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To determine putative effects of various protocols of propylthiouracil (PTU)-induced hypothyroidism on vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) levels in the atria of developing and adult female rats.
Perinatal hypothyroidism was induced by treating pregnant rats with 0.05% PTU in drinking water from late gestation till the age of 60 days (P-PTU). Adult rats were given PTU for 10, 30 or 70 days (PTU-10, PTU-30 and PTU-70, respectively). Corresponding age-matched controls were left intact (P-Cont, Cont-10, Cont-30 and Cont-70, respectively). Resting heart rate, serum total thyroxine concentration, body weight and atrial weight were determined in all animals. VIP-LI levels in tissue extracts were measured by radioimmunoassay.
The values of heart rate, serum total thyroxine, body weight and atrial weight showed that 10-day treatment did not suppress thyroid gland function completely. However, the remaining experimental protocols were sufficient to reach stable hypothyroid conditions. Thyroid hormone deficiency led to a significant increase in VIP-LI levels in both atria of PTU-30 and PTU-70 rats (P<0.01 versus corresponding controls). Interestingly, in P-PTU atria, VIP-LI reached significantly higher values than in rats treated with PTU for the same time during adulthood (PTU-70).
These results provide new evidence that hypothyroidism interferes with VIP-ergic innervation in rat heart atria. The impact of thyroid hormone deficiency on VIP-LI levels differed in P-PTU and PTU-70 rats suggesting that thyroid hormone may play an important part in the development of VIP-ergic innervation in rat heart atria.
The cardiovascular system is considered to be the main target for the action of thyroid hormones. Thyroid hormones influence cardiac function both directly at the cellular level and indirectly through changes in the peripheral circulation and energy metabolism, and by interacting with both divisions of the autonomic nervous system (1,2). Hypothyroidism is associated with decreased heart rate, reduced cardiac contractility, diminished cardiac oxygen consumption and increased peripheral resistance (3). Even in its subclinical form, hypothyroidism negatively affects the diastolic function of the human heart (4). Thyroid hormone deficiency leads also to disturbances in the function of the cardiac sympathetic innervation. In the hearts of hypothyroid rats, noradrenaline concentrations were found to be significantly decreased, which may indicate an increase in sympathetic neuronal activity (5). Increased plasma noradrenaline concentrations (6) and enhanced noradrenaline turnover in the heart (7) have also been found in hypothyroid rats. At the postsynaptic level, fewer myocardial adrenoceptors (both alpha and beta) have been reported repeatedly in thyroid hormone-deficient rats (5–9).
Fewer studies have dealt with the effect of hypothyroidism on cardiac parasympathetic innervation. Hypofunctional abnormalities in the parasympathetic nervous system have been reported in association with a reduction in the concentrations of serum thyroid hormones in humans (2). In addition, thyroid hormone deficiency resulted in increased density of muscarinic receptors in the rat heart (10,11).
Vasoactive intestinal polypeptide (VIP) is a 28-amino acid peptide, which has been identified in intracardiac parasympathetic nerve terminals and intrinsic cardiac neurons (12,13). VIP has been proposed to be involved in the regulation of coronary blood flow, and cardiac chronotopy and inotropy (14–16), ie, parameters that are substantially affected by hypothyroidism.
We have recently shown that VIP-like immunoreactivity (VIP-LI) levels in rat heart atria were substantially affected by hyperthyroidism. In addition, thyroid hormone seemed to interfere with the development of VIP-ergic innervation (17). Therefore, in the present study, we similarly investigated the effect of thyroid hormone deficiency on VIP-LI in the atria of albino rats. Various experimental protocols were used to compare the impact of hypothyroidism induced either prenatally or in adulthood.
Wistar female rats purchased from VELAZ (Czech Republic) and their offspring bred in the authors’ laboratory were used. Pregnant rats were housed individually with free access to food and water. After birth, litters were made up of eight to 10 each. All experiments were conducted in accordance with relevant guidelines of the Czech Ministry of Agriculture for scientific experimentation on animals.
Perinatal hypothyroidism was induced by treating pregnant rats with 0.05% propylthiouracil (PTU) in drinking water starting 15 days after mating and continuing through 60 postnatal days (P-PTU). Corresponding age-matched controls were left intact (P-Cont). Another group of animals was given PTU in drinking water from postnatal day 60 for 10, 30 or 70 days (PTU-10, PTU-30 and PTU-70, respectively). Control groups of rats were left intact and designated Cont-10, Cont-30 and Cont-70, respectively.
