Decreased hypothalamic growth hormone-releasing hormone content and pituitary responsiveness in hypothyroidism.

The effects of thyroidectomy (Tx) and thyroxine replacement (T4Rx) on pituitary growth hormone (GH) secretion and hypothalamic GH-releasing hormone (GRH) concentration were compared to define the mechanism of hypothyroid-associated GH deficiency. Thyroidectomized rats exhibited a complete loss of pulsatile GH secretion with extensive reduction in GRH responsiveness and pituitary GH content. Cultured pituitary cells from Tx rats exhibited reduced GRH sensitivity, maximal GH responsiveness, and intracellular cyclic AMP accumulation to GRH, while somatostatin (SRIF) suppressive effects on GH secretion were increased. Hypothalamic GRH content was also markedly reduced. T4Rx completely restored hypothalamic GRH content and spontaneous GH secretion despite only partial recovery of pituitary GH content, GRH and SRIF sensitivity, and intracellular cyclic AMP response to GRH. The results indicate multiple effects of hypothyroidism on GH secretion and suggest that a critical role of T4 in maintaining normal GH secretion, in addition to restoring GH synthesis, is related to its effect on hypothalamic GRH.

A non-acromegalic case of multiple endocrine neoplasia type 1 accompanied by a growth hormone-releasing hormone-producing pancreatic tumor.

Cases of acromegaly due to GHRHproducing pancreatic endocrine tumors have been reported. Here we present a case of a 31-yr-old nonacromegalic man with hyperparathyroidism and elevated serum IGF-I with normal serum GH levels. Serum GH was not suppressed below 1 ng/ml by the glucose tolerance test and increased in response to TR H and GHRH administration. Magnetic resonance imaging (MRI) revealed pituitary hyperplasia and an abdominal computed tomography (CT ) scan showed a tumor in the pancreatic tail. Plasma concentration of GHRH was elevated. Based on these clinical data, multiple endocrine neoplasia (MEN) type 1 was suspected. Three enlarged parathyroid glands were removed and a distal pancreatectomy was performed. Pathological examination of the parathyroid glands and pancreatic tumor showed nodular hyperplasia and a well-differentiated endocrine tumor, respectively, both compatible with MEN features. Immunohistochemistry revealed positive immunoreactivity for GHRH, SS , insulin, glucagon, chromogranin A, and pancreatic polypeptide in the pancreatic tumor. After pancreatic surgery, elevated levels of GHRH and IGF-I were normalized and pituitary hyperplasia definitely decreased in size. In cases of pituitary hyperplasia with elevated IGF-I, ectopic GHRH syndrome must be considered even if physical features of acromegaly are absent. It is also important to measure plasma GHRH concentrations in order to give a diagnosis.

Growth hormone (GH)-releasing factor stimulates hypothalamic somatostatin release: an inhibitory feedback effect on GH secretion.

GH-releasing factor (GRF) is a hypothalamic peptide that stimulates the secretion of pituitary GH. The possibility of feedback effects of GRF within the central nervous system was studied in conscious freely moving male rats with indwelling iv and intracerebroventricular (icv) cannulae. Animals were injected icv or iv with 10 ng-10 micrograms human (h) GRF(1-40)-OH (hGRF-40) or GRF(1-44)-NH2 (hGRF-44), and blood samples were obtained every 10-20 min from 1000-1400 h. GH secretion was pulsatile, with major secretory peaks at around 1200 h in most control animals. When 10 ng hGRF-40 were injected icv at 1100 h, immediately before the expected onset of the spontaneous GH secretory burst, GH secretion was suppressed during the following 2-h period. An iv injection of 10 ng hGRF-40 was without effect. In contrast, when 1 microgram hGRF-40 was injected icv or iv, plasma GH levels peaked at 20 and 10 min, respectively, and returned toward baseline shortly thereafter. The spontaneous GH secretory pulse after 1 microgram hGRF-40 (icv or iv) was suppressed in proportion to the magnitude of the GH secretory response to GRF (r = 0.78, p less than 0.01), and the prolongation of the interval between the injection of GRF and the subsequent spontaneous GH surge was directly related to the GH response to GRF (r = 0.85, p less than 0.001). The icv or iv injection of a larger dose of either hGRF-40 or hGRF-44 (10 micrograms) at 1100 h also resulted in marked and comparable increases in plasma GH levels, with peaks at 20 min (icv) and 10 min (iv) after injection. No changes in behavior or plasma glucose were observed up to 3 h after icv injection of any of the doses of hGRF-40 or of hGRF-44. The suppressive effect of centrally administered hGRF-40 (10 ng) on GH secretion was blocked by the iv administration of a specific antisomatostatin serum immediately before the injection of hGRF. These results demonstrate a dual action of GRF on spontaneous GH secretion and indicate the presence of an inhibitory feedback system within the central nervous system for the regulation of GH secretion which is mediated by hypothalamic somatostatin.

