the modulation of synaptic activity by HHIF could alter vascular smooth muscle tone.6 In nerve terminals, the calcium pump drives Ca2+ out and, together with the Na+-Ca2+ carrier of the plasma membrane, maintains cytosolic Ca2+ below 10-7 mol/L.7 8 Therefore, inhibition of the Ca2+ pump should produce an increase of the cytosolic free Ca2+ concentration. On the other hand, the entry of Ca2+ into nerve terminals and axons is controlled mainly by Ca2+ channels and by the Na+ gradient that reversibly drives the Na+-Ca2+ carrier.7 8 9 In this report, we present the results of our study on the effects of HHIF on the intrasynaptosomal free Ca2+ concentration and on Ca2+ fluxes across the synaptosomal plasma membrane.
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Methods |
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HHIF Extraction and Purification
HHIF was purified by successive chromatographic procedures from bovine hypothalamus and hypophysis as previously indicated.3 4 The inhibition by HHIF of the Na+,K+-ATPase activity from porcine kidney outer medulla was measured with a coupled enzyme assay,10 11 and 1 U was defined as the amount of HHIF required to produce 50% inhibition of the activity of 8 µg purified Na+,K+-ATPase.10 11
Preparation of Synaptosomes and Synaptosomal Plasma Membrane
Vesicles
Synaptosomes were prepared from female Wistar rat brains (200 to
250 g) following the protocol of Michaelis et al,12
as modified by García-Martín and
Gutiérrez-Merino.13 Plasma membrane vesicles were
prepared from synaptosomes by hypotonic lysis as previously described
in García-Martín and
Gutiérrez-Merino.13 The percentage of inverted
vesicles was estimated to be approximately 40% from measurements of
the Na+,K+-ATPase and
Ca2+,Mg2+-ATPase activities of these membranes,
as in García-Martín et al.14 Protein
concentration was measured by the method of Lowry et al15
using bovine serum albumin as standard.
Measurement of Intrasynaptosomal Ca2+
Concentration
The intrasynaptosomal free Ca2+ concentration
([Ca2+]i) was measured with the
fluorescent dye fura 2 following the method described in
Fontana and Blaustein16 with minor modifications. Isolated
synaptosomes were washed by centrifugation at
12 000g for 20 minutes in a Hanks-HEPES buffer (pH 7.4)
containing (mmol/L) Tris 4, HEPES 5, NaCl 137,
Na2HPO4 0.4, NaHCO3 4.2, KCl 5.4,
KH2PO4 0.4, CaCl2 1.25,
MgCl2 0.8, and glucose 10. Synaptosomes were incubated for
40 minutes at 37°C, with gentle shaking, in Hanks-HEPES buffer
containing 5 µmol/L fura 2–acetoxymethyl ester (AM). Control
synaptosomes containing 0.5% dimethyl sulfoxide, but no fura 2-AM,
were prepared in parallel and used for measurement of
autofluorescence. Synaptosomes were centrifuged at
12 000g for 20 minutes, resuspended in Hanks-HEPES buffer
without Ca2+, and stored on ice until used (within 2 to 3
hours). Fluorescence measurements were made in a dual
excitation wavelength spectrofluorimeter (4800C SLM Aminco), equipped
with a thermostatically regulated cell holder and magnetic stirrer, by
alternating the excitation wavelengths of 340 and 380 nm with an
emission wavelength of 510 nm. Fura 2 fluorescence signals were
calibrated at the end of each experiment by sequentially adding 0.3%
(vol/vol) Triton X-100 to determine maximum fluorescence
(Fmax) and 2.5 mmol/L EGTA to determine
minimum fluorescence (Fmin).
[Ca2+]i was calculated as described by
Grynkiewicz et al.17 Autofluorescence was measured
in synaptosomes not loaded with fura 2 and was always less than 10% of
the total fluorescence measured with fura 2–loaded
synaptosomes.
