Neuropeptides (1989) 13,59-64 0 Longman Group UK Ltd 1989

Effects of VIP and Helodermin on Thyroid Hormone Secretion in the Mouse

B. AHRBN*t and P. HEDNERS

Departments of *Surgery, tPharmacology, and #Medicine, Lund University, S-221 85, Lund, Sweden.

Abstract-The two peptides VIP (vasoactive intestinal peptide) and helodermin have both been shown to occur within the thyroid gland: VIP in intrathyroidal nerves and helodermin in the C-cells. Both peptides have previously been demonstrated to enhance the release of radioiodine from the prelabelled thyroid in viva. Since a considerable amount of radioioidine released from the thyroid under these conditions may be non-hormonal, we reexamined the effects of VIP and helodermin on thyroid hormone secretion by the use of the specific technique of studying the release of radioiodine bound to specific T4 antiserum in mice. We thereby found that anti-T4-bound radioiodine in T3-pretreated animals increased after intravenous injection of VIP (1.5 nmollanimal) to 280 -+ 24% (P < O.OOl), and after intravenous injection of helodermin (1.5 nmol/-animal) to 186 + 26% (P < 0.001) compared to 78 rt 5% in controls, As a comparison, the corresponding figure after injection of TSH (70 @/animal) was approximately 350% (P < 0.001). In contrast, in animals not pretreated with TJ, neither TSH, nor VIP helodermin significantly altered the plasma level of anti-T4-bound radioiodine. Also, VIP and helodermin did not change the plasma levels of free T4 in non-pretreated animals. In summary, the sensitive and specific technique of measuring the release of anti-T4-bound radioiodine in vivo after pretreatment with NA’251 and T3 detected a stimulation of the thyroid hormone secretion by VIP and helodermin. In contrast, less sensitive techniques, such as measuring the release of radioiodine in animals not pretreated with TB, or the determination of plasma levels of T4, could not detect this effect. We conclude that VIP and helodermin stimulate the secretion of thyroid hormones under in vivo conditions.

Introduction peptide)-immunoreactive nerves occurring both

The thyroid gland has been demonstrated to be around. blood vessels and between and- along

richly innervated by VIP (vasoactive intestinal thyroid follicles and originating from nerve cell bodies in juxtathyroid laryngeal ganglia (l-3).

VIP has been shown to enhance the radioiodine

Date received 7 July 1988 Date accepted 22 August 1988

release from labelled thyroid glands in viva in the mouse (1, 3-6), which could indicate that the

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intrathyroidal neuropeptide VIP can activate thyroid hormone secretion. This conclusion is, however, valid only if determinations of radio- iodine truely reflect those of thyroid hormones, which, however, is not always the case since a fraction of the plasma radioiodine is non- hormonal (7,8). It is in this context of significance, therefore, that, in rats, VIP changed the local thyroid blood flow without any concomitant changes in the plasma levels of T4 or T3 (9). This finding suggested that the main function of VIP within the thyroid is not to stimulate the thyroid hormone secretion but rather to regulate the local thyroid blood flow. However, this conclusion is in turn valid only if the determination of plasma levels of T4 and Ts is sensitive enough to observe alterations induced by the peptide. To solve the question whether VIP and other neuropeptides activate thyroid hormone secretion in viva, we recently developed a technique in mice for measuring the thyroid hormone secretion in vivo by the use of specific anti-T4 antiserum to bind the hormone-bound radioiodine released from labelled thyroids (9). This technique was found to be more sensitive than determination of plasma levels of T4, and more specific than the earlier used technique of measuring blood radioiodine accord- ing to McKenzie (10).

Therefore, we investigated in the present study the effects of VIP on both plasma T4 levels in non-pretreated mice and on the levels of radio- iodine bound to anti-T4 antiserum after labelling of the thyroid with 125I to explore whether VIP indeed stimulate thyroid hormone secretion in vivo or not. We also investigated the effects of helodermin with these two techniques. Heloder- min, which is a VIP-like peptide (11)) was recently demonstrated to occur within the thyroidal C-cells and, like VIP, to stimulate the release of radio- iodine from labelled thyroids according to the technique of McKenzie (12-13).

Materials and Methods

Animals. Female mice of the NMRI strain (Anticimex, Stockholm, Sweden), weighing 20-25 g, were used.

Experiments. In the first set of experiments (Fig.

l), normal mice were injected iv in a tail vein with either TSH (natural ovine TSH; Ferring AB, Malmo, Sweden, 70@J/animal), synthetic porcine VIP (vasoactive intestinal peptide; Bac- them Inc., Torrance, Ca, U.S.A.; 1.5 nmol/ animal), or synthetic helodermin (Peninsula Ltd., Belmont, Ca, U.S.A.; 1.5 nmoYanima1). Controls were given saline. The injection volume was 0.2ml. The peptides were dissolved in saline with the addition of 1 g/l gelatine to avoid adsorption to tubes. Blood (200~1) was taken immediately before the injection and 2h after from the orbital plexus. Plasma was separated and stored at -20” C for later analysis of the free T4 concentration by radioimmunoassay method (Amerlex T4), earlier shown to have slightly better performance than radioimmunoassay of total T4 concentrations (8).