Inhibitory effect of hypothalamic medial preoptic area somatostatin on growth hormone-releasing factor in the rat.

Possible inhibitory effects of somatostatin (SRIF) on GRF were studied by assessing spontaneous GH secretion and GRF content and release in adult male rats depleted of hypothalamic SRIF by anterolateral hypothalamic deafferentation (AHD) or electrolytic lesions in the medial preoptic area (MPO). Plasma GH levels were measured 7 days postoperatively every 20 min in conscious animals with indwelling iv cannulae. Median eminence SRIF was markedly reduced 8 days postoperatively in both AHD and MPO rats, as determined by immunohistochemistry and RIA (P less than 0.01). Although GRF immunoreactivity in the median eminence of AHD and MPO animals appeared well preserved immunocytochemically, hypothalamic GRF content by RIA was significantly decreased at 8 days (P less than 0.01). Spontaneous GH secretion was pulsatile in sham-operated animals. In contrast, basal GH levels in AHD and MPO animals were markedly elevated (P less than 0.01), and secretory pulses were absent. Intravenous injection of specific anti-GRF serum into MPO animals decreased the elevated plasma GH levels (P less than 0.01), indicating increased hypothalamic GRF secretion. GRF release from hypothalamic median eminence-arcuate nucleus complexes in vitro was significantly greater in AHD and MPO animals than in control animals 4 and 8 days postoperatively in response to 30 mM K+ (P less than 0.01), but not under basal conditions. These results suggest that hypothalamic medial preoptic area somatostatinergic neurons play a tonic inhibitory role in the regulation of GRF release and that GH hypersecretion observed after MPO and AHD is attributable to changes in both SRIF and GRF.

Effect of hypophysectomy on hypothalamic growth hormone-releasing factor content and release in the rat.

The possible regulation of hypothalamic GH-releasing factor (GRF) by GH was studied in untreated and GH-treated hypophysectomized rats by measurement of rat hypothalamic GRF content and release in vitro with a specific GRF RIA. Two weeks after hypophysectomy, animals not receiving hormone replacement showed a marked reduction in hypothalamic GRF content (to 28% of sham-operated control values; P less than 0.001). Replacement therapy with T4, cortisone, and testosterone for 9 days did not correct the decrease in hypothalamic GRF content in hypophysectomized rats, though the addition of GH therapy partially restored GRF levels (to 55% of control values; P less than 0.001). GRF release from incubated mediobasal hypothalamic fragments of hypophysectomized rats was decreased both basally and in response to 30 mM K+. This defect was completely corrected by prior GH treatment. The results suggest an impairment of GRF synthesis and release in the presence of GH deficiency.

Hypothalamic somatostatin mediates the suppression of growth hormone secretion by centrally administered thyrotropin-releasing hormone in conscious rats.

