Effects of glucocorticoids on pituitary hormonal responses to hypoglycemia. Inhibition of prolactin release.

Effects of Glucocorticoids on Pituitary Hormonal Responses to Hypoglyeemia. Inhibition of Prolactin Release* G. COPINSCHI,1 M. L'HERMITE,2 R. LECLERCQ,3 J. GOLSTEIN,4 L. VANHAELST,1 E. VIRASORO,1 AND C. ROBYN2 School of Medicine, Universities of Brussels, Brussels, Belgium: laboratory of Experimental Medicine; 2Human Reproduction Research Unit; ^Laboratory of Clinical Chemistry (present address: Laboratory, IMC, Anderlecht); institute of Interdisciplinary Research (LMN) pressed basal levels of ACTH and cortisol and the ACTH—but not the cortisol—response to hypoglyeemia. Both basal levels of prolactin and prolactin response to hypoglyeemia were significantly lowered but growth hormone response was not modified by administration of 1 mg of dexamethasone. The administration of larger doses of dexamethasone (1 mg every 6 h for 2 days) almost completely suppressed basal levels of ACTH, cortisol and prolactin, as well as the hypoglycemiainduced release of these hormones. In contrast, the growth hormone response to hypoglyeemia was only partially inhibited. These findings demonstrate that both basal secretion and hypoglycemia-induced release of prolactin, ACTH, cortisol and growth hormone are suppressible by glucocorticoids. (J Clin Endocrinol Metab 40: 442, 1975)

ABSTRACT. The characteristics of pituitary hormonal responses to insulin-induced hypoglyeemia were investigated in 16 normal men. In all subjects, levels of blood sugar fell below 35 mg/100 ml. A statistically significant increase in mean plasma levels of prolactin, ACTH, cortisol and growth hormone was observed. Prolactin levels increased in all subjects but one; individual peak values were 1.4—8.4 times greater than base levels. The kinetics of prolactin, GH and ACTH responses were similar; in particular, the onset of release (25 min) of prolactin, GH and ACTH was similar. After dexamethasone administration, insulin tolerance tests were repeated in a number of subjects using adequate amounts of insulin to achieve hypoglyeemia equivalent to that obtained in the control experiments. The administration of 1 mg of dexamethasone the evening before the test sup-

I

T has been recently demonstrated in normal man that insulin-induced hypoglyeemia will promote prolactin (1-3) as well as growth hormone (GH) (4) and corticotropin (ACTH) (5) release. Although the mechanisms involved are not completely understood, hypoglyeemia very likely acts at the hypothalamic (or suprahypothalamic) level (3-5). Several studies have shown that the GH as well as the ACTH response to hypoReceived August 27, 1974. The work was supported in part by grants from the Ford Foundation to Prof. P. O. Hubinont and from the Fonds de la Recherche Scientifique Medicale. This work was partially performed under contract of the Ministere Beige de la Politique Scientifique within the framework of the Association Euratom — University of Brussels — University of Pisa. * Presented in part at the seventh annual meeting of the European Society for Clinical Investigation, Rotterdam, The Netherlands, April 26-29, 1973. Requests for reprints should be addressed to Dr. G. Copinschi, Laboratoire de Medecine Experimental, Universite Libre de Bruxelles, Boulevard de Waterloo 115, B—1000 Bruxelles (Belgium).

glycemia can be suppressed by prior administration of dexamethasone (6-8), suggesting that the effects of glucocorticosteroids at the hypothalamo-pituitary level are not entirely specific to the adrenocorticotropic axis. No data exist about the possible influence of corticosteroids on prolactin release induced by hypoglyeemia. In the present study, we have investigated in normal man the characteristics— and particularly the kinetics—of prolactin response to hypoglyeemia. Corticotropic and somatotropic responses were monitored also. In addition, the effects of both low and high doses of dexamethasone upon this prolactin release have been explored, and compared with effects on the release of ACTH, cortisol and GH. Materials and Methods Sixteen normal men (no. 1-16), aged 15-47, were investigated after an overnight fast. An indwelling plastic catheter was inserted into a forearm vein at 8 AM and insulin "Actrapid" Novo (0.12 U/kg body wt) was injected at 9 AM.

