Influence of thyroid status on hepatic
1-adrenoreceptor
responsiveness
Francisco J.
Daza,
Roberto
Parrilla, and
Angeles
Martín-Requero
Department of Physiopathology and Human Molecular Genetics,
Centro de Investigaciones Biológicas, 28006 Madrid, Spain
 |
ABSTRACT |
The present work aimed to elucidate the
influence of thyroid functional status on the
1-adrenoreceptor-induced
activation of hepatic metabolic functions. The experiments were
performed in either a nonrecirculating liver perfusion system featuring continuous monitoring of portal pressure,
PO2, pCa, and pH, or isolated
hepatocytes from euthyroid, hyperthyroid, and hypothyroid rats.
Hypothyroidism decreased the
1-adrenergic stimulation of
respiration, glycogen breakdown, and gluconeogenesis. These effects
were accompanied by a decreased intracellular
Ca2+ mobilization corroborating
that those processes are regulated by the
Ca2+-dependent branch of the
1-adrenoreceptor signaling
pathway. Moreover, in hyperthyroid rats the
1-adrenergic-induced increase in cytosolic Ca2+ was enhanced,
and glucose synthesis or mobilization was not altered. The thyroid
status influenced neither the
1-adrenergic
stimulation of vascular smooth muscle contraction nor the
1-agonist-induced intracellular
alkalinization and protein kinase C (PKC) activation. Thus the distinct
impairment of the Ca2+-dependent
branch of the
1-adrenoreceptor
signaling pathway by thyroid status provides a useful tool to
investigate the role played by each signaling pathway,
Ca2+ or PKC, in controlling
hepatic functions.
hyperthyroidism; hypothyroidism;
1-adrenergic agonists; protein
kinase C; cytosolic Ca2+; intracellular pH
 |
INTRODUCTION |
SOME HORMONES, like glucocorticoids or
triiodothyronine, may exert a permissive action on the target cells'
responsiveness to other hormones (5, 19, 21). In this sense there are numerous studies illustrating the role played by thyroid hormones on
the adrenergic activation of metabolism (3, 16, 30). The following
observations might have justified this type of studies: 1) similarity of the thyrotoxicosis
to the increased adrenergic activation,
2) effectiveness of adrenergic
blockers in the treatment of hyperthyroidism, and
3) finally, the observations in
hypothyroidism of changes indicative of decreased adrenergic
responsiveness. The studies on the influence of thyroid status on
1-adrenergic regulation of
hepatic metabolism are controversial. Hypothyroidism has been reported
to decrease the
1-agonist-induced activation of
phosphorylase a and
Ca2+ release in liver cells (23).
In contrast, other authors failed to detect any effect of
hypothyroidism on the
1-adrenergic-mediated actions,
although they observed a marked inhibition of the hepatic responses to
the vasoactive peptides vasopressin and angiotensin II (6, 12). On
these grounds, we found it of interest to investigate the role played
by thyroid status on hepatic
1-adrenergic responsiveness.
Hepatic
1-adrenoreceptor
stimulation elicits a complex variety of hemodynamic, respiratory,
ionotropic, and metabolic responses (5, 28, 29). Recent work supports
the existence of at least two branches in the hepatic
1-adrenoreceptor signaling
pathway that can operate independently (4). One of them is PKC mediated and Ca2+ independent, and the
other is Ca2+ dependent and PKC
independent (4). The present work aimed to investigate the influence of
thyroid status on the hepatic
1-adrenoreceptor-mediated
responses. Our results indicate that the thyroid hormones control a
postreceptor step(s) in the hepatic
1-adrenergic signaling
pathways. Hypothyroidism attenuated some of the hepatic
1-adrenergic responses, and
this effect was accompanied by a decreased
1-adrenergic-induced
intracellular Ca2+ mobilization.
Hyperthyroidism did not change the
1-agonist-mediated mobilization
of intracellular Ca2+, and
carbohydrate synthesis and mobilization were not impaired. Neither
hypothyroidism nor hyperthyroidism altered the
1-agonist-induced stimulation
of PKC activity. It is concluded that thyroid hormones modulate the
Ca2+-sensitive PKC-independent
branch of the
1-adrenergic
signaling cascade in rat liver.
 |
MATERIALS AND METHODS |
Animals.
Male Wistar rats were maintained under controlled lighting and
temperature conditions with standard laboratory chow and tap water ad
libitum until surgery. All the animals were treated in a manner that
complied with the European guidelines for the care and use of
laboratory animals. Hyperthyroidism was induced in rats by daily
intraperitoneal injections of 15 µg of sodium
L-triiodothyronine (L-T3)/100 g
of body mass for 7 days. The T3
solution (250 µg/ml) was prepared in 0.1 N NaOH. Hypothyroid rats
were obtained by thyroid-parathyroidectomy of rats of ~150 g. They
were used 3 wk later, when their body mass was 200-250 g. Serum
L-T3
levels, measured using a commercial radioimmunoassay kit (Biomerieux, France), were 1.53 ± 0.11 and 14.86 ± 0.7 nM in euthyroid and hyperthyroid rats, respectively, and were below the limit of detection for the assay (0.2 nM) in hypothyroid rats.
