J. Endocrinol. Invest. 8: 103, 1985

Effect of 1 ,25-dihydroxyvitamin D3 and nifedipine on prolactin release in normal man

D. Verbeelen*., L. Vanhaelst*, M. Fuss**, and A.C. Van Steirteghem* *Department oJ Medicine and Radioimmunoassay Laboratory, Academisch Ziekenhuis (VUB) 1090 Brussels, and **Department of Medicine, H6pital Brugmann (ULB), 1020 Brussels, Belgium

ABSTRACT. In normal man 1,25 (OHh-vitamin Os [1,25 (OH)2D] increases both basal and TRH­stimulated prolactinemia; this effect is completely reversible by the calcium antagonist nifedipine. Similarly the 1,25 (OH)2D-induced hypercalcemia is totally inhibited by nifedipine. Thesefindings suggest that both biological effects of 1,25 (OH)2D are mediated by calcium-dependent mecha­nisms.

INTRODUCTION

Receptors for 1,25 (OH)2-vitamin 0 3 [1,25 (OHhO] have been identified in the rat adenohypophysis and in PRL-secreting cell lines (1-3). In vitro studies have revealed that 1,25 (OHhO influ­ences PRL secretion (4, 5) and we have previously shown that, when administered to patients with chronic renal failure, 1,25 (OHh 0 causes a decrease of plasma PRL (6) .. Other studies have shown that PRL release can be influenced by the calciotropic hormones, para­thyroid hormone (7-9) and calcitonin (10) and even by the serum calcium concentration itself (11-17). The present study was undertaken to examine the effect of 1 ,25 (OHhO on plasma PRL concentrations in healthy male volunteers.

MATERIALS AND METHODS

Subjects Five normal male volunteers aged 23-36 yr participat­ed in the study. Throughout the investigation period they ate their usual diets, had unrestricted activity and did not take any medication besides those administered for the study.

Drugs 1,25 (OH)20 (Rocaltrol®, Roche), 0.5 /Jg, was taken at 08:00, 16:00 and 23:00 h. Nifedipine (Adalat®, Bayer), two capsules of 10 mg, was taken at 08:00, 13:00, 18:00 and 23:00 h.

TRH test Each subject received 200 /Jg TRH as an iv bolus injection at 09:00 h. Serial blood samples were ob-

Key·words: 1.25 (OH)2 vit 03. prolactin. nifedipine. calcium antagonists.

Correspondence: Dr. O. Verbeelen, Dept of Medicifle, Academisch Ziekenhuis VUB, Laarbeeklaan 101, B- t 090 Brussels, Belgium.

Received April 4, 1984; accep1ed October 31, 1984.

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tained for PRL determination 15 min before, at the start and every 15 min after TRH injection during 1 h.

Study protocol

The study protocol was designed subchronically in order to observe an effect of 1 ,25 (OHhO on calcemia besides the possible influence on PRL release.

Experiment 1: Blood was taken before and after 7 days treatment with 1,25 (OH)20 (1.5 /JgI day) for the deter­mination of calcium, total protein, PRL, phosphate and 1,25 (OH)20 concentrations. A TRH test was performed at the onset and at the end of the treatment period.

Experiment 2: Blood was taken before and after two days of nifedipine (80 mg/day) administration and after 7 days of combined treatment with nifedipine (80 mg/day) and 1,25 (OHhO (1.5/Jg/day). The same blood parameters were measured as in experiment 1. TRH tests were performed together with each blood sampling. A time lag of at least 6 weeks separated both experi­ments.