442

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PROLACTIN RESPONSE TO HYPOGLYCEMIA Blood samples were obtained 60 and 30 min before injection, at the time of injection, and at frequent intervals for 180 min after the injection. In 6 subjects (no. 1-6), the insulin tolerance test was repeated 3-6 days later, after pretreatment with a single oral dose of 1 mg dexamethasone at 11 PM the previous evening. The dose of insulin was increased to 0.20 U/kg in order to attain a degree of hypoglycemia similar to that obtained during the control experiment (6). In 4 subjects (no. 1-4), a third insulin tolerance test was performed 3-10 months later, following oral pretreatment with higher doses of dexamethasone (1 mg every 6 h for the 2 preceding days; an additional dose of 1 mg at 8 AM the day of the test). To obtain adequate hypoglycemia, the amount of insulin employed was 0.25 U/kg. Blood sugar was measured using a colorimetric procedure adapted to the Technicon AutoAnalyzer (9). Plasma cortisol was determined by a competitive protein-binding radioassay (10). Plasma levels of GH (11) and of TSH (12), were estimated by radioimmunoassay. The sensitivity of the TSH assay was 0.6 fxU/ml. All TSH samples were measured in the same assay. Plasma prolactin was determined by a radioimmunologic method (13,14) involving a doubleantibody procedure. Prolactin levels were evaluated by reference to a serum pool, rich in prolactin, which was arbitrarily considered to contain 1 U of immunoreactive prolactin per ml; 1 mU of this standard preparation corresponds to 2.3 milliampouls of the standard prolactin preparation no. 71/167 of the Medical Research Council, Great Britain. Highly purified ovine prolactin (LER-860-2) labeled with I25I was used as the tracer, and the rabbit anti-ovine prolactin serum no. 770 as the antibody. No cross-reaction was found with GH (up to 500 ng of human growth hormone HS 1394 per test tube), human chorionic somatomammotropin (up to 10 fig of HPL 717340 per test tube) or human chorionic gonadotropin (up to 2500 IU per test tube). The sensitivity of the prolactin assay ranged between 10 and 15 mU/ml plasma. The coefficient of variation of replicate determinations of plasma samples averaged 8% within a single assay and 25% between assays (13). All samples of the first two series of experiments were measured in the same assay. Plasma levels of ACTH were measured without

extraction by a radioimmunologic method (15) derived from that reported by Berson and Yalow (16), using a specific and highly potent antiserum (17). Native human ACTH (Li) was used for the standard curve. The point on the standard curve corresponding to a concentration of 10 pg/ml plasma was significantly and systematically different from the zero point of this standard curve (t = 14.99; P < 0.001; n = 13). ACTH and TSH measurements were only performed in a certain number of experiments. Unless otherwise stated, all data were submitted to a variance analysis with calculation of within-subject and between-subjects variations by F tests (18). When, according to the Bartlett

80 70 SO . mg/IOOml 40

90 0 TOO

600 SOO

400 300 O

SO

40 30 20 10 0 20

s 0 1OOr

ACTH pg/ml

78

0

iO TIME

tO (mm)

FIG. 1. Mean values of blood sugar, and of plasma prolactin, GH, cortisol and ACTH during control studies in 16 normal men (8 subjects for ACTH). Vertical bars denote standard errors of the mean.


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test (18), the variances exhibited significant heterogeneity, analyses were performed on log transforms.

Results Control studies. Results are summarized in Fig. 1. Individual data for blood sugar and plasma prolactin are given in Table 1. Blood sugar levels fell markedly after the injection of insulin, reaching a minimal value at 25 min. Individual minimal levels, ranging from 14 to 34 mg/100 ml, were observed 20 to 35 min after the insulin injection. TSH values (recorded in subjects no. 1-6) remained stable throughout the experiment and no response to hypoglycemia could be evidenced (see Fig. 2).