Na+,K+-ATPase Activity
The composition of the assay medium used to measure the
Na+,K+-ATPase activity was 50 mmol/L
2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic
acid (TES) (pH 7.4), 20 mmol/L KCl, 100 mmol/L NaCl, 2
mmol/L MgCl2, 2 mmol/L ATP, 2 mmol/L
ß-mercaptoethanol, 5 mmol/L sodium azide, 0.42 mmol/L
phospho(enol)pyruvate, 0.22 mmol/L NADH, 17.5 µg pyruvate
kinase/mL, 15 µg lactate dehydrogenase/mL, and 20 µg synaptosomal
protein/mL. The Na+,K+-ATPase activity was
determined as the ATPase activity inhibited by 1 mmol/L
ouabain.18
Light-Scattering Measurements
Ionic permeabilities were measured following the
light-scattering method as described in García-Martín
et al14 and Escudero and
Gutiérrez-Merino.19 Synaptosomal plasma membrane
vesicles previously dialyzed against 5 mmol/L TES (pH 7.4) at
4°C for 4 hours were mixed with 5 mmol/L TES (pH 7.4) and
100 mmol/L CaCl2. The final protein concentration was
0.1 mg/mL. Light-scattering intensity at 400 to 410 nm was measured at
25°C as a function of time with a spectrofluorimeter (model 650-40,
Hitachi/Perkin-Elmer). Analysis of the slow recovery phase of
the light-scattering data was carried out as indicated in
García-Martín et al14 and Escudero and
Gutiérrez-Merino.19 Briefly, the experimental data
were fitted to the sum of two exponentials
 | (1) |
are the scattering intensities of the
vesicle at the peak after mixing, at time t, and at
equilibrium after mixing, respectively. D1 and
D2 are the diffusion coefficients of the cation
through two distinct channels in the membrane or two different
conformational states of the same channel with different permeability
properties, and A is the fraction of cation channels of type
1 or in conformational state 1. The transformed data were fitted by
linear regression analysis to obtain the best values for
D.
Measurement of Ca2+ Influx Using the
Fluorescence of Chlorotetracycline
Na+-Ca2+ exchange was measured
using the fluorescence of chlorotetracycline (CTC) as described
by García-Martín et al.8 14 Synaptosomal
plasma membrane vesicles were preequilibrated by incubation at 37°C
with 5 mmol/L TES (pH 7.4), 0.1 mol/L NaCl, and 50 µmol/L
CTC. Na+ gradient–dependent Ca2+ transport was
initiated by diluting (35-fold) these vesicles in an isosmotic assay
medium containing 5 mmol/L TES (pH 7.4); 50 µmol/L CTC;
50 µmol/L CaCl2; and 0.1 mol/L KCl, 0.1 mol/L NaCl,
or 0.1 mol/L choline chloride as indicated in every experimental
series. Protein concentration was 45 µg/mL. CTC fluorescence
was measured at 37°C as a function of time with a spectrofluorimeter
(model 650-40, Hitachi/Perkin-Elmer), with excitation and emission
wavelengths of 380 and 520 nm, respectively. The data were fitted by
nonlinear regression analysis to the following exponential
equation8 14 :
 | (2) |
, Ft,
and F0 are the fluorescence intensities
at times , t, and 0 of the kinetic process, respectively;
and k is the rate constant of the transport process.
Measurements of the Fluorescence Polarization
Fluorescence polarization measurements were carried out
at 30°C with a spectrofluorimeter (model 650-40,
Hitachi/Perkin-Elmer) using diphenylhexatriene as the
fluorescence probe, with excitation and emission wavelengths of
360 and 440 nm, respectively, as previously
described.6 14
Materials
Phospho(enol)pyruvate, ATP, bovine serum albumin, TES,
LaCl3, ß-mercaptoethanol, phenylmethylsulfonyl
fluoride, fura 2-AM, and choline chloride were obtained from
Sigma Chemical Co. NADH, pyruvate kinase (200 IU/mg at 25°C), and
lactate dehydrogenase (550 IU/mg at 25°C) were purchased from
Boehringer Mannheim. All other chemicals used were of
analytical grade and obtained from Merck.
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Results |
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Changes in [Ca2+]i Induced by HHIF
To determine the effect of HHIF on [Ca2+]i, we used synaptosomes loaded with fura 2. The [Ca2+]i of synaptosomes in the absence of HHIF was 150±25 nmol/L. Fig 1
shows the
effect of increasing HHIF concentrations on
[Ca2+]i. HHIF produced an increase in
[Ca2+]i that depended on HHIF concentration.