Laboratory methods Calcium was measured with atomic absorption spec­trometry and total protein with the biuret method. Calci­um concentration was corrected for total protein. Phosphate was determined with an automated colori­metric method (molybdenum blue with ferrous sulfate as reducing agent). Reference range in our laboratory: calcium 8,80-10.00 mg/dl; phosphate 3.0-4.5 mg/dl. 1,25 (OHhO was determined by RIA after previous extraction and purification of the steroid (18). Sensitivi­ty of the assay is 3 ng/l and the reference range is 26-50 ng/l. PRL was determined using reagents from IRE (Fleurus, Belgium) (6). Standard PRL used in this assay was equilibrated against MRC preparation 75/504. The detection limit is 23 mU II. Intra- and interassay variation coefficients measured were less than 1 0%. Normal range of PRL in males is 59~235 mU II. The increment of PRL afterTRH

D. Verbeelen, L. Vanhaelst, M. Fuss, et a/.

Table 1 - Effect of/he administration of 1,25 (OHh-vitamin 0 3

during one week on serum calcium, phosphate, 1,25 (OHh-vi­tamin 0 3 and PRL in 5 normal males!.

Pretreatment

Calcium 9.7± 0.1 (mg/dl)

Phosphate 3.5± 0.5 (mg/dl)

1,25 (OH)2 viI. 0 3 44 ±11 (ng/I)

PRL 138 ±48 (mU/I)

1Results are expressed as mean ± SO. 2p < 0.05 using Student's t test.

1000

800

...J

~ 600 E

...J a:: Cl.

400

200

o

-~5 ----- control

TRH ,

b

---125 (OH),D

3'0

1,25 (OH)2-vitamin 03

10.2 ± 0.52

3.8± 0.6

106 ± g2

301 ± 542

do min

Fig. 1 - Effect of 1,25 (OHh-vitamin 0 administration on basal and TRH-stimulated prolactinemia.

administration, was calculated from the area under the curve, using the PRL concentration at time a as base­line value. Results were expressed as mean ± SD. Statistical significance was calculated using Student's t test for paired values.

RESULTS Table 1 shows that after 1,25 (OH)2D administration serum calcium, 1,25 (OH)2D and basal PRL concentra­tions rise significantly. The PRL response to TRH is significantly increased at all times (Fig. 1). The area under the curve of the TRH-induced plasma PRL re­sponse is 15,773± 2,675 mU min 1-1 pretreatment, com-

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Table 2 - Effect of the administration of nifedipine (80 mg / day) for two days and nifedipine (80 mg/day) plus 1,25 (OHh-vi­tamin 0 3 (1.5 pg/day) for 7 days on serum calcium, phos­phate, 1,25 (OHh-vitamin 0 3 and PRL in 5 normal males!.

Pretreatment Nifedipine

Calcium 9.8± 0.3 9.8± 0.3 (mg/dl)

Phosphate 3.4± 0.7 3.5± 0.4 (mg/dl)

1,25 (OH)2 47 ± 5 50 ± 9 viI. 0 3 (ng/I)

PRL 140 ±30 131 ±17 (mU/I)

1 Results are expressed as mean ± SO. 2p < 0.05 using Student's t test.

1000

800

...J

~ 600 E

...J a:: Cl.

400

200

o

TRH ,

I 15

.---.. control min

().----{) nifedipine

I!r-----6 nifedipine + 1,25 (OH)20

I 30

Nifedipine + 1,25 (OH)2-vitamin 0 3

9.8± 0.4

3.4± 0.4

94 ± 72

129 ± 16

Fig. 2 - Effect of 1,25 (OH)2-vitamin 0 administration on basal and TRH-stimulated prolactinemia on nifedipine + 1,25(OH)2-vitamin 0 3 administration.

pared to 24,916 ± 2,941 mU min r1 under 1,25 (OH)2D. Those areas are significantly different (p < 0.05). Table 2 shows that nifedipine does riot influence serum calcium, 1,25 (OH)2D and PRL. Under simultaneous administration of 1,25 (OH)2D and nifedipine, only se­rum 1 ,25 (OH)2D concentrations increase significantly, while serum calcium and PRL remain unchanged. . The PRL responses to TRH under nifedipine alone, or under simultaneous nifedipine and 1 ,25 (OH)2D admin­istration are not different from pretreatment levels (Fig. 2).

