Immunoreactive corticotropin-releasing hormone levels in discrete hypothalamic nuclei of genetically obese Zucker rats.

The levels of immunoreactive corticotropin-releasing hormone (ir-CRH) were measured in discrete hypothalamic nuclei and the median eminence of obese Zucker rats and their lean littermates. More than 90% of total hypothalamic ir-CRH was detected in the median eminence in both obese and lean rats. Though ir-CRH levels in the paraventricular nucleus of obese rats tended to be lower than those of their lean littermates, no significant difference of ir-CRH levels was observed in any hypothalamic nuclei studied between obese and lean rats. However, ir-CRH levels in the median eminence of obese rats were significantly lower than those of their lean littermates (5263 +/- 438 pg/tissue vs. 7050 +/- 473 pg/tissue, P < 0.05). These results suggest that the hypoactive hypothalamic CRH tonus would play some role in the phenotypic expression of obesity in the genetically obese Zucker rats.

Chimeric studies of the extracellular domain of the rat thyrotropin (TSH) receptor: amino acids (268-304) in the TSH receptor are involved in ligand high affinity binding, but not in TSH receptor-specific signal transduction.

A series of chimeric TSH-LH/CG receptors were constructed by substituting homologous segments of the extracellular domain of the rat TSH receptor with corresponding segments of rat LH/CG receptor: C1 (amino acids 37-123 substituted), C2 (91-112), C3 (173-234), C4 (233-266), C5 (268-304), C6 (112-305) and C7 (36-404). After transfection in Cos-7 cell, TSH- and LH/CG-receptor activities of these chimeras were evaluated and compared with those of deletion mutants involving the same residues [Kosugi et al. Thyroid 1:321 (1991)]. Western blot analyses revealed that most of the chimeric receptor proteins were normally synthesized and integrated in the membrane of transfected Cos-7 cells: an antibody to a TSH receptor specific synthetic peptide (residues 352-366) identified 170-190kDa and 90-100kDa TSH receptor structures in the plasma membrane fractions of Cos-7 cells transfected with wild-type TSH receptor cDNA and the C1 to C6 chimeras, but not C7 or wild LH/CG receptor cDNA. Despite this, no receptor except C5 exhibited any significant TSH receptor activities either in [12I]TSH binding or in cAMP responses to TSH and thyroid-stimulating antibodies (TSAbs) from Graves' patients. The chimeric receptor C5 exhibited only low affinity TSH binding (Kd = 3.5 x 10(-8) M), as did its counterpart the M2C mutant with residues 268-304 deleted. However, unlike M2C, C5 demonstrated a significant cAMP response to TSH as well as to TSAbs. The cAMP increase in response to TSH in the wild type receptor was observed at 10(-11) M TSH. In C5 the response was first evident at 10(-10) M TSH, but the maximum cAMP stimulation by TSH and TSAbs in C5 (EC50 = 6.7 x 10(-10) M) was approximately the same as the wild type receptor (EC50 = 1.5 x 10(-10) M). Inhibition of either TSH- or TSAb- stimulated cAMP increase by thyroid-stimulating blocking antibodies (TSBAbs) was also preserved in C5. These results suggest that amino acids 268-304 do not include an important determinant required for signal transduction, since a significant cAMP response to TSH and TSAbs was observed in the C5 receptor with these residues substituted. Additionally, these residues appear to be involved in ligand high affinity binding because high affinity TSH binding was lost in the chimeric receptor C5.

Potassium accelerates urinary sodium excretion during salt loading without stimulating atrial natriuretic polypeptide secretion.