A slight decrease of plasma levels of ACTH (estimated in subjects no. 1-3 and 7-11) was observed from - 6 0 to 15 min. After 25 min, plasma values increased rapidly to reach a plateau from 35 to 60 min (F = 68.0; P < 0.001); thereafter, ACTH levels decreased rapidly towards basal values. Circulating levels of cortisol also decreased from - 6 0 to 15 min. A progressive increase was observed after 25 min, to reach a mean peak value at 75 min (F = 59.9; P < 0.001); thereafter, cortisol values slowly decreased. Plasma levels of GH changed little for 25 min after insulin injection, then increased rapidly to reach a mean peak value at 60 min (F = 207.8; P < 0.001). Plasma values of prolactin were fairly stable for 25 min,

TABLE 1. Individual values of blood sugar and of plasma prolactin during control studies in 16 normal men (no. 1-16) Minutes

40

45

-60

-30

0

15

20

25

1

89

97

91

42

25

25

26

32

36

40

31

32

31

51

41

62

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

97 98 100 99 80 73 76 76 75 73 71 81 82 69 81

99 93 99 97 73 75 76 75 78 53 75 77 85 73 79

95

49

34

27

54

69 51 43 36 43 29 41 43 40 31 56 57 48 44

58 35 27 26 32 21 29 33 28 26 41 43 35 28

44 29 26 26 26 14 26 22 19 15 29 31 26 25

36 34 42 48 48 42 33 39 34 30 30 35 30 22 34

52

90 100 95 74 77 75 74 78 63 72 73 85 72 81

28 37 28 43 28 28 24 36 21 23 23 27 31 23 25

40 52 54 50 44 36 42 43 46 30 49 38 31 46

54 59 50 53 41 39 38 46 45 28 56 43 33 45

57 67 54 55 53 33 38 41 53 49 32 57 47 40 46

60 74 52 51 52 37 31 41 53 50 31 60 56 47 52

67 76 60 59 60 37 40 41 59 55 28 68 68 61 57

71 80 63 72 68 37 41 52 64 57 31 71 73 66 63

80 86 90 77 47 60 60 77 63 45 •70 79 65 71

92 91 90 95 82 62 71 74 82 61 43 78 76 55 79

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

179 221 298 286 368 511 424 331 75 409 312 317 612 139 93 377

166 117 263 249 340 612 380 450 60 348 258 182 381 179 32 275

220 123 227 175 284 541 378 458 44 326 282 86 597 159 37 204

190 130 218 206 269 505 331 167 18 483 312 244 760 122 44 323

177 91 189 198 397 503 380 235 38 227 163 232 938 142 35 295

160 108 188 235 542 425 344 276 75 440 292 176 796 109 51 264

1,501

1,417

1,204

1,210

1,347

1,282

120 163 828 475 797 561 511 25 239 448 650

165 143 700 500

131 192 567 415

1,058

1,137

661 453 90 306 286 700

132 171 592 507 763 813 650 65 462 346 538

510 470 65 344 456

726 334 48 346 320 720

151 127 437 438 761 648 466 70 290 306 660

135 117 498 390 745 610 511 84 290 368 479 — 181 124 546

Subject

30

35

50

60

75

90

120

180

Blood sugar mg/100 ml

76

Plasma prolactin mlj/ml

485 109 149 277 328 676 353 448 65 299 274 240

719 97 145 402 611 925 414 232 75 241 280 510

1,046

1,629

1,596

1,724

1,374

1,239

1,400

1,400

79 71 392

243 20 433

371 21 717

454 58 510

386 83 658

523 112 591

322 157 595

369 85 565

905 174 156 557 597 1,194

557

815 83 133 260 280 415 362 311 '