The effect and mechanism of action of central TRH on the regulation of GH secretion was studied in conscious male rats with indwelling intraatrial and intracerebroventricular (icv) cannulae. Plasma GH was measured every 10-20 min from 1000 h-1400 h by repeated blood sampling. In animals that received saline iv or icv, GH secretion was pulsatile, with peak hormone levels occurring at 1120-1200 h. TRH (10 micrograms), injected icv at 1100 h, inhibited spontaneous GH secretion, and mean plasma GH levels remained suppressed (less than 20 ng/ml) for at least 3 h after injection. In contrast, an iv injection of the same dose of TRH at 1100 h did not significantly affect spontaneous GH secretion. Intravenous injection of human GH-releasing factor [1-40] (hGRF, 1 micrograms) at 1100 h in animals injected 5 min earlier with saline (10 microliters, icv) stimulated GH release, with peak values (748 +/- 63 ng/ml, mean +/- SE) observed 10 min after injection. However, animals injected icv with TRH (10 micrograms) 5 min before the iv injection of hGRF exhibited an attenuated GH response to hGRF (peak values, 115 +/- 28 ng/ml; P less than 0.001 vs. saline icv + hGRF). The inhibition of GH secretion by central TRH was abolished by pretreatment of animals with antisomatostatin serum (0.5 ml, iv) but not with normal serum (P less than 0.001). These results suggest an inhibitory role of central TRH in the regulation of spontaneous GH secretion in the rat that is mediated by stimulation of hypothalamic somatostatin.

Pre- and postnatal developmental changes in hypothalamic content of rat growth hormone-releasing factor.

The ontogenesis of hypothalamic GH-releasing factor (GRF) in pre- and postnatal rats was examined by means of a specific rat GRF RIA. Whereas GRF content was undetectable (less than 10 pg/hypothalamus) on day 17 of gestation, it increased to 30-65 pg/hypothalamus during days 18-20. During postnatal life, hypothalamic GRF content increased more rapidly during days 20-50 than during days 0-20 or 50-90. GRF content was 900-1300 pg/hypothalamus in 50- to 90-day-old rats, and there was no consistent sex difference during postnatal life. Hypothalamic somatostatin levels, as measured by RIA, showed a developmental pattern similar to that of rat GRF. GRF immunoreactivity in hypothalamic extracts from fetal as well as adult rats exhibited HPLC retention times identical to that of synthetic rat GRF. Administration of antirat GRF serum produced a significant decrease in plasma GH levels in fetal rats on day 21 of gestation and in newborn pups 4 h after birth. Passive immunization against GRF caused a more marked suppression of plasma GH (75-85%) 6-9 h after birth and on postnatal day 3. The results demonstrate that immunoreactive GRF is present in measurable levels in the hypothalami of fetal and newborn rats, is chemically indistinguishable from synthetic rat GRF, and exhibits biological effects as early as day 21 of fetal life.

Modulation of somatostatin binding to rat pituitary membranes by exogenously administered growth hormone.

The effect of exogenously administered GH on somatostatin (SRIF) receptor regulation was studied in rat anterior pituitary membranes. A single class, high affinity specific receptor for SRIF was identified by binding studies with [125I-Tyr11]SRIF [binding capacity (mean +/- SD), 129.4 +/- 23.3 fmol/mg protein; binding affinity, 2.8 +/- 0.6 X 10(10) M-1]. A single injection of rat GH (150 micrograms) caused a significant reduction in capacity, but not in affinity, of SRIF receptors 2 and 6 h after injection (mean decrease, 23% and 24%, respectively) from that in controls. In contrast, mean SRIF binding capacity 24 h after a single injection of rat GH was increased 48% above control values, but affinity was unaffected. Measurement of membrane SRIF content indicated that these changes could not be explained by alterations in receptor occupancy. When rat GH was injected repeatedly for 3 days (150 micrograms/rat X day), the mean binding capacity, though not the affinity, of SRIF receptors was decreased 23% from that in controls 24 h after the last injection. The results can be explained by stimulation of SRIF release from the hypothalamus by GH and somatomedins, with subsequent internalization of the pituitary plasma membrane SRIF receptor. They suggest yet another level of neuroendocrine regulation of GH secretion.