Liver perfusion and isolation of liver cells.
Livers from rats of different thyroid status were perfused with
Krebs-Ringer bicarbonate (KRB) buffer in a flow-through system. Technical details were similar to those previously described (5). Routinely, the perfusion flow rate was adjusted to 30 ml/min. A 30-min
equilibration period was allowed routinely before the infusion of
substrates. Substrates and phenylephrine were administered diluted in
buffer. Oxygen consumption was determined by measuring the
PO2 arteriovenous difference with a
Clark-type oxygen electrode. pH, pK, and pCa were
continually monitored in the outflow perfusate by use of selective
electrodes. The calibration of the electrodes was carried out under the
same conditions used to perfuse the organs, by varying the
concentration of each ion in the perfusate buffer within the expected
range of variations in the actual experiments. The portal vein pressure
was measured with a pressure transducer, model Hugo Sach Elektronik
(March-Hugstetten, Germany). The amplified analog signals from the
different sensors were digitized in a Hewlett-Packard analog-to-digital
converted model HP3421A, and the data were imported into a desk
computer through an IEEE-488 communication port for further processing.
Liver cells from fed rats were obtained by perfusion of the hepatic
vascular system with collagenase following previously described
protocols (5).
Measurement of cytosolic
Ca2+ and
intracellular pH.
Cytosolic free Ca2+ was determined
using the fluorescent probe fura 2, as previously described (5). Cells
were loaded with the intracellular indicator by a 15-min incubation
with fura 2-acetoxymethyl ester (fura 2-AM, 5 µM) in the presence of
0.0012% Pluronic F-127 followed by a 30-min incubation in the standard
incubation buffer [modified KRB containing 20 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4, 15 mM glucose, 1.3 mM
CaCl2, and 0.2% defatted bovine serum albumin (BSA)] to allow for complete deesterification of the indicator. After three washes with fresh buffer, portions of the
cell suspension (
20 mg wet wt) were transferred to 2 ml of the
incubation buffer in a cuvette prewarmed at 37°C.
Fluorescence measurements were carried out using a Perkin-Elmer LS-50 B
spectrofluorimeter equipped with a fast-filter accessory that minimizes
the interval delay between changing wavelengths. The excitation
wavelengths were 340 and 380 nm and the emission wavelength was 510 nm.
Calibration of the fluorescence signal was carried out by the method of
Grynkiewicz et al. (15) with a dissociation constant, or
Kd, of 224 nM.
Intracellular pH was determined using the intracellular trappable
fluorescent pH indicator
2'-7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF)
(24). The experimental details for loading hepatocytes with BCECF were
as previously described (28). Fluorescence of BCECF was determined as
described above, with the excitation wavelengths 495 and 440 nm and the
emission wavelength 530 nm. Calibration of the fluorescent signal was
carried out using the nigericin/K+
method of Thomas et al. (27). The 495/440 ratio was a linear function
of pH over the range of 6.8-7.6 pH units.
PKC activity.
To determine the activity of PKC, hepatocytes (100 mg wet wt/ml) were
incubated in KRB buffer, pH 7.4, containing 2% Ficoll-70 for 15 min in
the absence or the presence of 1 µM phenylephrine. The cells were
collected by centrifugation, resuspended, and homogenized in ice-cold
buffer A [20 mM
tris(hydroxymethyl)aminomethane (Tris), pH 7.5, 2 mM EDTA, 0.5 mM
ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 2 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, 0.25 M sucrose, 20 µg/ml trypsin inhibitor, and 0.04 mM
leupeptin]. Homogenates were centrifuged at 12,000 g for 20 min. The supernatants were
used as the cytosol fraction, and the pellets were homogenized in
buffer A containing 1% Triton X-100,
incubated at 4°C for 30 min, and centrifuged at 12,000 g for 30 min. The supernatant was used
as solubilized membrane fraction. Cytosol and solubilized plasma
membranes were applied to DEAE-cellulose columns (1 ml) equilibrated
with buffer A without sucrose. Elution
was performed with 0.1 M NaCl in buffer
A. PKC was assayed by measuring the incorporation of
32P from
[
-32P]ATP into
histone (type III, Sigma Chemical, St. Louis, MO) essentially as
described (17). This approach measures all the isoforms of PKC. The assay mixture (200 µl)
contained 1 mM Tris, pH 7.5, 7.5 mM
MgCl2, 1.5 mM
CaCl2, 50 µg histone, 25 µg
phosphatidylserine, 1 µg 1,2-dioctanoylglycerol, 0.05 mM ATP, 0.25 µCi [
-32P]ATP,
and 50 µl of the enzyme preparation. Basal activity was obtained by
replacing CaCl2 and phospholipids
with 5 mM EGTA. The mixture was incubated at room temperature for 10 min. The reaction was stopped by the addition of 2 ml of 5%
trichloroacetic acid containing 10 mM
H3PO4
and 200 µg of BSA. The precipitates were collected and washed on
Whatman GF/A glass fiber filters and counted in a liquid scintillation
counter.