DISCUSSION The data presented in this study suggest a stimulatory effect of 1,25 (OHbD on PRL release in normal sub­jects, completely suppressible by simultaneous nifedi­pine intake. To explain the rise of plasma PRL during 1,25 (OH)2D intake, two possible mechanisms must be examined, namely a direct action of 1,25 (OH)2D on the pituitary and an indirect one via 1,25 (OHhD induced increase of serum calcium. It has been shown that hypercalce­mia results in a decrease of PRL release (12-17), and therefore an indirect effect of 1,25 (OH)2 on PRL is unlikely. On the other hand the demonstration of 1,25 (OH)2D specific receptors in rat pituitary cells (1, 3) and in clonal pituitary cell lines (2, 5) rather suggests a direct effect of 1,25 (OH)2D on PRL release. In cultured cell lines the effect of 1 ,25 (OHhD is modu­lated by the TRH concentration of medium (5). Low TRH concentrations result in an inhibitory effect of 1 ,25 (OHhD on PRL release while high TRH concentrations cause an opposite effect. Murdoch and Rosenfield (5) have suggested that the dual effect of 1,25 (OH)2D observed in vitro may also apply in vivo. Since in chron­ic renal failure there are low TRH and low 1 ,25 (OH)2D increasing the plasma concentration of 1,25 (OH)2D in this condition could cause a decrease of basal PRL release. This is in accordance with our previous find­ings, namely that administration of 1,25 (OHhD in chronic renal failure results in a decrease of plasma PRL (6). On the contrary, in the normal physiological state, 1,25 (OHhD administration could cause a rise of plasma PRL, as is observed in the present study. We further show that the calcium-antagonist nifedipine does not influence basal nor TRH-induced PRL re­lease, but inhibits the effect of 1,25 (OHbD on PRL secretion. The lackof effect of calcium antagonists on basal and TRH-induced prolactinemia is in accor­dance with previous in vivo studies (12, 19) and with mosl in vitro experiments (20-24). Some in vitro studies show however an inhibtory effect of high-dose verapamil on TRH induced PRL release (25), probably because TRH causes two effects on Ca++ fluxes: 1 ) an acute release of Ca++ into the medi­um from the superficial cell compartment and 2) a Ca++ uptake into the intracellular compartment from extracellular and previously released intracellular Ca++. Only the latter is influenced in vitro by calcium antagonists (25, 26). It is notestablished to what extent those mechanisms are responsible in vivo for TRH-in­duced PRL release. On the other hand, we show that calcium antagonists clearly suppress the 1 ,25 (OHhD­induced rise of PRL. Therefore the effect of 1,25 (OHhD must be mediated by an intracellular Ca++ influx, since it can be inhibited by calcium antagonists. These data indicate that the release of PRL must be under the control of different Ca:++ -mediated mecha­nisms. The effect of 1,25 (OH)2D on calcemia was not ob-

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1,25 (OH)2-vitamin 0 3 and prolactin

served when nifedipine and 1 ,25 (OH)2D were adminis­tered simultaneously. Since the serum concentration of 1,25 (OHhD was markedly elevated, this might suggest that this effect of 1,25 (OH)2D is inhibited by calcium antagonists. An inhibitory effect of verapamil on 1 aOH vit D-induced bone release in vitro has been described (27), thus demonstrating that calcium antagonists can antago­nize some of the biological effects of hydroxylated vita­min 0 metabolites. In conclusion, our data suggest that 1,25 (OH)2D can influence PRL release in vivo' and that this effect is mediated by calcium dependent mechanisms. Other biological effects of 1 ,25 (OHbD could also be mediat­ed by calcium-dependent mechanisms since the 1,25 (OHhD-induced rise of serum calcium disappears when nifedipine and 1,25 (OH)2D are simultaneously administered.

ACKNOWLEDGMENTS The 1.25 (OH)2 vitamin 0 was a gift from Roche N.Y. Belgium and Nifedipine from Bayer N.v. Belgium. This work was performed thanks to grant 3.0033.82 from the Belgian Medical Research Council (FGWO).

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