Possible involvement of gamma-aminobutyric acid in growth hormone release induced by a Met5-enkephalin analog in conscious rats.

The interaction between opioid peptides and gamma-aminobutyric acid (GABA) in regulating GH secretion was studied in unanesthetized male rats with chronically implanted intraatrial catheters. GH secretion was pulsatile with GH bursts (mean +/- SE, 259.1 +/- 70.3 ng/ml) occurring at regular intervals (mean +/- SE, 3.6 +/- 0.2 h) in control rats. When a potent met5-enkephalin analog, FK 33-824 ([D-Ala,MePhe4,Met(O)-ol]enkephalin; 10 micrograms/100 g BW), was injected iv in the interval between two anticipated spontaneous GH bursts, plasma GH abruptly increased to a peak value of 716.0 +/- 144.9 ng/ml 20 min after the injection, and the following spontaneous GH burst appeared at longer intervals than expected (5.0 +/- 0.4 h, P less than 0.025 vs. control). The plasma GH increase induced by FK 33-824 was blunted by a specific opiate antagonist, naloxone (125 micrograms/100 g BW, iv), and the following spontaneous GH bursts occurred at the same time as in controls. The plasma GH response to FK 33-824 was significantly inhibited by two GABA antagonists, picrotoxin (0.3 mg/100 g BW, iv) and bicuculline (60 micrograms/100 g BW, iv), with peak GH values of 24.6 +/- 6.4 and 136.4 +/- 35.2 ng/ml, respectively (P less than 0.01 vs. FK 33-824 alone). The following natural GH bursts were also inhibited by these GABA antagonists (50.1 +/- 21.3 and 35.3 +/- 9.6 ng/ml; P less than 0.01 vs. control). These findings suggest the possible involvement of GABA in GH release induced by opioid peptides in the rat.

Role of alpha-adrenergic mechanism in regulating tonic luteinizing hormone release in conscious ovariectomized rats.

The role of brain catecholamines in controlling tonic LH secretion was studied in unanesthesized, freely moving, ovariectomized rats. Pretreatment with reserpine (10 mg/kg), given 24 h before the experiment, significantly suppressed pulsatile LH secretion in these rats. Intraperitoneal administration of clonidine (1 mg/kg), an alpha-adrenergic stimulating agent, significantly raised plasma LH levels in reserpine-treated rats. On the other hand, the administration of apomorphine (1.5 mg/kg), a dopamine receptor-stimulating agent, had no effect on the plasma LH levels. These findings suggest an excitatory role of noradrenaline, operating through alpha-adrenergic receptors, in pulsatile LH secretion in ovariectomized rats.

Interrelation between somatostatin output from the mediobasal hypothalamus and plasma growth hormone levels in conscious rats: effects of glucagon administration.

The interrelationship between SRIF output from the mediobasal hypothalamus and plasma GH levels was studied in conscious male rats using the push-pull perfusion technique in combination with repeated blood samplings. The MBH was perfused with artificial cerebrospinal fluid at the rate of 30 microliter/min, and blood samples were collected every 20 min from 1000-1700 h. In control animals, which received injection of acidified saline at 1100 h into the lateral ventricle, two large episodes of spontaneous GH secretion occurred regularly at around 1200 and 1540 h, and troughs occurred around 1400 h. In contrast, SRIF levels from mediobasal hypothalamus perfusate fluctuated at random, ranging from 10-116 pg/ml, with a mean value of 39.2 pg/ml. Mean SRIF levels at 1200 and 1540 h (43.4 +/- 9.0 and 24.4 +/- 4.2 pg/ml, respectively; n = 8) were not different from those at 1400 h (39.9 +/- 12.2 pg/ml). When glucagon (10 micrograms/rat) was injected at 1100 h, plasma GH levels decreased and remained low until 1600 h, whereas perfusate SRIF levels were elevated and remained high for the period. In these animals, the mean plasma GH levels during 1120-1540 h were lower than those in control rats [17.2 +/- 2.4 ng/ml (n = 9) vs. 143.4 +/- 17.5 ng/ml (n = 8); P less than 0.01]. In contrast, the mean SRIF levels in glucagon-treated rats were higher than those in controls [112.5 +/- 15.9 pg/ml (n = 9) vs. control 40.1 +/- 4.3 pg/ml (n = 8); P less than 0.01]. These results suggest that SRIF plays a role in tonic inhibition of GH release in response to the intracerebroventricular injection of glucagon in conscious rats, although SRIF plays, if any, a minor role in regulating episodic GH secretion.