Metabolite analysis.
Perfusate samples were analyzed immediately after their collection and
assayed spectrophotometrically by use of previously described enzymatic
procedures (1).
Materials.
All the reagents were of the highest purity available; most were
obtained from Sigma Chemical. The enzymes and BCECF-AM were obtained
from Boehringer Mannheim.
[
-32P]ATP (5,000 Ci/mmol) was obtained from Amersham (UK). EIPA
[(5-N-ethyl-N-isopropyl)-amiloride] was obtained from Research Biochemical (Natick, MA).
Statistics.
Comparison between groups was performed by analysis of variance (ANOVA)
followed by the Newman-Keuls multiple comparison test. Differences were
considered to be significant at P < 0.05.
 |
RESULTS |
Influence of thyroid status on the
1-adrenergic stimulation of
hepatic functions.
Figure 1 shows the effects of the
1-agonist phenylephrine on
oxygen consumption, glucose output,
H+ and
Ca2+ changes in the outflow
perfusate, and portal pressure in perfused livers from fed euthyroid,
hyperthyroid, or hypothyroid rats. In agreement with
previous observations (4, 5, 28, 29), the
1-adrenergic stimulation
produced an increase of respiration, enhanced glucose mobilization,
increased H+ and
Ca2+ release, and vascular
resistance. The lack of glucose release in livers from hyperthyroid
rats (see Fig. 1) is an effect of virtual depletion of
glycogen stores (7). The outflow of protons in livers from fed rats is
almost totally accounted for by its cotransport with lactate (10, 28);
thus the apparent inhibitory effect of hyperthyroidism on
1-agonist-induced extracellular acidification (see Fig. 1) is nothing but the consequence of the virtual absence of glycogenolysis. The phenylephrine-induced
acidification of the outflow perfusate reflects, in these conditions,
the activation of the
Na+/H+
exchanger. Another change associated with hyperthyroidism was a
decrease in the
1-agonist-induced
Ca2+ release. Hypothyroidism
decreased the
1-adrenergic
stimulation of respiration, Ca2+
release, and glucose output by 43, 50, and 35%, respectively, compared
with the same
1-adrenergic
responses in livers from euthyroid rats (Fig. 1). The
1-agonist-induced increase in
portal pressure was not perturbed by the thyroid status. These changes were reversed by in vivo thyroid hormone replacement (results not
shown).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 1.
Influence of thyroid status on hepatic
1-adrenergic responsiveness.
Livers from eu-, hyper-, and hypothyroid rats, fed ad libitum, were
perfused with Krebs-Ringer bicarbonate buffer, pH 7.4, until a steady
rate of oxygen consumption was attained. Then phenylephrine (1 µM PE)
was administered diluted in medium for the time indicated. Data shown
for glucose output are means of 6 experiments. Traces are from
representative experiments. Averages (means ± SE) of sustained
changes after addition of PE in livers from eu-, hyper, and hypothyroid
rats were, respectively: for oxygen consumption, 267 ± 26, 236 ± 20, and 153 ± 23 µatom · 100 g body
wt 1 · h 1;
for pH, 0.064 ± 0.002, 0.01 ± 0.007, and
0.058 ± 0.01 units; for portal pressure, 2.2 ± 0.2, 2.1 ± 0.1 and 2.0 ± 0.3 mmHg; and for
Ca2+ concentration
([Ca2+]), 0.04 ± 0.01, 0.01 ± 0.006, and 0.02 ± 0.002 mM.
|
|
Influence of thyroid status on the
1-adrenoreceptor-induced
responses in livers from starved rats.