Control TSH VIP HD

Fig 1 Changes in plasma levels of T4 in mice after the iv injection of TSH (70 pkJ/animal), VIP (1.5 nmol/animal), helodermin (HD; 1.5 nmollanimal), or saline. Each group consisted of 20 animals. Values are the mean rt SEM of the % ratio of plasma concentrations at 2 h after injection and before.

In the second and third experimental series (Figs 2-3) the mice had been given an iodine-poor diet (Astra-Ewos, Sodertllje, Sweden) for 10 days. Then each animal was injected with 16 &?i NA’251 (Radiochemical Center, Amersham, England) ip 48 h before the experiments, and with L-T3 (British Drug Houses Ltd., Poole, England), 1 &animal, SC 48. 24, and 3h before the experiments. At the time of experiment, the mice were injected iv with TSH and/or VIP or helodermin at the dose levels given above. The volume loads were 0.2ml (SC and iv) and 0.1 ml (ip), respectively. In the fourth experimental series (Fig 4), lastly, the mice were

EFFECI-S OF VII’ AND HELODERMIN ON THYROID HORMONE SECRETION IN THE MOUSE 61

600

ln

2 Control TSH VIP TSH+VIP

Fig 2 Changes in blood levels of ‘**iodine bound to anti-T4 in mice pretreated with Ts after the iv injection of TSH (70 @J/animal) or VIP (1.5 nmolIanima1) alone or together, or saline. Each group consisted of 20 animals. Values are the mean + SEM of the % cpm ratio of radioactivity at 2h after injection and before

x 500

!i!

E 400 f?

T 5 300

.’

5 200 ,.

q

0

,. z 100 ,.

a n

ui N Control TSH HD TSH+HD

Fig 3 Changes in blood levels of ‘*‘iodine bound to anti-T4 in mice pretreated with Ts after the iv injection of TSH (70 t.LJ/animal) or helodermin (HD; 1.5 nmol/animal) alone or together, or saline. Each group consisted of 20 animals. Values are the mean + SEM of the % cpm ratio of radioactivity at 2h after injection and before.

pretreated as in the second and third series, except that no T3 pretreatment was given. The mice were then injected iv with TSH, VIP, or helodermin. In all the three last experimental series, blood (100 ~1) was sampled immediately before and at 2 h after the iv injections. The blood radioactivity bound to specific anti-T4 antiserum was then determined (8). Plasma was separated and 50~1

EJ Control TSH VIP HD

Fig 4 Changes in blood levels of 125iodine bound to anti-T4 in mice not pretreated with Ts after the iv injection of TSH (70 lN/animal), VIP (1.5 nmol/animal), helodermin (HD; 1.5 nmopmin), or saline. Each group consisted of 20 animals. Values are the mean + SEM of the % cpm ratio of radioactivity at 2h after injection and before.

was mixed with 200~1 anti-T4 antiserum (Milab, Malmo, Sweden) diluted to 1:50 in barbiturate buffer supplemented with bovine serum albumin. Then 200 l.i.1 &anilinoaphtalene-1-sulfonic acid (ANS; 1 mg/ml) were added, and the mixture was incubated at 4” C overnight. Goat antirabbit immunoglobin antiserum (Milab; 500 Al), diluted 1:50 in barbiturate buffer with 5% polyethylene glycol, was added to each tube. After 30 min, the tubes were centrifuged at 2000 x g at 4°C for 15 min, the supernatant was discarded and the pre- cipitate was counted in an Autogamma spec- trometer.

statistics

The results are expressed as the quotient between values in samples taken at 2 hours after injection and before times 100 = % ratio. Means + SEM are given. Student’s t-test was used for statistical analysis. Thereby, the results in an experimental group was compared to its control group.

Results

Effects of TSH, VIP, and helodermin on plasma levels of free T4 The ratio between plasma free T4 levels in samples taken at 2h after an iv injection of TSH (70 lJLJ/animal) and before was 99 + 4% compared to

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78 f 5% in controls (P < 0.01). In contrast, the corresponding ratio after injection of VIP (1.5 nmol/animal) or helodermin (1.5 nmoYanima1) was not different from that in controls (Fig 1). Thus, neither VIP nor helodermin increased plasma T4 levels.

Effects of TSH and VIP on anti-T4 bound radio- activity

The blood radioiodine bound to anti-T4 increased to 375 f. 37% of the preinjection level after injection of TSH (P < O.OOl), and to 280 f 25% after injection of VIP (P < O.OOl), compared to 80 Z!X 5% in controls. After the combined injection of TSH and VIP, radioiodine levels bound to anti-T4 had increased to 570 + 41% (P < 0.001) (Fig 2).

Thus, VIP enhanced the release of radioiodine bound to anti-Td.