Effects of glucagon, Neurotensin, and vasoactive intestinal polypeptide on somatostatin release from perifused rat hypothalamus.

The effects of neuropeptides on the release of immunoreactive somatostatin (SRIF) from the rat hypothalamus were examined in vitro using a perifusion system. Twelve hypothalamic halves of male rats were placed on a Sephadex G-25 column and continuously eluted with Krebs-Ringer bicarbonate buffer, poH 7.4, at 37 C. A high potassium concentration (56 mM) stimulated SRIF release in a calcium-dependent manner. The infusion of glucagon (10(-7), 5 x 10(-7), and 10(-6) M) resulted in a dose-related increase in the release of SRIF. Neurotensin (10(-6) M) also stimulated SRIF release, whereas vasoactive intestinal polypeptide (10(-7) and 10(-6) M) inhibited SRIF release. SRIF release was not affected by cholecystokinin-octapeptide (10(-6) M), cholecystokinin-tetrapeptide (10(-6) M), or tRH (10(-6) M). These findings suggest that SRIF release from the rat hypothalamus is influenced by glucagon, neurotensin, and vasoactive intestinal polypeptide.

Effect of prostaglandin E1 on vasoactive intestinal polypeptide release from the hypothalamus and on prolactin secretion from the pituitary in rats.

In order to elucidate the mechanisms by which prostaglandin (PG) affects PRL secretion, the effect of PGE1 on vasoactive intestinal polypeptide (VIP) release from the rat hypothalamus was examined by determining plasma VIP levels in rat hypophysial portal blood in vivo and VIP release from the perifused hypothalamus in vitro. Intraventricular injection of PGE1 (1 and 5 micrograms/rat) caused a 2- to 3-fold increase in the concentration of plasma VIP in hypophysial portal blood in anesthetized rats. The flow rate of portal blood was slightly increased after the injection of PGE1. VIP release from the perifused rat hypothalamus was stimulated by high potassium levels (56 mM). The infusion of PGE1 (10 microM) resulted in a significant increase in VIP release from the hypothalamus in vitro. Both these responses were calcium dependent. The intraventricular injection of PGE1 (1 and 5 micrograms/rat) resulted in a dose-related increase in peripheral plasma PRL levels in the rat. These findings suggest that PGE1 plays a stimulatory role in regulating VIP release from the hypothalamus into hypophysial portal blood and causes PRL secretion from the pituitary in rats.

Stimulation by serotonin of vasoactive intestinal polypeptide release into rat hypophysial portal blood.

The effect of serotonin (5-HT) on plasma vasoactive intestinal polypeptide (VIP) levels in hypophysial portal blood was studied in urethane-anesthetized rats. Portal blood was collected by the parapharyngeal approach and plasma VIP was determined by radioimmunoassay. Mean (+/- SE) basal plasma VIP level was 1799 +/- 232 pg/ml, which was slightly decreased during the control experiments in which physiological saline was injected either intraventricularly or intravenously. Intraventricular injection of 5-HT (2 and 10 micrograms/rat) resulted in a significant increase in plasma VIP concentrations within 20 min. Intravenous injection of L-5-hydroxytryptophan (5-HTP, 1 mg/100 g BW), a precursor of 5-HT, also caused an increase in VIP concentrations in hypophysial portal plasma. The flow rate of hypophysial portal blood did not change throughout the experiments. These findings suggest that 5-HT stimulates VIP release from the median eminence into the hypophysial portal vessels in the rat.