The carbohydrate stores of livers from 48-h-starved rats are totally
depleted of glycogen, and therefore the
1-agonist-induced variations in
the outflow perfusate pH are due to
H+ released by means other than
cotransport with metabolites like lactate (28). Thus we used livers
from starved rats to investigate the eventual effect of
1-agonist stimulation on
extracellular acidification and also the gluconeogenic responses of
livers perfused with substrate amounts of a gluconeogenic precursor. In
the absence of exogenous substrates,
1-adrenoreceptor activation
produced its characteristic effects in stimulating respiration,
Ca2+ and
H+ release, increased portal
pressure, and a small and transient stimulation of glucose release
(Fig. 2). The thyroid status did not
influence the
1-agonist-induced
changes in pH (Fig. 2), indicative of a normal activation of the plasma
membrane
Na+/H+
exchanger (5, 28). The
1-agonist-induced
Ca2+ release was similarly
affected in livers from hypo- or hyperthyroid rats. Figure
3 depicts the
1-adrenergic stimulation of
gluconeogenesis in livers from euthyroid, hyperthyroid, and hypothyroid
rats perfused with pyruvate as the gluconeogenic precursor. The oxygen
uptake (Fig. 3) is plotted as changes above the basal rates that varied (in µatom · 100 g body
wt
1 · h
1)
from 897 ± 94 in livers from euthyroid rats to 1,140 ± 38 in livers from hyperthyroid rats or 561 ± 32 in livers from
hypothyroid rats. These variations are consistent with the known effect
of thyroid status on the basal rate of oxygen consumption (25). In
livers from hyperthyroid rats, neither the basal nor the
1-agonist-induced increase in
gluconeogenesis was altered (Fig. 3). In contrast, the
1-adrenergic stimulation of
gluconeogenesis was seriously impaired in livers from hypothyroid rats,
an effect that was accompanied by stoichiometric changes in the
stimulation of respiration (Fig. 3).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 2.
Influence of thyroid status and starvation on hepatic
1-adrenergic responsiveness.
Livers from 48-h-starved rats were perfused as described in Fig. 1.
Averages (means ± SE) of sustained changes after PE addition in
livers from eu-, hyper- and hypothyroid rats were, respectively: for
oxygen consumption, 270 ± 18, 227 ± 30, and 123 ± 9 µatom · 100 g body
wt 1 · h 1;
for pH, 0.019 ± 0.01, 0.026 ± 0.006, and
0.019 ± 0.002 units; for portal pressure, 2.6 ± 0.1, 2.6 ± 0.2, and 2.4 ± 0.3 mmHg; and for
[Ca2+], 0.027 ± 0.01, 0.012 ± 0.004, and 0.018 ± 0.004 mM.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
Influence of thyroid status on basal and
1-adrenergic-induced
stimulation of gluconeogenesis. Livers from eu-, hyper-, and
hypothyroid rats, starved for 48 h, were perfused as described in Fig.
1. When steady rates of oxygen consumption were obtained in the
presence of 1 mM pyruvate, PE (1 µM) was added for times indicated by
the boxes. Data shown for oxygen consumption are representative traces
of 4 experiments. Averages (means ± SE) of sustained changes
after addition of PE in livers from eu-, hyper-, and hypothyroid rats
were, respectively, for oxygen consumption, 295 ± 17, 262 ± 28, and 129 ± 28 µatom · 100 g body
wt 1 · h 1,
and for glucose production, 68 ± 6, 57 ± 8, and 16 ± 3 µmol · 100 g body
wt 1 · h 1.
|
|
The glycogenolytic responses, or the variations in
Ca2+ fluxes across the plasma
membrane in response to
Ca2+-mobilizing agents other than
1-adrenergic agonists, like the vasoactive peptides vasopressin or angiotensin II, were also altered in
livers from hypothyroid rats (results not shown). These observations are consistent with the fact that the
1-adrenegic agonist and the
vasoactive peptide receptors share some intracellular signaling events.
Influence of thyroid status on the agonist-induced changes on
intracellular
Ca2+
mobilization, pH, and PKC activation.
A rise in the cytosolic free Ca2+
concentration
([Ca2+]i)
is the best characterized effect of hepatic
1-adrenergic stimulation, and
it has been made responsible for some of the functional responses. However, recent work has demonstrated that PKC activation may account
for some of the agonist-induced hepatic responses, such as the
intracellular alkalinization that follows the activation of the
Na+/H+
exchanger (4, 5, 28). Thus we have investigated whether the functional
changes associated with the thyroid status were accompanied by
variation in these parameters.
Figure 4 depicts the influence of thyroid
status on the intracellular Ca2+
responses to calcium-mobilizing agonists in isolated liver cells. Cells
from euthyroid rats have a resting
[Ca2+]i
of 213 ± 20 nM; thyroid hormone deficiency did not alter the basal
cytosolic free Ca2+ levels (204 ± 4 nM), whereas increased resting
[Ca2+]i
(418 ± 9 nM) was found in cells from hyperthyroid rats.
Hyperthyroidism enhanced the sustained phase of the
Ca2+ response without
significantly affecting the peak levels of cytosolic free
Ca2+, regardless of the
Ca2+-mobilizing agonist with which
the cells were stimulated (Fig. 4). In contrast, hypothyroidism
diminished the rises in
[Ca2+]i
evoked by all agonists (Fig. 4).

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 4.