70

343 292 360 1,051

370 19 462


PROLACTIN RESPONSE TO HYPOGLYCEMIA

445

then increased rapidly to reach a mean peak value at 45 min (F = 27.5; P < 0.001), and remained elevated until 90 min. This increase occurred in all subjects but one (no. 3); individual peak values, 1.4-8.4 times greater than the basal (0 min) levels, were observed 35 to 75 min after insulin injection. Effects of a single dose of dexamethasone. Results obtained in subjects no. 1-6 with and without dexamethasone pretreatment are summarized in Fig. 2. For ACTH, data were obtained in 5 subjects (no. 1-5) after dexamethasone pretreatment, and in 8 subjects (no. 1-3, and 7-11) in control studies (Fig. 2). Thus, only in 3 subjects were ACTH results obtained simultaneously in both experiments. Therefore, group comparisons (t test) were performed to compare dexamethasone and control studies for this hormone. Individual data for blood sugar and plasma prolactin after a single dose of dexamethasone are shown in Table 2. The hypoglycemic action of insulin was slightly delayed after dexamethasone pretreatment, since the mean minimum value was found at 30 min instead of 25 min in control studies. However, the degree of hypoglycemia was similar in both series of experiments. Values obtained at 20, 25 and 30 min in control experiments were not statistically different from those observed at 25, 30 and 35 min after dexamethasone pretreatment (F = 0.4; P > 0.20). Basal values of TSH were not modified after dexamethasone (F = 0.1; P > 0.20). They changed little for 40 min after insulin injection, then decreased rapidly to reach a mean minimal value at 45 min (F = 18.1; P < 0.001). Basal values of ACTH were completely suppressed after dexamethasone. A slight but significant elevation occurred after 35 min, to reach a mean peak level at 45 min (F = 8.2; P < 0.01). However, the ACTH response to hypoglycemia was markedly inhibited by the dexamethasone pretreatment: values of plasma ACTH between 40

FIG. 2. Mean values of blood sugar, and of plasma TSH, prolactin, GH, cortisol and ACTH in 6 normal men, during control experiments (black circles) and after a single dose of dexamethasone (open circles) (for ACTH, values for 8 subjects in control studies and for 5 subjects after dexamethasone). Vertical bars denote standard errors of the mean.

and 60 min, as well as increments at 40-60 min over 0 min levels, were significantly lower in dexamethasone than in control experiments (t test significant at least at the 0.05 level).


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TABLE 2. Individual values of blood sugar and of plasma prolactin in 6 normal men (no. 1-6) after a single dose of dexamethasone Minutes Subject

-60

-30

108 107 105 108 110 78

108 107 94 105 107 76

20

15

25

30

45

35

50

60

75

90

120

180

79 69 68 61 73 66

93 74 78 67 94 72

102 88 89 87 98 72

620 150 167 433 417 180

533 97 144 322 355 146

318 98 127 112 331 120

Blood sugar rug/100 ml 66 58 55 67 62 51

106 103 89 102 104 78

48 50 40 56 48 46

27 37 29 48 36 30

30 28 25 34 27 25

40 28 26 31 28 28

55 46 33 4.3 44 40

66 42 48 57 55 49

66 48 53 58 60 49

54 55 55 55 64 52

1,257 96 202 243 355 134

145 165 553 582 187

67 57 59 58 74 59

Plasma prolactin mU/ml 308 131 306 127 242 327

191 139 124 106 255 244

147 119 178 70 185 278

153 141 116 163 297 272

283 112 137 101 342 244

186 85 142 174 312 201

202 100 143 135 431 113

374 97 128 157 269 110

INSULIN IV.

•SO

-JO

0

30

SO

90

IK

ISO

l«0

TIME (min)

FIG. 3. Mean values of blood sugar, and of plasma prolactin and GH in 4 normal men, during control experiments (black circles), after a single dose of dexamethasone (open circles) and after a 2-day dexamethasone pretreatment (triangles). Vertical bars denote standard errors of the mean. (ACTH and cortisol values are not shown since they were indistinguishable from zero after the 2-day dexamethasone pretreatment).