From a Hill plot of these data, a K0.5 value of
5±0.5 U/mL and a Hill coefficient of 1.1 are obtained. We reported
previously that HHIF strongly binds to synaptosomal membranes, and
therefore, HHIF concentrations should be referred to as free
HHIF.6 The apparent partition coefficient
(Kp) for HHIF binding to synaptosomal plasma
membranes is 0.08 (µg
protein/mL)-1.6 Using this
Kp value, we obtained a
K0.5 value for free HHIF of 0.3 U/mL.
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The increase in [Ca2+]i induced by HHIF
could be a result of activation of Ca2+ influx and/or
inhibition of the Ca2+ efflux. We have already described
the inhibitory effect of HHIF on the Ca2+ pump
of synaptosomes. In an attempt to elucidate the mechanisms by which
HHIF induced the increase of [Ca2+]i shown in
Fig 1
, we undertook a systematic study of the effects of HHIF on
Ca2+ fluxes across the plasma membrane of synaptosomes.
Effects of HHIF on Calcium Fluxes
The Ca2+ influx through the
Na+-Ca2+ exchanger was continuously monitored
by using the fluorescence of CTC in synaptosomal plasma
membrane vesicles preloaded with 0.1 mol/L NaCl. Figs 2A and 3A show the effects of various HHIF
concentrations on the time dependence of the fluorescence
intensity of CTC. HHIF stimulated several-fold the rate of
Ca2+ influx, with both 0.1 mol/L KCl (Fig 2) and 0.1 mol/L
choline chloride in the external medium (Fig 3). We performed control
experiments to determine the Ca2+ uptake in the absence of
Na+ gradient (ie,
[Na+]i=[Na+]o=0.1
mol/L). In these latter experimental conditions, the vesicles did not
significantly accumulate Ca2+. Table 1 shows
the kinetic parameters of the
Na+-Ca2+ exchange obtained from fitting the
experimental data to an exponential equation, as shown in
"Methods" and in García-Martín et
al.14 HHIF increased the rate constant of Ca2+
influx with both depolarized and nondepolarized plasma membrane
vesicles (in K+ medium and in choline chloride medium,
respectively).
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),
0.5 (
), 1 (
), and 2 (
). Data were obtained in a
typical experimental series. Results were confirmed with at least three
different preparations of synaptosomal plasma membrane vesicles.
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The Na+ gradient across the plasma membrane drives the
Na+-Ca2+ exchange.7 8 Therefore,
we studied the effects of HHIF on Na+,K+-ATPase
activity. Fig 4 shows that HHIF inhibited the
Na+,K+-ATPase activity of synaptosomal plasma
membrane vesicles. From the Hill plot of these data, an apparent
K0.5 value of 0.76 U/mL and a Hill coefficient
of 0.98±0.05 were obtained. The free HHIF concentration corresponding
to this K0.5 value, once corrected for binding
of HHIF to the plasma membrane, was 0.3 U/mL.
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We examined whether the inhibition of the Na+,K+-ATPase is a reversible process. For this, we chose an HHIF concentration that inhibited 70% of ATPase activity (50 U/mL HHIF and 0.25 g/L synaptosomal protein). After removal of HHIF by dialysis for 4 hours at 4°C against 5 mmol/L TES (pH 7.4), 2 mmol/L ß-mercaptoethanol, 0.3 mol/L sucrose, and 0.5 mmol/L phosphatidylcholine, the activity was still 70% inhibited. This result suggests that inhibition is likely related to protein denaturation.
The permeability of membrane vesicles to cations and anions can be
measured with the kinetics of light-scattering after a controlled
osmotic shock, as shown in García-Martín et
al14 and Escudero and
Gutiérrez-Merino.19 We studied the effects of HHIF
on the passive Ca2+ influx across the plasma membrane of
synaptosomes using this method. Fig 5A shows the
dependence of the time course of the light-scattering intensity of
vesicles on different HHIF concentrations after an osmotic shock with
0.1 mol/L CaCl2. These results show that HHIF increases the
permeability of these membranes to Ca2+ in a
concentration-dependent manner. The semilogarithmic plot of the
experimental data (Fig 5B) allows for estimation of the rate constant
of this process, as indicated in "Methods." The plots
presented in Fig 5 show that the data can be satisfactorily
fitted to the sum of two exponentials, therefore indicating that
the diffusion of Ca2+ across synaptosomal plasma membrane
proceeds through two kinetically different pathways. The kinetic
parameters obtained from the best fit of the experimental
data by least-squares regression analysis are listed in Table 2. HHIF produced a large increase of the passive
permeability to CaCl2 of the slow and fast processes.