Influence of thyroid status on 1-adrenoreceptor
and vasoactive peptide-induced Ca2+ mobilization
in liver cells. Loading of hepatocytes with fura-2 and measurement of
cytosolic Ca2+ were performed as described in
MATERIALS AND METHODS. Each trace is representative of
observations from 6 different cell preparations. Average peak
intracellular [Ca2+]
([Ca2+]i) values (means ± SE) after
hormone stimulation were, for PE, 120 ± 21, 140 ± 15 and 71 ± 9 nM; for vasopressin (VP), 180 ± 24, 187 ± 14 and 81 ± 8 nM; and for angiotensin II (ANG II), 180 ± 18, 205 ± 30, and 76 ± 7 nM in hepatocytes from euthyroid,
hyperthyroid, and hypothyroid rats, respectively. Mean values of peak
Ca2+ levels in cells from hypothyroid rats were found
to be significantly different from those obtained in cells from
euthyroid rats incubated under identical conditions (P < 0.01).
|
|
The intracellular Ca2+ response to
Ca2+-mobilizing agents is the net
effect of enhanced Ca2+
mobilization from intracellular stores and
Ca2+ influx from the extracellular
medium (11); therefore, we further investigated the influence of
thyroid status on both processes. The inflow of
Ca2+ was appraised as the increase
in
[Ca2+]i
observed in cells incubated in nominally calcium-free medium in the
presence of the endomembrane
Ca2+-adenosinetriphosphatase
inhibitor thapsigargin (26) after the normal extracellular level of
Ca2+ was restored. As shown in
Fig. 5, the basal and stimulated rates of
Ca2+ inflow observed in cells from
hyperthyroid rats were greater than those observed in control cells.
Hypothyroidism did not significantly affect the rate of
Ca2+ entry (Fig. 5).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5.
Influence of thyroid status on basal and
Ca2+-mobilizing agonist-induced stimulation of
Ca2+ inflow. Loading of cells with fura 2 and
fluorescence measurement were performed in nominally
Ca2+-free medium. Depletion of internal
Ca2+ was accomplished as described in MATERIALS
AND METHODS in presence of 0.5 µM thapsigargin. Values shown
are average maximal changes (means ± SE) obtained after addition of
1.3 mM CaCl2 to medium, in the
absence or the presence of 10 µM PE.
* P < 0.001, significantly
different from mean values of liver cells from euthyroid rats incubated
in either the absence or the presence of PE.
|
|
The fact that neither basal
[Ca2+]i
nor Ca2+ inflow from the
extracellular medium was altered in hepatocytes isolated from
thyroid-parathyroidectomized rats makes unlikely eventual changes on
the functioning of liver channels, secondary to parathyroid hormone
deficiency, as it has been reported for voltage-gated
Ca2+ channels in excitable tissues
(22).
Figure 6 depicts the influence of thyroid
status on the
1-agonist or
vasoactive peptide-induced changes in intracellular pH. In agreement
with previous reports (4, 5, 28),
1-adrenoreceptor activation
produced an intracellular alkalinization. The activation of vasoactive
peptide receptors also led to intracellular alkalinization (Fig. 6).
This receptor-mediated intracellular alkalinization was prevented by
preincubating the cells with the
N+/H+
antiporter blocker EIPA (18) (results not shown). Neither
hyperthyroidism nor hypothyroidism influenced the
Ca2+-mobilizing receptor-induced
intracellular alkalinization (Fig. 6). It has been previously shown
that activation of the
Na+/H+
antiporter is under PKC control (16, 20). For this reason, we studied
the influence of thyroid status on the
1-agonist-induced stimulation
of PKC activity. Table 1 shows how the
addition of Ca2+-mobilizing
agonists to liver cells from euthyroid rats, as well as from hyper- or
hypothyroid rats, increased the membrane-associated PKC activity and
decreased proportionally the cytosolic PKC activity, indicating the
translocation of the protein to the membrane activation sites. Thus the
observation that thyroid status does not influence the agonist-induced
activation of PKC activity (Table 1) is consistent with the lack of
effects on intracellular pH (Fig. 6) and also with the postulate that
PKC plays a major role in the activation of the antiporter in liver
cells. However, it should be pointed out that the present data cannot
rule out the possibility that thyroid hormones could regulate the
expression or the enzymatic properties of selective PKC isoforms.
Further work will be required to elucidate this point.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 6.
Influence of thyroid status on
Ca2+-mobilizing agonist-induced
intracellular alkalinization. Loading of hepatocytes with
2'-7'-bis(carboxyethyl)-5,6 carboxyfluorescein and
measurement of intracellular pH were performed as described in
MATERIALS AND METHODS. Each trace is
representative of observations from 6 different cell preparations.
