Molecular and Cellular Endocrinology, 9 (1977) 223-226 @Elsevier/North-Holland Scientific Publishers, Ltd.

LANTHANUM INHIBITION OF CALCITONIN SECRETION AND CALCIUM

UPTAKE IN PORCINE THYROID SLICES

J. Thomas PENT0 Division of Pharnzacodyr~amics arld Toxicology, College of Pharrrzacy. University of Oklahoma, Health Sciences Center. Oklahoma City, Oklahoma 73190, II S.A.

Received 7 July 1977;accepted 23 August 1977

The influence of lanthanum on theophylline-induced calcitonin secretion and 45Ca uptake by slices of porcine thyroid was examined in the present study. Theophylline (4 mM) produced a significant rise in calcitonin release; however, tissue uptake of 45Ca was not affected. In the presence of 10 mM lanthanum 45Ca uptake was reduced in both the control and theophylline- treated tissue and theophylline-stimulated calcitonin secretion was abolished. It is concluded that the calcium ion is necessary for theophylline-mediated calcitonin secretion.

Keywords; calcium-mediated hormone secretion; lanthanum antagonism; porcine thyroid.

The calcium ion is an established calcitonin secretagogue and appears to be the

most potent among the alkaline earth cations in stimulating calcitonin secretion (Pento et al., 1974). Although a number of endocrine systems require the presence of calcium for normal secretion, the calcitonin and parathyroid hormone secretory systems are unique in that circulating levels of ionic calcium appear to be the primary secretory control signal (Rubin, 1970). In addition to calcium, other bio- chemicals such as gastrin and cyclic adenosine 3’,5’-monophosphate (cyclic AMP) have been shown to stimulate calcitonin secretion both in vivo and in vitro (Care et al., 1970; Bell and Queener, 1974: Selawry et al., 1975), and it has been suggest- ed that these agents may function to modify calcium-regulated calcitonin release (Care et al., 1970; Bell and Queener, 1974). However, it is not clear whether these biochemicals stimulate calcitonin secretion directly or if their action is mediated by the calcium ion.

Lanthanum is a trivalent cation which is known to displace calcium in a number of biological systems. Accordingly, this cation has been used as a calcium antago- nist to study the function of calcium in various physiological processes (Weiss, 1974). Therefore, the purpose of the present study was to examine the influence of lantha- num on calcium uptake and theophylline-stimulated calcitonin secretion by slices

of porcine thyroid glands.

223

224 J. T. Pen to

MATERIALS AND METHODS

Tissue incubation Fresh porcine thyroid glands were obtained from a local abattoir and tissue slices

were prepared as previously described (Orme and Pento, 1976). The medium used for tissue incubation contained 146 mM NaCl, 1 mM MgS04, 4.75 mM KCl, 1.25 mM CaClz, 5.6 mM glucose, 0.025 PCi 4s Ca and 20 mM Tris-HCl. Lanthanum (as LaCls) was added at a concentration of 10 mM without precipitation and the pH

was adjusted to 7.4. This concentration of lanthanum has been observed to reduce calcium uptake by other calcium-dependent tissues (Weiss, 1974). 100 f 10 mg of

thyroid slices were weighed into 10 ml Erlenmeyer flasks containing 3 ml of incu- bation media. Incubations were conducted under an atmosphere of 95% 0, and 5% CO* on a gyrorotary incubator (New Brunswick Scientific) at 37°C for 1 h. After incubation the tissue and media were immediately separated.

4sCa determination The tissue was solubilized in 1 ml of a tissue solubilizer (Soluene 100, Packard

Instruments Co.) and the media was stored at -20°C for subsequent assay. Tissue and media 4sCa concentrations were determined by liquid scintillation spectrome- try. Sample quenching was determined by use of the counter external standard and counting times were automatically adjusted to obtain a counting error of less

than 1%.

Calcitonin radioimmunoassay Incubation media calcitonin levels were measured by means of a specific porcine

calcitonin radioimmunoassay which has been described elsewhere (Pento et al., 1973). Pure porcine calcitonin (Lot 4688C-140A, Lederle Laboratories) was used for labeling with 12’1 and as the assay standard. Equilibrium incubations were con- ducted at 4°C for 6 days in a 0.05 M Verona1 buffer containing 0.02 M disodium EDTA and 0.5% bovine albumin. Bound and free labeled fractions were separated by talc adsorption. Each calcitonin determination was performed in duplicate on several dilutions of incubation medium. The assay data were processed by com- puter (IBM Systems/360) using an algorithm which has been described (Pento et al., 1974).