Suppressive effect of prostaglandin (PG)D2 on pulsatile luteinizing hormone release in conscious castrated rats.

The effect of intraventricular administration of prostaglandin (PG)D2 on pulsatile LH release was studied in castrated conscious rats. The administration of 5 micrograms of PGD2 into the lateral ventricle inhibited pulsatile discharge of LH secretion, in contrast to the stimulatory effect of PGE2. Intraventricular administration of 13,14-dihydro-15-keto-PGD2, a metabolite of PGD2, had no significant effect. Intravenous administration of 100 micrograms of PGD2 caused only a slight decrease in LH secretion. Intravenous administration of naloxone, a specific opiate antagonist, blocked the suppressive effect of PGD2 on Lh release. These results suggest that PGD2 plays an inhibitory role in pulsatile LH secretion in castrated male rats and that opiate receptors are involved in the PGD2-induced inhibition of LH secretion.

Immunoreactive vasoactive intestinal polypeptide in rat hypophysial portal blood.

Vasoactive intestinal polypeptide (VIP) was measured by RIA in the hypophysial portal blood of rats after total hypophysectomy. The mean (+/- SE) VIP concentrations were 1332 +/- 171 pg/ml (range, 300-3868 pg/ml) under urethane anesthesia and 1735 +/- 707 pg/ml under pentobarbital anesthesia. The concentrations of VIP in peripheral plasma were, in most animals, less than 100 pg/ml. The secretion rate of VIP was not considerably changed during the blood collection. Immunoreactive VIP in hypophysial portal blood was identical to authentic VIP on gel chromatography. These findings suggest that VIP secreted from the hypothalamus may modulate pituitary function via the portal circulation. (Endocrinology 108: 395, 1981)

Production and secretion of parathyroid hormone-related protein in pheochromocytoma: participation of an alpha-adrenergic mechanism.

We studied the relation between calcium metabolism and serum PTH-related protein (PTHrP)-like immunoreactivity (PRP-LI) in 12 patients with benign pheochromocytoma, 10 of whom had not received any medication before admission. Basal serum PRP-LI levels were elevated in 7 of these 10 untreated patients. After tumor resection, serum PRP-LI became undetectable in all 10 patients examined. Serum calcium decreased (P < 0.01), and serum phosphorus increased significantly (P < 0.05) after tumor resection. Serum PTH increased in 5 of 7 patients postoperatively. alpha-Adrenoceptor-blocking agents given as preoperative treatment (n = 6) decreased elevated PRP-LI levels in 5 patients, had no effect in the remaining patient with undetectable levels, and tended to decrease serum calcium levels that were normal in 5 of 6 patients and elevated in 1, while serum phosphorus levels showed reciprocal changes. In the remaining 2 patients who had received alpha-adrenoceptor-blocking agents before admission, serum PRP-LI was undetectable. Tissue PRP-LI concentrations were high in all resected tumors (n = 8). Western blot analysis of tumor extracts showed multiple bands at about 14, 16, 21, 27, and 34 kilodaltons, which differed among tumors. Northern blot analysis of PTHrP mRNA (n = 5) and immunohistochemistry (n = 5) showed positive findings. These findings suggest that PTHrP is synthesized in pheochromocytoma and released into the general circulation, probably via an alpha-adrenergic mechanism, and that changes in serum PRP-LI levels are fairly positively associated with those in serum calcium levels in patients with pheochromocytoma.

Stimulation of growth hormone release by vasoactive intestinal polypeptide from human pituitary adenomas in vitro.