Effects of TSH and helodermin on anti-T4 bound radioactivity

After injection of TSH, the blood radioiodine bound to anti-T, had increased to 305 +- 40% (P < 0.001) and after injection of helodermin it had increased to 186 + 26% (P < 0.001) compared to 79 + 4% in controls. The combined injection of TSH and helodermin increased the radioiodine levels bound to anti-T4 to 402 + 81% of the preinjection level (P < 0.001) (Fig 3). Thus, also helodermin enhanced the release of radioiodine bound to anti-T+

Effects of TSH, VIP, and helodermin on anti T4 bound radioactivity in animals not pretreated with

T3

In mice pretreated with I*?, but not with Ts, the blood radioactivity bound to anti-T4 was not significantly affected by TSH, VIP, or helodermin (Fig 4).

Discussion

We have earlier shown that VIP enhances the release of radioiodine from the prelabelled thyroid gland in vivo in mice (1,4-6). This suggests that the intrathyroidal neuropeptide VIP can stimulate thyroid hormone secretion (5, 14). However, the technique of measuring radioiodine release from

labelled thyroids has been criticized since a con- siderable fraction of the measured radioiodine is not hormone-bound (15). Therefore, other tech- niques must also be used before establishing whether VIP indeed stimulates thyroid hormone secretion. Thereby, however, Huffman and Hedge (9) recently found that VIP in rats did not increase the plasma levels of Td, though it induced thyroid vasodilatation. This casted doubts whether VIP indeed stimulates thyroid hormone secretion under physiological conditions. Therefore, we have in the present study reexamined the effects of VIP on thyroid hormone secretion by the use of our newly developed technique of measuring the fraction of radioiodine released from prelabelled thyroid glands that is bound to specific anti-T4 antiserum. This technique has been shown to be more specific than the conventional method to determine the total radioiodine according to McKenzie (lo), since it is only the hormone-bound fraction of radioiodine that is measured (8). Furthermore, the anti-Td-technique detects a thyroid hormone release with better sensitivity and precision than measuring total or free Tq. Probably this is due to the fact that a release of unlabelled thyroid hormones comes on top of quite a large preexisting pool of circulating Ta while a release of radiolabelled hormones in animals prelabelled with 1251 and T3 to suppress endogenous TSH adds to a very low level of 1251-Th in the circulation and will be easier to detect (8). Consequently, we found in the present study that VIP, at the dose level used, could not increase the plasma levels of Tq. In contrast, the peptide could markedly enhance the levels of radioiodine bound to anti-T4. We conclude therefore that VIP indeed stimulates the secretion of thyroid hormones but that the method of measuring plasma levels of Td has a too low sensitivity to detect this effect under the conditions of the experiments. In analogy, VIP, like TSH at a low dose level, could not induce any significant increase in radioiodine levels in animals not pretreated with T3, since their basal levels of radioiodine were high.

VIP has earlier been demonstrated to exert the following actions as signs of a stimulation of the thyroid follicular cells: 1) stimulation of radio- iodine release from labelled thyroids in vivo in the mouse (1,4-6, 14)) 2) colloid droplet formation in

EFFECTS OF VIP AND HELODERMIN ON THYROID HORMONE SECRETION IN THE MOUSE 63

mouse thyroids (1)) 3) formation of cyclic AMP in References both mouse thyroids (l), rat thyroids (16, 17) and in human thyroid cells (18-20), 4) release of T4 from both human thyroid cells (18, 20) and per- fused canine thyroid (21), 5) stimulation of P-naphthylamidase activity in guinea pig thyroids (22), and 6) increase in plasma levels of T4 after intravenous injection in rats (23). Hence, together with our present findings a vast amound of data suggests that VIP indeed can stimulate the folli- cular cells to induce secretion of T4. Such a conclusion is also strongly corrobated by the demonstration of VIP receptors on the follicular cell membrane (24).

In the present study we also demonstrate that the peptide helodermin stimulated thyroid hormone secretion as measured by the release of radioiodine bound to anti-T4. Helodermin was originally demonstrated in the venom of Gila monster (25), and found to consist of a 35 amino acid sequence with structural similarities to VIP (11). Helodermin-like immunoreactivity has sub- sequently been demonstrated in various mam- malian organs, for example in the brain, gut, and salivary glands (26,27), and helodermin has been shown to exert VIP-like effects and to bind VIP- receptors in the exocrine pancreas (28, 29). Recently, the peptide was demonstrated in the thyroid C-cells (12)) and found to induce release of radioiodine from prelabelled thyroids in vivo in mice according to the technique of McKenzie (13). We confirm in the present study the original findings by Grunditz et al. (12) that helodermin stimulates thyroid hormone secretion. Since helo- dermin is a VIP-like peptide (11)) it is not unlikely that the same follicullar cell receptors are activated when these two peptides are administered. How- ever, considering their different localization, VIP to nerves (1) and helodermin to C-cells (12), it is likely that the stimulatory action of these two peptides represent different regulatory functions.

Acknowledgements

The authors are grateful to Lena Kvist and Leonor Cantera for technical assistance. This study was supported by the Swedish Medical Research Council (Grant No 14X-6834), by Crafoor- dska Stiftelsen, Lund, and by grants from the Medical Faculty, Lund University, Lund.

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