813 217 154 234 350 102

1,024 126 176 496 296 192

1,039 117 201 503 263 161

Basal values of cortisol were also completely suppressed after dexamethasone. However, a marked increase of cortisol values was observed after 35 min, to reach a mean peak level at 60 min (F = 29.9; P < 0.001). Values of plasma cortisol between 45 and 90 min were significantly lower in dexamethasone than in control studies (F = 353.9; P < 0.001), but increments over 0 min levels at 45-90 min were not statistically different in control and in dexamethasone experiments (F = 3.6; P > 0.05). The GH pattern was quite similar in control and in dexamethasone studies (F = 1.1; P > 0.20). In particular, mean maximal values, reached at 60 min, were almost identical (F = 1.1; P > 0.20). Basal levels of prolactin were diminished after dexamethasone (F = 5.3; P < 0.05). They changed little for 35 min after insulin injection, then increased to reach a mean peak value at 60 min (F = 6.0; P < 0.05). This increase occurred in all subjects but one (no. 6); individual peak values, 1.6 to 6.6 times greater than the 0 min levels, were observed between 40 and 60 min. However, values of plasma prolactin between 40 and 90 min, as well as increments at 40-90 min over 0 min levels, were


447

PROLACTIN RESPONSE TO HYPOGLYCEMIA significantly lower in dexamethasone than in control studies (respectively F = 15.4; P < 0.001; and F = 14.6; P < 0.001). Effects of a two-day dexamethasone pretreatment. Results obtained in subjects no. 1-4 in control studies and after both schedules of dexamethasone administration are summarized in Fig. 3. Individual values of blood sugar and of plasma prolactin after the high dose of dexamethasone are shown in Table 3. After the 2-day pretreatment with dexamethasone, the hypoglycemic effect of insulin was further delayed and the mean minimum value was only reached at 45 min. However, the degree of hypoglycemia was similar to that obtained in the other two series of experiments. Minimal levels (35, 40, 45 min) were not statistically different from minimal values recorded in the other two groups of studies (F = 0.01; P > 0.20). Plasma levels of ACTH (recorded only in subjects 2-4) and of cortisol were completely suppressed and no response to hypoglycemia could be observed. A significant increase occurred in GH values after 15 min, to reach a mean peak level at 40 min (F = 14.0; P < 0.001). However, the response of plasma GH to hypoglycemia was markedly inhibited: values between 30 and 50 min were significantly lower than maximal values

(45-90 min) of the other two series of experiments (F = 6.6; P < 0.05). Basal levels of prolactin were markedly inhibited (F = 44.6; P < 0.001) and no significant elevation was observed following insulin-induced hypoglycemia. Discussion In a preliminary report, we had shown in normal man that insulin-induced hypoglycemia systematically provokes a significant prolactin release, provided the magnitude of the blood sugar fall is sufficient (3). The present data confirm this observation in a larger sample of normal subjects. An increase of at least 40% over basal values occurred in all but one of the 16 normal men investigated. It is of interest that the subject who had no prolactin response to hypoglycemia in control experiments presented a lesser degree of hypoglycemia (nadir at 34 mg/100 ml) (Table 1). This observation appears to indicate that the standard insulin tolerance test, which is used as a clinical tool for assessment of pituitary GH and ACTH reserve, might also become a very useful test for the clinical investigation of prolactin secretion. The frequent blood sampling performed in the present experiment afforded a precise evaluation of the patterns of hormonal pituitary responses to hypoglycemia. The comparison of prolactin, GH and ACTH

TABLE 3. Individual values of blood sugar and of plasma prolactin in 4 normal men (no. 1-4) after a two-day dexamethasone pretreatment (1 mg every 6 h) Minutes Subject

-60

-30

0

15

20

25

30

35

40

45

50

60

75

90

120

180

34 28 36 45

48 55 50 63

64 74 60 70

75 82

84 88 86 96

94 95 92 97

64 2 90 37

68 6 92 25

102 5 80 44

71 9 142 25

70

55 8 81 40

Blood sugar mg/100 in 1

1 2 3 4

99 92 92 106

104 97 94 107

101 96 88 107

78 69 66 79

69 58 55 68

62 49 44 57

56 30 37 50

46 28 31 36

44 27 25 28

36 27 29 29

70 .