The effects of vasoactive intestinal polypeptide (VIP), TRH, dopamine, and rat median eminence extract on GH release from GH-secreting pituitary adenomas were studied in vitro using a sensitive superfusion method. Dispersed pituitary tumor cells obtained from three patients with acromegaly were placed in a superfusion column, and the amounts of GH in the superfusate were determined. The addition of VIP (10(-6) M) to the perfusion system resulted in a marked increase in GH release in all three cases, and a dose-response relationship in VIP (10(-8) 10(-6) M) induced GH secretion was observed in one case studied. TRH (10(-7) M) and median eminence extract (1 equivalent/ml) also caused an abrupt and marked increase in GH release in all of the experiments. The infusion of either dopamine (10(-7) M) or bromocriptine (10(-7) M) inhibited GH secretion. These results suggest that VIP as well as TRH stimulate GH secretion by a direct action on GH-secreting pituitary tumor cells in at least some acromegalic patients.

Increased serum immunoreactive parathyroid hormone-related protein levels in chronic hypocalcemia.

To elucidate possible negative feedback regulation of circulating PTH-related protein (PTHrP) by serum calcium levels, we measured serum immunoreactive PTHrP (iPTHrP) by a specific RIA for PTHrP-(1-34) in patients with hypocalcemia due to PTH deficiency or resistance. Serum iPTHrP levels were not detectable (< 4 pmol/L) in 9 of 11 patients with postoperative hypocalcemia who presented with transient tetany, in 1 patient with hypocalcemia due to hypomagnesemia induced by cisplatin treatment, in normal subjects (n = 60), or in 1 normocalcemic patient with pseudopseudohypoparathyroidism. In contrast, the other 2 patients with postoperative hypocalcemia who had had hypocalcemic symptoms for longer periods (6 months and 3 yr, respectively) showed increased iPTHrP levels (6.3 and 5.3 pmol/L). All 6 patients with idiopathic hypoparathyroidism showed undetectable or low PTH, but increased iPTHrP, ranging from 6.5-19.5 pmol/L (mean +/- SD, 10.8 +/- 4.8 pmol/L). Elevated serum iPTHrP levels (7.4 and 8.1 pmol/L) were also found in both patients with pseudohypoparathyroidism type I. When chronic and profound hypocalcemia in these patients was normalized by treatment with 1 alpha-hydroxyvitamin D3, the elevated serum iPTHrP levels were normalized (undetectable, < 4 pmol/L) in all 6 patients examined. These results suggest that chronic and profound hypocalcemia and/or vitamin D deficiency can stimulate endogenous PTHrP secretion via a negative feedback mechanism, although elevated iPTHrP does not normalize the decreased serum calcium levels.

Two novel mutations in the coding region for neurophysin-II associated with familial central diabetes insipidus.

Familial central diabetes insipidus is an autosomal dominant disease caused by a deficiency of arginine vasopressin (AVP). We previously reported three distinct mutations in the AVP gene in Japanese familial central diabetes insipidus pedigrees that result in a substitution of Ser for Gly57 in the neurophysin-II (NPII) moiety of the AVP precursor, a substitution of Thr for Ala at the COOH-terminus of the signal peptide, and a deletion of Glu47 in the NPII moiety. In this study, we analyzed the AVP gene in two pedigrees by direct sequencing of the polymerase chain reaction-amplified DNA and found two novel mutations in exon 2, which encodes the central part of the NPII moiety of the precursor. The mutation in one pedigree was a C to A transition at nucleotide position 1891, which replaces Cys67 (TGC) with stop codon (TGA). As the premature termination eliminates part of the COOH domain of the NPII moiety and the glycoprotein moiety, the conformation of the truncated protein is likely to be markedly different from that of normal precursor. In another pedigree, a G to T transversion was detected at nucleotide position 1874, which substitutes polar Trp (TGG) for hydrophobic Gly62 (GGG). It is possible that mutated NPII molecules, as a consequence of a conformational change, cannot bind AVP or self-associate to form higher oligomer complexes. Interestingly, all mutations we have identified to date, with the exception of the signal peptide mutation, are located in exon 2, suggesting the importance of the highly conserved central part of the NPII molecules and/or the NPII moiety in the precursor for AVP synthesis.