Influence of thyroid status on hepatic alpha 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present work aimed to elucidate the influence of thyroid functional status on the alpha 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 alpha 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 alpha 1-adrenoreceptor signaling pathway. Moreover, in hyperthyroid rats the alpha 1-adrenergic-induced increase in cytosolic Ca2+ was enhanced, and glucose synthesis or mobilization was not altered. The thyroid status influenced neither the alpha 1-adrenergic stimulation of vascular smooth muscle contraction nor the alpha 1-agonist-induced intracellular alkalinization and protein kinase C (PKC) activation. Thus the distinct impairment of the Ca2+-dependent branch of the alpha 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; alpha 1-adrenergic agonists; protein kinase C; cytosolic Ca2+; intracellular pH

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha 1-adrenergic regulation of hepatic metabolism are controversial. Hypothyroidism has been reported to decrease the alpha 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 alpha 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 alpha 1-adrenergic responsiveness.

Hepatic alpha 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 alpha 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 alpha 1-adrenoreceptor-mediated responses. Our results indicate that the thyroid hormones control a postreceptor step(s) in the hepatic alpha 1-adrenergic signaling pathways. Hypothyroidism attenuated some of the hepatic alpha 1-adrenergic responses, and this effect was accompanied by a decreased alpha 1-adrenergic-induced intracellular Ca2+ mobilization. Hyperthyroidism did not change the alpha 1-agonist-mediated mobilization of intracellular Ca2+, and carbohydrate synthesis and mobilization were not impaired. Neither hypothyroidism nor hyperthyroidism altered the alpha 1-agonist-induced stimulation of PKC activity. It is concluded that thyroid hormones modulate the Ca2+-sensitive PKC-independent branch of the alpha 1-adrenergic signaling cascade in rat liver.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 (approx 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(beta -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 [gamma -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 [gamma -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. [gamma -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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Influence of thyroid status on the alpha 1-adrenergic stimulation of hepatic functions. Figure 1 shows the effects of the alpha 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 alpha 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 alpha 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 alpha 1-agonist-induced Ca2+ release. Hypothyroidism decreased the alpha 1-adrenergic stimulation of respiration, Ca2+ release, and glucose output by 43, 50, and 35%, respectively, compared with the same alpha 1-adrenergic responses in livers from euthyroid rats (Fig. 1). The alpha 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).


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Fig. 1.   Influence of thyroid status on hepatic alpha 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 alpha 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 alpha 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 alpha 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, alpha 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 alpha 1-agonist-induced changes in pH (Fig. 2), indicative of a normal activation of the plasma membrane Na+/H+ exchanger (5, 28). The alpha 1-agonist-induced Ca2+ release was similarly affected in livers from hypo- or hyperthyroid rats. Figure 3 depicts the alpha 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 alpha 1-agonist-induced increase in gluconeogenesis was altered (Fig. 3). In contrast, the alpha 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).


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Fig. 2.   Influence of thyroid status and starvation on hepatic alpha 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.


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Fig. 3.   Influence of thyroid status on basal and alpha 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 alpha 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 alpha 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 alpha 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).


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Fig. 4.   Influence of thyroid status on alpha 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).


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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 alpha 1-agonist or vasoactive peptide-induced changes in intracellular pH. In agreement with previous reports (4, 5, 28), alpha 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 alpha 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.


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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.

                              
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Table 1.   Effect of thyroid status on Ca2+-mobilizing agent-mediated stimulation of PKC activity

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta -adrenoreceptors, and the hepatic studies are scarce.

The results of this study demonstrate that thyroid status modulates hepatic alpha 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 alpha 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 alpha 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 alpha 1-adrenergic stimulation of gluconeogenesis by thyroid hormones.

Recent work has demonstrated that signals arising from the hepatic alpha 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 alpha 1-adrenoreceptors by T3.

Both hyperthyroidism and hypothyroidism decreased the alpha 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 alpha 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 alpha 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 alpha 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 alpha 1-adrenergic-induced intracellular Ca2+ mobilization was impaired gives further support to the proposed relationship between alpha 1-adrenoreceptor-induced intracellular Ca2+ mobilization and the stimulation of gluconeogenesis (4, 5, 8, 13).

Neither hyper- nor hypothyroidism impaired the alpha 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 alpha 1-adrenergic-stimulated functions, ranging from totally dependent, such as vascular resistance, to almost independent, like gluconeogenesis. Thus the finding that neither the alpha 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 alpha 1-adrenoreceptor responsiveness by interacting with the Ca2+-dependent PKC-insensitive branch of its signaling pathway. T3 could act in controlling hepatic alpha 1-adrenoreceptor function by changing the alpha 1-adrenoreceptor density (23). However, because hepatic responses to Ca2+-mobilizing agents other than alpha 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 alpha 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 alpha 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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Endocrinol Metab 273(6):E1065-E1072
0193-1849/97 $5.00 Copyright © 1997 the American Physiological Society




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