87

Plasma prolactin inll/mil 1 2 3 4

101 15 56 54

28 15 33 49

56 10 98 66

41 9 75 54

85 7 90 31

49 11 81 38

122 8 59 22

67 7 46 23

88 9 84 42

50 7 102 36

9

61 40


448

COPINSCHI ET AL.

patterns demonstrates a remarkable similarity in the kinetics of these hormonal secretions. In particular, it must be stressed that, on an average, the onset of release of the 3 pituitary hormones during control studies occurred at the same time (25 min). This strongly suggests that the insulininduced prolactin release is mediated, as for GH (4) and ACTH (5), by a stimulation of hypothalamic centers. Hypoglycemia would probably either stimulate the secretion of a prolactin-releasing hormone (PRH) or decrease the secretion of a prolactin-inhibiting hormone (PIH). On the other hand, TSH release is not stimulated by hypoglycemia. This confirms previous results of Besser et al. (19), and adds further evidence to the already described (20) dissociation between TSH and prolactin secretions. The significance of the transient decrease in TSH values observed, in the present investigation, during insulin hypoglycemia after dexamethasone pretreatment, remains unclear. Although the degree of hypoglycemia was similar in the three series of the present experiments, the slope of blood sugar fall was slightly flatter in the high dexamethasone group (Fig. 3). Although this might have influenced the hormonal responses, it seems unlikely as the nadir sugar level was virtually identical in all studies. Thus, the present observations strongly suggest that glucocorticoid administration may inhibit and probably suppress both basal secretion and hypoglycemia-induced release of prolactin, ACTH and GH. Although in the present investigation, basal levels of TSH were not decreased by a single dose of dexamethasone, it has been demonstrated in other studies that different administration schedules of glucocorticoids may inhibit TSH secretion under basal conditions (21). Finally, it has also been shown that /3-MSH secretion may be inhibited by glucocorticoids (22). This confirms the hypothesis

JCE & M • 1975 Vol 40 • No 3

(8) that the inhibitory action of glucocorticoids on hypothalamo-pituitary secretion is far from being specific to the corticotropic axis. The efficiency of the inhibitory effect of glucocorticoids on hypothalamo-pituitary hormonal release is probably dependent on both the dose administered and the duration of administration (8). The present results show that a single administration of 1 mg of dexamethasone the evening before the test inhibited basal secretion of ACTH, cortisol and prolactin as well as hypoglycemia-induced ACTH and prolactin release. On the contrary, growth hormone and cortisol responses to hypoglycemia were not significantly reduced by this single dose of dexamethasone. This dissociation between ACTH and cortisol patterns is not surprising, since it has been shown—both in vitro (23) and in vivo (24-26)—that there is an apparent linear relationship between the logarithm of ACTH concentration and adrenal secretion of cortisol (a more exact representation is probably a saturation curve (27)). Thus, reduction in basal levels of ACTH resulted in a marked decrease in basal cortisol levels, whereas the significantly lessened ACTH response to insulin-induced hypoglycemia evoked a cortisol response nearly equivalent to that which was seen without dexamethasone suppression. On the other hand, the administration of higher doses of dexamethasone for two days almost completely suppressed basal levels of ACTH, cortisol and prolactin and virtually abolished the hypoglycemia-induced release of these hormones. Growth hormone response to hypoglycemia, which was not altered by prior administration of 1 mg of dexamethasone, was significantly inhibited by the higher doses. In conclusion, the present study demonstrates that both basal secretion and hypoglycemia-induced release of prolactin are suppressed by prior glucocorticoid ad-


PROLACTIN RESPONSE TO HYPOGLYCEMIA ministration. The sensitivity of this feedback mechanism appears to be greater than that for growth hormone. Acknowledgments We are indebted to Dr. C. H. Li who kindly supplied the human ACTH, and to Dr. R. Depieds for the generous gift of ACTH antiserum. We wish to thank the National Pituitary Agency, Endocrinology Study Section, and the National Institute of Arthritis and Metabolic Diseases, for supplying the reagents used for GH and TSH assays (GH antiserum was prepared by Dr. S. A. Berson and Dr. R. S. Yalow); and Dr. G. D. Niswender for the ovine reagents used for prolactin assays. We are most grateful to Drs. Ph. Vague, Ch. Oliver and H. Brauman for helpful advice and discussions during the development of the radioimmunoassay of ACTH.