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), 3 (
), and 4 (
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We considered the possibility of changes in the aggregation state of synaptosomal plasma membrane vesicles in these experimental conditions, which could produce misinterpretation of these results. To experimentally test this possibility, we carried out control experiments in which vesicles (25 µg/mL) were incubated with different HHIF concentrations in the absence of osmotic shock. No significant changes of the light-scattering of vesicles were observed in these experimental conditions, indicating that these HHIF concentrations did not produce aggregation of synaptosomal plasma membrane vesicles nor any significant membrane micellization.
Effect of HHIF on the Order Parameter of the Plasma
Membrane Vesicles
HHIF behaves as a lipophilic compound with a high partition
coefficient for synaptosomal membranes (see above and Reference 66 ).
Therefore, we considered the possibility that the effects of HHIF on
calcium fluxes across synaptosomal plasma membrane could be due to
changes in the fluidity of the membrane of synaptosomes. In the
presence of a protein concentration of 120 µg/mL, the order
parameter varied from 0.634±0.008 in the absence of HHIF
to 0.6±0.006 in the presence of 10 U/mL HHIF. These results show that
HHIF does not significantly alter the fluidity of the membrane at
concentrations that modulate Ca2+ fluxes.
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Discussion |
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The results reported in this article show that HHIF increased [Ca2+]i. [Ca2+]i was measured with the Ca2+ indicator fura 2, which has been widely used to measure free [Ca2+]i in synaptosomes under resting and activated conditions.16 20 A similar effect of HHIF has been demonstrated in cultured rat mesangial cells.5 Accordingly, an increase of Ca2+ concentration in platelets of patients with essential hypertension21 and in smooth muscle cells of spontaneously hypertensive rats22 has been reported.
Several mechanisms may contribute to this effect because various
transport systems contribute to maintain the cytosolic free
Ca2+ concentration in the submicromolar range. The
possibility that the rise in [Ca2+]i could be
due to micellization of the plasma membrane is excluded by the
light-scattering measurements. Another possibility is that inhibition
of the Na+,K+-ATPase could account for the rise
in [Ca2+]i. This has been shown to be the
case for smooth muscle cells, as ouabain induced a sustained increase
in [Ca2+]i in these cells that is inhibited
by verapamil.23 24 This is consistent
with a rise of [Ca2+]i caused by
Ca2+ influx through voltage-sensitive Ca2+
channels activated upon membrane depolarization. Meyer-Lehnert
et al23 have isolated a ouabainlike factor that increases
[Ca2+]i in vascular smooth muscle cells
through activation of voltage-sensitive Ca2+ channels.
However, in most cells, the inhibition of the Na+ pump
results in a very small depolarization, which is unlikely to
significantly activate voltage-sensitive Ca2+
channels.25 In this article, we show that HHIF increased
the passive Ca2+ influx across the plasma membrane of
synaptosomes independently of the inhibitory effect on the
Na+,K+-ATPase activity. These findings are
consistent with those reported by Goto et al26 in
smooth muscle cells. The results obtained in our study gave a value for
the apparent rate of passive Ca2+ influx of 0.033
min-1. This value is consistent with
the experimental results of passive Ca2+ efflux from
synaptosomes preloaded with 45Ca2+ reported by
Gill et al27 and also with the rate of passive
Ca2+ influx measured with light-scattering in an earlier
work from this laboratory.14 Taking an internal volume of
synaptosomal plasma membrane vesicles of 3 to 7 µL/mg
protein,28 for a physiological
gradient of Ca2+, and 0.1 µmol/L and 1 mmol/L
free Ca2+ inside and outside the vesicles, respectively, we
obtained a maximum rate of passive Ca2+ flux of 0.1 to 0.2
nmol/mg protein per minute. The data presented in Fig 5 and the
parameters listed in Table 2 show that HHIF increases the
rate of passive Ca2+ influx at lower concentrations than
those needed to inhibit the Ca2+ pump.6 The
HHIF concentrations should be corrected for HHIF binding to the plasma
membrane. For a free HHIF concentration of approximately 0.45 U/mL, the
Ca2+ influx is 1.06 to 2.12 nmol/mg protein per minute, a
value 10- to 11-fold higher that the control value.