Statistical analysis Student’s t-test for nonpaired data was used to compare individual group means.

Statistical comparisons which resulted in P values of less than 0.05 were considered significantly different.

RESULTS

The results of the present study are presented in fig. 1. Theophylline (4 mM) produced a significant (P < 0.025) rise in calcitonin secretion. The addition of 10

Lanthanum inhibition of calcitorlin secrctiort 225

r 1

I A 15Ca CT 45Ca CT 45Ca

Theophylline Lanthanum THEOPHYLLINE

(4mM) (10mM) (4lyAb

LAWHlH~,UUM

25 1

CT ’ Control

Fig. 1. Influence of theophylline and lanthanum on calcitonin (CT) secretion and 45Ca uptake

in vitro. Each vertical bar represents the mean media calcitonin concentration (open bar) or

total tissue 45Ca (shaded bar) from 5 incubation flasks t SE.

mM lanthanum to the incubation medium did not alter basal calcitonin secretion; however, it completely abolished the theophylline-stimulated calcitonin release (P< 0.01). In this experiment 45Ca uptake by the thyroid slices was not altered by theophylline administration. Lanthanum, on the other hand, significantly reduced 45Ca uptake in both the control and theophylline-treated tissue (P < 0.01, P < 0.005, respectively) and produced a greater reduction in 4sCa uptake in the control group than in the theophylline-treated group (P < 0.05).

DISCUSSION

The results of the present study indicate that lanthanum inhibition of calcium uptake by porcine thyroid slices abolishes theophylline-mediated calcitonin secre- tion. These data are consistent with the results of Selawry et al. (1975) who ob- served a marked reduction in the calcitonin secretory response in human thyroid slices to cyclic AMP, theophylline and pentagastrin in calcium-free media.

The secretagogue action of theophylline on the calcitonin secretory system is believed to be due to an accumulation of intracellular cyclic AMP since theophyl- line is known to produce an inhibition of cytoplasmic cyclic nucleotide phospho- diesterase (Care et al., 1970). Accordingly, both theophylline and dibutyryl cyclic AMP have been observed to enhance calcitonin release (Care et al., 1970; Bell and

226 J.T. Pento

Queener, 1974; Selawry et al., 1975). Since calcium is known to be iirvolved in

cyclic AMP activity within the cell (Rubin, 1974), it is possible that lanthanum abohshed theophylline-induced calcitonin secretion by reducing calcium availabil-

ity within the secretory C-cell. The lack of an observable increase in 45Ca uptake in the theophylline-treated tissue, which has previously been observed for penta- gastrin-stimulated calcitonin secretion in a similar in vitro system (Pento, 19761,

appears to be inconsistent with this concept. However, since the C-cell population represents a small fraction of the total cell mass in the mammalian thyroid, it is

possible that calcium uptake by the C-cells was obscured by calcium movements in the thyroid follicular cells.

An alternative explanation is that, in addition to tissue uptake of extracellular

calcium, ~an~anum may influence the intracellular tran~ocation of a calcium pool

which is necessary for theophy~ine-stimulated secretion. Although it is generally believed that lan~anum does not cross the cell membrane because of its valency,

Hodgson et al. (1972) have reported the binding of lanthanum to intracellular com- ponents in rat myometria1 cells. Further, since theophy~ine-induced calcitonin secretion in human thyroid slices was reported to be reduced, but not abolished, in a calcium-free medium (Selawry et al., 1975), it may be possible that lanthanum

inhibits calcitonin release by a mechanism which is unrelated to the lanthanum-

induced alteration in 45Ca uptake observed in the present study. Whatever the exact

mechanism of lanthanum action at the cellular level, the results of this study clearly

indicate that lanthanum abolished theophylline-stimulated calcitonin release and suggest that the calcium ion is involved in cyciic nucleotide-induced calcitonin

secretion in the porcine thyroid.

ACKNOWLEDGEMENTS

The author gratefully acknowledges the generous supply of porcine thyroids by Mr. T.F. Andreskowski of Wilson and Co. and the technical assistance of Charles

Anderson and Helen Wuertele. This study was supported in part by NSF Grants GB-43214 and PCM 76-03624.

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