References 1. Noel, G. L., H. K. Suh, J. G. Stone, and A. G. FrantzJ Clin Endocrinol Metab 35: 841, 1972. 2. Friesen, H., B. R. Webster, P. Hwang, H. Guyda, R. E. Munro, and L. Read, J Clin Endocrinol Metab 34: 192, 1972. 3. Copinschi, G., M. L'Hermite, L. Vanhaelst, R. Leclercq, O. D. Bruno, J. Golstein, and C. Robyn, C R Acad Sci [D] (Paris) 275: 1419, 1972. 4. Roth, J., S. M. Glick, R. S. Yalow, and S. A. Berson, Metabolism 12: 577, 1963. 5. Landon, J., W. Wynn, and V. H. T. James, J Endocrinol 27: 183, 1963. 6. Moses, A. M., and M. Miller, Metabolism 18: 376, 1969. 7. Nakagawa, K., Y. Horiuchi, and K. Mashimo, J Clin Endocrinol Metab 32: 188, 1971. 8. Von Werder, K., S. Hane, and P. H. Forsham, Norm Metab Res 3: 171, 1971. 9. Hoffman, W. S.J Biol Chem 120: 51, 1937. 10. Leclercq, R., G. Copinschi, and J. R. M. Franckson, Rev Fr Etud Clin Biol 14: 815, 1969.

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11. Virasoro, E., G. Copinschi, O. D. Bruno, and R. Leclercq, Clin Chim Ada 31: 294, 1971. 12. Golstein, J., and L. Vanhaelst, Clin Chim Ada 49: 141, 1973. 13. L'Hermite, M., P. Delvoye, J. Nokin, M. Vekemans, and C. Robyn, In Boyns, A. R., and K. Griffiths (eds.), Prolactin and Carcinogenesis, Alpha Omega Alpha, Cardiff, 1972, p. 81. 14. Nokin, J., M. Vekemans, M. L'Hermite, and C. Robyn, Br MedJ III: 561, 1972. 15. Virasoro, E., G. Copinschi, and O. D. Bruno, In Radioimmunoassay and Related Procedures in Medicine, vol. I, International Atomic Energy Agency, Vienna, 1974, p. 323. 16. Berson, S. A., and R. S. Yalow, J Clin Invest 47: 2725, 1968. 17. Cros, G., P. Vague, C. Oliver, and R. Depieds, C R Soc Biol (Paris) 164: 1289, 1970. 18. Snedecor, G. W., Statistical Methods Applied to Experiments in Agriculture and Biology, The Iowa State University Press, Ames, Iowa, 1956, p. 291. 19. Besser, G. M., J. G. Ratcliffe, J. R. Kilborn, B. J. Ormston, and R. Hall J Endocrinol 51: 699, 1971. 20. L'Hermite, M., C. Robyn, J. Golstein, G. Rothenbuchner, J. Birk, U. Loos, M. Bonnyns, and L. Vanhaelst, Horm Metab Res 6: 190, 1974. 21. Wilber, J. F., and R. D. Utiger.7 Clin Invest 52: 1099, 1973. 22. Abe, K., W. E. Nicholson, G. W. Liddle, D. N. Orth, and D. P. Island, J Clin Invest 48: 1580, 1969. 23. Saffran,- M., and A. V. Schally, Endocrinology 56: 523, 1955. 24. Landon, J., V. H. T. James, M. J. Wharton, and M. Friedman, Lancet II: 697, 1967. 25. McDonald, R. K., A. R. Sollberger, P. S. Mueller, and M. H. Sheard, Proc Soc Exp Biol Med 131: 1091, 1969. 26. Leclercq, R., G. Copinschi, and O. D. Bruno, Horm Metab Res 4: 202, 1972. 27. Dallman, M. F., and F. E. Yates, Ann N Y Acad Sci 156: 696, 1969.


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Mechanisms of dihydroergotoxine's effect on prolactin release.

Pancreatic and pituitary hormonal responses to insulin-induced hypoglycaemia during muscarinic cholinergic blockade in man.