A rise in [Na+]i could increase
[Ca2+]i by decreasing the rate of
Ca2+ efflux mediated by the
Na+-Ca2+ exchanger.29 Synaptosomes
and plasma membrane vesicles transport Ca2+ ions at the
expense of the Na+ gradient across these membranes via
Na+-Ca2+ exchange.7 8 The exchange
system can mediate Ca2+ fluxes in either direction across
the cell membrane, depending on the prevailing electrochemical
gradients for Na+. We have shown that HHIF stimulates the
Na+-Ca2+ exchange about 2.5- to 3-fold with
respect to the control values at approximately the same concentration
that inhibits 50% Na+,K+-ATPase activity (0.3
U/mL free HHIF, after correction for membrane binding). Considering
this inhibition, the stimulation of the
Na+-Ca2+ exchanger could be smaller than
expected because after dissipation of Na+ gradient, the
exchanger does not operate. Although the occurrence of
Na+-Ca2+ exchange activity has been
demonstrated in large vessels,29 its existence in the
precapillary resistance vessels remains uncertain,30 31
and therefore, its role in the increase of vascular tone in the
presence of an inhibitor is unlikely.32 In
addition, HHIF inhibits the plasma membrane
Ca2+,Mg2+-ATPase, as we have reported
previously.6 This effect may explain by itself the
observed increase in [Ca2+]i (Fig 1). It has
been reported that the Ca2+ pump of erythrocytes from
humans with essential hypertension and from spontaneously hypertensive
rats has specific activities lower than the pump of healthy
individuals.33 34 This result is in agreement with Goto et
al,26 35 who have reported that a factor purified from
human urine inhibited the efflux of 45Ca2+ in
smooth muscle cells.
HHIF is a lipophilic molecule, which partitions between membranes and aqueous phases,4 6 36 and is particularly enriched in some brain areas where the concentration of HHIF (in units per gram of wet tissue) estimated from the amount of HHIF recovered are as follows: bovine hypophysis, 2.5; bovine hypothalamus, 0.424 ; and whole brain homogenate, 0.36.37 Thus, the possibility that HHIF acts as a local mediator in the nervous system seems most plausible, thereby exerting its effects through modulation of Ca2+ metabolism in nerve terminals, as discussed above. In fact, the sympathoexcitation and hypertension described by Huang and Leenen38 with the central administration of ouabain in rats seem to be replicated by HHIF (unpublished observations, 1996). However, it should be noted that the concentration of HHIF (in units per gram of wet tissue) in peripheral tissues reaches values of 0.4 (bovine adrenal gland), 0.22 (bovine spleen), 0.21 (bovine pancreas), 0.18 (bovine kidney), 0.21 (human placenta), and 1 U/100 mL in human blood bank plasma.4 Therefore, HHIF circulates in human plasma, and the concentration of free HHIF in some peripheral tissues is in the range of HHIF concentrations that modulate the Na+,K+-ATPase, the Ca2+-pumps, and [Ca2+]i (this article and also References 6, 36, and 396 36 39 ).
In summary, these results show that HHIF increases [Ca2+]i with the same K0.5 value (0.3 U/mL of free HHIF) that inhibits the Na+,K+-ATPase. This concentration is approximately half the K0.5 value of inhibition of the Ca2+ pump6 and stimulates the passive Ca2+ influx and Na+-Ca2+ exchange fourfold and twofold, respectively.
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Acknowledgments |
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This work was partially supported by financial aid from the Spanish FIS (grant 94/0497), DGICYT (grant PB91-0311), and Junta de Extremadura (grant EIA94-21) as well as Dr Mercedes Ricote fellowships from FIS and the Rich Foundation.
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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 March 18, 1996; first decision April 24, 1996; accepted July 22, 1996.
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