One day before dissection, rats were placed in a small chamber with electrodes located in its floor, and their heart rates were recorded electrocardiographically. Heart rate was measured repeatedly until stable values were reached. The average of these measurements is given.
Rats were killed by decapitation and their hearts were rapidly excised. Tissues were rinsed with ice-cold 155 mmol/L NaCl and placed on ice-cold buffer containing 0.1 mmol/L EDTA, 5.0 mmol/L Tris-HCl, pH 7.4, and 0.25 mol/L sucrose. Hearts were cleaned of connective tissue and fat, and left atria with interatrial septum and right atria were dissected. Immediately after dissection, tissues were frozen on dry ice and weighed. The samples were then placed in 0.1 mol/L HCl containing 100 μmol/L EDTA and 0.01% Na2S2O5 1:10 (weight:volume) and briefly pulverized. Test tubes with tissues were heated in a water bath at 95°C for 15 min and then cooled on ice. The content of the tubes was homogenized for 30 s in an Ultra-Turrax homogenizer (IKA Labortechnik, Germany). The homogenate was centrifuged at 10,000 g at 4°C for 20 min. The supernatant was neutralized with 1 mol/L Tris-base and centrifuged again at 5000 g at 4°C for 15 min. The clear supernatant was aspirated, lyophilized and stored at −70°C until it was radioimmunoassayed.
Total thyroxine concentrations in the plasma were assayed by commercial diagnostic kits (Total Thyroxine RIA, Immunotech, Czech Republic) with a lower detection limit of 25 nmol/L. Commercial kits (Phoenix Pharmaceuticals, USA) were also used for assessing VIP-LI levels in tissue extracts. Lyophilized extracts were dissolved in 250 μL of assay buffer. Assay tubes were set up in duplicate, each containing 100 μL of an unknown sample or the standard plus 100 μL of rabbit antipeptide serum. After incubation (20 h) at 4°C, 100 μL of the tracer solution was added to each tube and the tubes were incubated for a further 20 h. Bound radioactivity was separated by adding goat antirabbit immunoglobulin G serum and centrifugation.
Recovery was assessed in another set of measurements (n=7) by addition of exogenous VIP at the time of heating in HCl. About 65% of the added exogenous VIP was detected in the final extract. The results were not corrected for recovery. Intra-assay variation did not exceed 10%.
Tissue content of VIP is expressed in ng/g tissue wet weight and total serum thyroxine level in nmol/L. Results are reported as mean ± SEM with levels of significance calculated by analysis of variance with post hoc pairwise comparisons determined by BMDP statistical software (BMDP Statistical Software, Inc, USA) or Student’s t test where appropriate. P≤ 0.05 was considered to be significant.
Body weight, weight of both atria, serum total thyroxine concentration and resting heart rate were determined in all PTU-treated rats and their respective controls to evaluate thyroid status. Values obtained are shown in Table 1. Body weight and atrial weight of PTU-treated rats were significantly reduced (P<0.01) 10 days after the onset of treatment, and the differences between PTU-treated rats and controls progressed till the end of the investigated period. In the group of P-PTU rats, body weight did not exceed 45 g at the age of 60 days. Serum total thyroxine concentrations were significantly lower (P<0.01) 10 days after the onset of treatment (PTU-10), and they fell below the detection limit of the method in all remaining PTU-treated rats. Heart rate was significantly (P<0.01) decreased in all PTU-treated animals, and this change progressed with respect to the duration of PTU treatment. The lowest heart rates were reached in PTU-70 rats (295±7 beats/min) and P-PTU rats (295±6 beats/min).
VIP-LI levels in the right atria did not differ among all groups of the control rats and ranged from 1.38±0.08 ng/g in Cont-10 to 1.42±0.08 ng/g in Cont-30 (Figure 1). VIP-LI levels were significantly higher in the control left atria than in the right atria (P<0.05), ranging from 1.96±0.13 ng/g in Cont-70 to 2.02±0.12 ng/g in Cont-30. No significant differences were found among VIP-LI levels in the control left atria (Figure 2).
In PTU-treated rats, VIP-LI levels in the right atria increased after 30 and 70 days of treatment, being significantly different from their respective controls (P<0.01). However, PTU-70 VIP-LI levels (2.38±0.23 ng/g) did not differ significantly from the value obtained from the right atria of PTU-30 rats (2.25±0.17 ng/g) (Figure 1).
In the left atria, PTU treatment had the same effect as in the right atria, ie, VIP-LI levels were significantly higher than in their corresponding controls, being 2.87±0.14 ng/g in PTU-30 left atria and 2.94±0.17 ng/g in PTU-70 left atria (Figure 2).