Basal intracellular pH (pHi)
mean ± SE values were 7.05 ± 0.019, 7.08 ± 0.013, and
7.09 ± 0.007 for hepatocytes from euthyroid,
hyperthyroid, and hypothyroid rats, respectively.
|
|
 |
DISCUSSION |
The relationship between thyroid function and adrenergic state has been
a subject of attention by many researchers because of similarities in
thyroid and adrenergic functional states and the ability of adrenergic
blockers to ameliorate the hyperthyroid states. However, most of these
studies have been focused on the
-adrenoreceptors, and the hepatic
studies are scarce.
The results of this study demonstrate that thyroid status modulates
hepatic
1-adrenergic
responsiveness. In this work we have investigated a number of
parameters, such as energy production, ion fluxes across the plasma
membrane, Ca2+ mobilization, and
intracellular pH, and their correlation with rate changes in
glycogenolysis or gluconeogenesis. Our results are in close agreement
with the reported decrease in
1-adrenergic stimulation of
glycogen phosphorylase a in liver
cells from hypothyroid rats (23). However, our results seem to be in
conflict with previous work by Corvera et al. (6) and Garcia-Sainz and
Hernandez-Sotomayor (12). The reason for this is not clear at present.
These authors used rats in which hypothyroidism was induced
pharmacologically, rather than by surgical thyroidectomy, and they used
epinephrine instead of the
1-agonist phenylephrine. The
use of different precursors for glucose synthesis (lactate and
dihydroxyacetone or pyruvate in this work) might perhaps
explain the different sensitivity of the
1-adrenergic stimulation of
gluconeogenesis by thyroid hormones.
Recent work has demonstrated that signals arising from the hepatic
1-adrenoreceptor follow at least two independent
pathways, one PKC mediated and
Ca2+ independent and the other
Ca2+ sensitive and PKC independent
(4). On these grounds our investigation was oriented toward the
elucidation of the role played by these two signaling pathways in the
control of the hepatic
1-adrenoreceptors by
T3.
Both hyperthyroidism and hypothyroidism decreased the
1-adrenergic-induced sustained
release of Ca2+ in perfused liver.
However, the significance of this finding most probably differs in each
case. In hyperthyroid livers the
1-adrenergic-induced
intracellular mobilization of Ca2+
was not altered (Fig. 4), and both basal and stimulated
Ca2+ inflow from the extracellular
medium was enhanced (Figs. 4 and 5). Thus the decrease in the
1-adrenergic-induced hepatic
sustained release of Ca2+ in
hyperthyroidism seems to primarily reflect the increased rate of
Ca2+ inflow. In contrast, in
livers from hypothyroid animals, the decrease in the
1-adrenergic-induced sustained
release of Ca2+ could be accounted
for, at least in part, by the decreased ability to mobilize
Ca2+ from the intracellular stores
(Figs. 1, 2, and 4). The finding that glycogenolysis or gluconeogenesis
was perturbed only in livers from hypothyroid animals in which the
1-adrenergic-induced
intracellular Ca2+ mobilization
was impaired gives further support to the proposed relationship between
1-adrenoreceptor-induced
intracellular Ca2+ mobilization
and the stimulation of gluconeogenesis (4, 5, 8, 13).
Neither hyper- nor hypothyroidism impaired the
1-adrenergic or vasoactive
peptide-induced extracellular acidification or intracellular
alkalinization (Figs. 2 and 6). Because the PKC activation induced by
these agonists was not impaired by the thyroid status either, these
observations corroborate the postulated relationship between the
receptor-mediated activation of PKC and stimulation of processes such
as the
Na+/H+
exchange (5, 16, 20). Previous work in which the state of activation of
PKC was manipulated by use of pharmacological and physiological
substrates or enzyme inhibitors (29) demonstrated the variable degree
of dependency on PKC activity among the
1-adrenergic-stimulated functions, ranging from totally dependent, such as vascular resistance, to almost independent, like gluconeogenesis. Thus the finding that
neither the
1-adrenergic
stimulation of vascular resistance (Figs. 1 and 2) nor the
agonist-mediated translocation of PKC toward the plasma membrane (Table
1) was influenced by the thyroid status adds further support to the
proposed relationship between these two hepatic responses (29). The
intact agonist-induced stimulation of PKC in liver cells from
thyroidectomized rats, together with reduced phospholipase C activation
(monitored as decreased rises in cytosolic free
Ca2+), supports the proposed
role of Ca2+-mobilizing agents in
increasing the diacylglycerol generation from the hydrolysis of
phospholipids other than phosphoinositides (9).
The mechanism by which thyroid hormones enhance
Ca2+ inflow in liver cells cannot
be ascertained by the present data. However, the observation that the
agonist-mediated PKC activation and mobilization of intracellular
Ca2+ were not impaired in
hepatocytes from hyperthyroid rats rules out an action of thyroid
hormones in control of second messenger-operated Ca2+ channels (2). Further work
will be needed to elucidate which type(s) of
Ca2+ channels are sensitive to
thyroid hormone regulation.
In conclusion, thyroid hormones modulate hepatic
1-adrenoreceptor responsiveness
by interacting with the
Ca2+-dependent PKC-insensitive
branch of its signaling pathway.
T3 could act in controlling
hepatic
1-adrenoreceptor
function by changing the
1-adrenoreceptor density (23).
However, because hepatic responses to
Ca2+-mobilizing agents other than
1-agonists were also influenced by the thyroid status (Fig. 4), it seems plausible to conclude that a
postreceptor event(s), shared by
Ca2+-mobilizing receptors, must be
the target for the T3 action. The interaction of T3 with the
1-adrenoreceptor signaling
pathway provides a basis for the understanding of its effect in
controlling carbohydrate metabolism. Moreover, hypothyroidism emerges
as a unique physiopathological model to further evaluate the
independent regulation of the two branches of the
1-adrenoreceptor signaling pathway.
 |
ACKNOWLEDGEMENTS |
The skillful technical assistance of M. J. Arias-Salgado is greatly
appreciated, and we are highly indebted to Dr. D. Ibarreta for the
language revision of the manuscript.
 |
FOOTNOTES |
This work has been supported in part by grants from Fondo de
Investigaciones Sanitarias (94/0224) and from Dirección General de Investigación Científica y Técnica (PB93/0163
and PB94/1544).
Address for reprint requests: A. Martín-Requero, Velazquez 144, 28006 Madrid, Spain.
Received 14 March 1997; accepted in final form 13 August 1997.
 |
REFERENCES |
1.
Bergmeyer, H. U.
Methods in Enzymatic Analysis. New York: Academic, 1975.
2.
Berridge, M. J.
Inositol trisphosphate and calcium signalling.
Nature
361:
315-325,
1993[Medline].
3.
Bilezikian, J. P.,
and
J. N. Loeb.
The influence of hyperthyroidism and hypothyroidism on
and
-adrenergic receptor systems and adrenergic responsiveness.
Endocr. Rev.
4:
378-388,
1983[Abstract].
4.
Butta, N.,
A. Martín-Requero,
E. Urcelay,
R. Parrilla,
and
M. S. Ayuso.
Modulation of the hepatic
1-adrenoreceptor responsiveness by colchicine.
Br. J. Pharmacol.
118:
1797-1805,
1996[Abstract].
5.
Ciprés, G.,
N. Butta,
E. Urcelay,
R. Parrilla,
and
A. Martín-Requero.
Impaired protein kinase C is associated with decreased hepatic
1-adrenoreceptor responsiveness in adrenalectomized rats.
Endocrinology
136:
468-475,
1995[Abstract].
6.
Corvera, S.,
S. M. T. Hernandez-Sotomayor,
and
J. A. García-Saínz.
Modulation by thyroid status of cyclic AMP-dependent and Ca2+ dependent mechanisms of hormone action in rat liver cells. Possible involvement of two different transduction mechanisms in
-adrenergic action.
Biochim. Biophys. Acta
803:
195-205,
1984.
7.
Diamant, S.,
E. Gorin,
and
E. Shafrir.
Enzyme activities related to fatty-acid synthesis in liver and adipose tissue of rats treated with triiodothyronine.
Eur. J. Biochem.
26:
553-559,
1972[Medline].
8.
Exton, J. H.
Molecular mechanisms involved in
1-adrenergic responses.
Mol. Cell. Endocrinol.
23:
233-264,
1981[Medline].
9.
Exton, J. H.
Signaling through phosphatidylcholine breakdown.
J. Biol. Chem.
265:
1-4,
1990[Abstract/Free Full Text].
10.
Fafournoux, P.,
C. Demigné,
and
C. Rémésy.
Carrier-mediated uptake of lactate in rat hepatocytes.
J. Biol. Chem.
260:
292-299,
1985[Abstract/Free Full Text].
11.
Fasolato, C.,
B. Innocenti,
and
T. Pozzan.
Receptor-activated Ca2+ influx: how many mechanisms for how many channels.
Trends Pharmacol. Sci.
15:
77-83,
1994[Medline].
12.
Garcia-Sainz, J. A.,
and
S. M. T. Hernandez-Sotomayor.
Adrenergic regulation of gluconeogenesis: possible involvement of two mechanisms of signal transduction in
1-adrenergic action.
Proc. Natl. Acad. Sci. USA
82:
6727-6730,
1985[Abstract].
13.
Garrison, J. C.,
M. K. Borland,
V. A. Florio,
and
D. A. Twible.
The role of calcium ion as a mediator of the effects of angiotensin II, catecholamines, and vasopressin on the phosphorylation and activity of enzymes in isolated hepatocytes.
J. Biol. Chem.
254:
7147-7156,
1978[Abstract].
14.