Interestingly, in P-PTU right atria, VIP-LI levels reached 3.31±0.19 ng/g, which was significantly higher than in the group of rats treated with PTU for the same time during adulthood (PTU-70) (Figure 3). In the left atria of P-PTU rats, VIP-LI levels were also significantly higher (4.45±0.25 ng/g) than in those of PTU-70 animals (Figure 3).
In the present study PTU administration to young adult rats led to significant changes in the measured basal parameters. It is, however, evident that treatment lasting 10 days was not sufficient to suppress thyroid function completely because all of the changes with the exception of heart rate progressed till day 30 of the treatment. Serum total thyroxine concentrations, attenuation in the growth rate and atrial weight gain were comparable in rats treated for 30 and 70 days (PTU-30 and PTU-70, respectively), whereas heart rate reached its minimum in PTU-70 rats. In perinatal hypothyroidism extended to adulthood (P-PTU rats), thyroid hormone deficiency produced large deficits in body weights and weights of both atria. These findings are in accordance with previous reports (18,19). Thus, we can conclude that the PTU treatment used in the present study was appropriate to induce severe hypothyroidism with its typical symptoms.
Only a few studies concerning the effects of dysthyroid status on tissue VIP-LI levels in adult animals have been reported. The lack of thyroid hormones was shown to cause an increase in VIP-LI levels in the rat adrenal gland (20), as well as in VIP mRNA levels in the hypothalamus and in the anterior pituitary of the same species (21,22). In contrast, no changes in VIP mRNA concentrations were observed in the cerebral cortex of the hypothyroid animals (23).
The present study shows that VIP-LI levels in the atria increased similarly in adult rats treated with PTU for either 30 or 70 days. Interestingly, in rats rendered hypothyroid from prenatal life till adulthood (P-PTU), the impact of thyroid hormone deficiency on VIP-LI levels in the atria was expressed even more than in PTU-70 animals despite the same duration of treatment. It is well known that thyroid hormones modulate neuronal functions in both adult and developing animals. However, reports of the effect of hypothyroidism on developing cardiac innervation focused exclusively on its sympathetic division. Transient perinatal hypothyroidism resulted in lasting deficits in noradrenaline turnover rate associated with decreased noradrenaline concentration in the heart (24). In contrast, reduced cardiac noradrenaline concentration was linked rather to the increased activity of adrenergic innervation in adult hypothyroid rats (7). Thus, thyroid hormones have been considered to play a crucial part in establishing the time course of development of sympathetic nerve pathways to the heart and adjusting the final level of their activity in mature animals (24).
The results of our recent studies indicate that the impacts of transient and sustained neonatal hyperthyroidism on VIP-LI levels in the heart atria do not differ substantially (17). Hyperthyroidism induced for nine postnatal days led to a significant decrease in VIP-LI levels in both atria of adult rats. Only slightly lower values of VIP-LI were found in rats treated with thyroxine for 60 postnatal days. With respect to these observations we assume that the early neonatal period may be critically important in the development of VIP-ergic innervation.
The present study also shows that hypothyroidism induced in various periods of life results in significant changes in VIP-LI levels in both atria. The detection of the maximum effect in animals treated with PTU perinatally is in accordance with our previous hypothesis. We can conclude that the ontogeny of VIP-ergic innervation, in addition to sympathetic innervation, most probably requires a sufficient concentration of thyroid hormones during the critical developmental period.
Not only is VIP a neurotransmitter of parasympathetic and nonadrenergic noncholinergic intrinsic cardiac neurons but also it may serve important trophic functions. The peptide has been reported to cause trans-synaptic activation of tyrosine hydroxylase within sympathetic ganglia (25), and to promote survival of rat sympathetic neurons and neurite outgrowth of PC12 cells derived from a rat pheochromocytoma (26).
It therefore may be questioned whether the increase in VIP-LI levels in the atria of hypothyroid rats is linked to the increase in the sympathetic nervous system activity reported in adult thyroid hormone-deficient animals (7).
This is the first report of the effects of thyroid hormone deficiency on VIP-LI levels in rat heart atria. The mechanisms underlying these effects (altered gene expression, release and degradation of the peptide) remain to be elucidated.
Supported by the Grant Agency of the Czech republic (grant No 305/97/0046) and by the Czech Ministry of Education (Research Project No MSM 111400001). A preliminary communication (27) was presented at the 2nd FEPS Congress, Prague, June 30 to July 4, 1999.