Grinstein, S.,
S. Cohen,
J. D. Goetz,
and
A. Rothstein.
Osmotic and phorbol ester-induced activation of Na+/H+ exchange: possible role of protein phosphorylation in lymphocyte volume regulation.
J. Cell Biol.
101:
269-276,
1985[Abstract].
15.
Grynkiewicz, G.,
M. Poenie,
and
R. Y. Tsien.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J. Biol. Chem.
260:
3440-3450,
1985[Abstract].
16.
Irvine, W. J.,
and
A. D. Toft.
The diagnosis and treatment of thyrotoxicosis.
Clin. Endocrinol.
5:
687-707,
1976[Medline].
17.
Kikawa, U.,
Y. Takai,
R. Minakuchi,
S. Inohara,
and
Y. Nishizuka.
Calcium-activated, phospholipid-dependent protein kinase from rat brain. Subcellular distribution, purification, and properties.
J. Biol. Chem.
257:
13341-13348,
1982[Free Full Text].
18.
Lesburg, C.,
S. Li,
E. J. Cragoe, Jr.,
and
R. C. Deth.
Influence of amiloride derivatives on
1-adrenergic receptor-induced contractions of the rabbit aorta.
J. Pharmacol. Exp. Ther.
253:
530-536,
1990[Abstract].
19.
Malbon, C. C.,
and
J. R. Hadcok.
Evidence that glucocorticoid response elements in the 5'-noncoding region of the hamster
2-adrenergic receptor gene are obligate for glucocorticoid regulation of receptor mRNA levels.
Biochem. Biophys. Res. Commun.
154:
676-681,
1988[Medline].
20.
Moolenaar, W. H.,
R. Y. Tsein,
P. T. Van Der Saag,
and
S. W. De Laat.
Na+/H+ exchange and cytoplasmic pH in the action of growth factors in human fibroblasts.
Nature
304:
645-648,
1983[Medline].
21.
Oppenheimer, J. H.,
H. L. Schwartz,
C. N. Maiash,
W. B. Kinlaw,
N. C. W. Wong,
and
H. C. Freake.
Advances in our understanding of thyroid hormone action at the molecular level.
Endocr. Rev.
8:
288-308,
1987[Medline].
22.
Pang, P. K.,
R. Wang,
J. Shan,
E. Karpinski,
and
C. G. Beinshin.
Specific inhibition of long-lasting L-type calcium channels by synthetic PTH.
Proc. Natl. Acad. Sci. USA
87:
623-627,
1990[Abstract].
23.
Preiksaitis, H. G.,
W. H. Kan,
and
G. Kunos.
Decreased
1-adrenoceptor responsiveness and density in liver cells of thyroidectomized rats.
J. Biol. Chem.
257:
4321-4327,
1982[Abstract/Free Full Text].
24.
Rink, T. J.,
R. Y. Tsien,
and
T. Pozzan.
Cytoplasmic pH and free Mg2+ in lymphocytes.
J. Cell Biol.
95:
189-196,
1982[Abstract].
25.
Seitz, H. J.,
M. J. Müller,
and
S. Soboll.
Rapid thyroid-hormone effect on mitochondrial and cytosolic ATP/ADP ratios in the intact liver cells.
Biochem. J.
227:
149-153,
1985[Medline].
26.
Thaastrup, O.,
P. J. Cullen,
B. K. Drobak,
M. R. Hanley,
and
A. P. Dawson.
Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase.
Proc. Natl. Acad. Sci USA
87:
2466-2470,
1990[Abstract].
27.
Thomas, J. A.,
R. N. Buschbaum,
A. Zimniak,
and
E. Racker.
Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ.
Biochemistry
18:
2210-2218,
1979[Medline].
28.
Urcelay, E.,
N. Butta,
G. Ciprés,
A. Martín-Requero,
M. S. Ayuso,
and
R. Parrilla.
Functional coupling of Na+/H+ and Na+/Ca2+ exchangers in the
1-adrenoreceptor-mediated activation of hepatic metabolism.
J. Biol. Chem.
269:
860-867,
1994[Abstract/Free Full Text].
29.
Urcelay, E.,
N. Butta,
C. G. Manchón,
G. Ciprés,
A. Martín-Requero,
M. S. Ayuso,
and
R. Parrilla.
Role of protein kinase C in the
1-adrenoreceptor-mediated responses of perfused rat liver.
Endocrinology
133:
2105-2115,
1993[Abstract].
30.
Williams, R. S.,
and
R. J. Lefkowitz.
Thyroid hormones regulation of
-adrenergic receptors. Studies in rat myocardium.
J. Cardiovasc. Pharmacol.
1:
181-189,
1979[Medline].
AJP Endocrinol Metab 273(6):E1065-E1072
0193-1849/97 $5.00
Copyright © 1997 the American Physiological Society