1. Effects of potassium (K) supplementation (100 mEq/day) on urinary sodium (Na) excretion and on the secretion of atrial natriuretic polypeptide (ANP) during salt loading (350 mEq/day) were studied in 12 healthy salt-resistant normotensives under strictly controlled metabolic ward conditions. 2. Urinary volume and Na excretion on the first day of the high salt period (HSP) were significantly greater in the K-supplemented group (KG) than in the control group (CG). 3. There was a significant gain in bodyweight after salt loading in both groups, with a significantly greater gain in CG on the second day of HSP. Haematocrit decreased significantly during salt loading in both groups, the degree of which was significantly greater in CG. 4. Plasma norepinephrine decreased significantly during salt loading in both groups, the degree of which was significantly less in KG than in CG. A significant increase in plasma ANP was observed in CG on and after the second day of HSP, while a significant increase in plasma ANP was observed on the fifth day of HSP in KG. 5. These findings indicate that K supplementation accelerates diuresis and natriuresis, resulting in moderate suppression of volume expansion induced by salt loading and that this accelerated diuresis and natriuresis is not a result of the action of ANP.

Biphasic changes in hypothalamo-pituitary-adrenal function during the early recovery period after major abdominal surgery.

Regulatory mechanisms of the hypothalamo-pituitary-adrenal (H-P-A) axis during and after major abdominal surgery were studied in a group of patients who underwent upper abdominal surgery. We first examined the general profile of the changes of the H-P-A axis from the day before surgery to the seventh day after surgery. On the day of surgery, plasma levels of CRH, ACTH, and cortisol were all significantly elevated after skin incision (phase I). During the next 2 days, plasma cortisol levels remained significantly elevated, and the both plasma CRH and ACTH levels were suppressed below the control levels obtained on the day before surgery (phase II). Several additional studies, carried out to analyze the mechanism that maintains the high plasma cortisol levels, revealed the following features of the H-P-A axis during phase II. Plasma free cortisol levels in this phase were higher than those during the preoperative period. The exogenously administered hydrocortisone clearance rate in phase II did not differ from that observed on the day before surgery. Dexamethasone administration resulted in a decrease in plasma cortisol levels similar to that observed preoperatively. Conversely, the ACTH-stimulated cortisol increase was significantly greater in phase II than that observed preoperatively. These results suggest that during and after major surgical stress, the H-P-A axis undergoes a biphasic change in the pattern of the stress response and during the second phase, not the continuous hypothalamo-pituitary drive but the increased adrenal responsiveness to ACTH is responsible at least in part for maintaining the elevated plasma cortisol level.

Chronic effects of interleukin-1 on hypothalamus, pituitary and adrenal glands in rat.

To assess the chronic effects of interleukin-1 (IL-1) and IL-2 on the hypothalamo-pituitary-adrenal axis in vivo, we administered recombinant human (rh) IL-1 alpha, rhIL-1 beta or rhIL-2 (2.0 micrograms/day) repetitively to adult male rats for 10 days. In rhIL-1 beta-treated rats, adrenocorticotropic hormone-like immunoreactivity (ACTH-LI) of the anterior pituitary appeared to increase first on day 3 followed by an increase of corticotropin-releasing hormone (CRH)-LI both in the hypothalamus and in the adrenal gland after day 7. At the end of the 10-day treatment, wet weights of the adrenal glands of rhIL-1 beta-treated rats increased significantly compared with those of control rats. Plasma ACTH levels in rhIL-1 beta-treated rats at the sampling time continued to be elevated throughout the experimental period. Under the same experimental design, rhIL-1 alpha increased plasma ACTH levels at the sampling time without changes in adrenal weight or in the peptide contents investigated. The same amount of rhIL-2 had no effect on these measured variables during the 10-day treatment. These data indicate that the repetitive administration of IL-1 beta resulted in chronic effects in the hypothalamo pituitary-adrenal axis to increase the activities in these organs during the treatment and, moreover, IL-1 possibly has a positive direct effect on the CRH-containing cells in the adrenal glands.

Radiofrequency thermotherapy for malignant liver tumors.

Inoperable malignant liver tumors have been treated by radiofrequency hyperthermia at Kyoto University Hospital since 1983. In this study, clinical hyperthermia for malignant liver tumor was evaluated for 67 tumors in which we could measure intratumor temperatures. Of the 67 tumors, 41 were hepatocellular carcinomas (HCC), six cholangiocarcinomas, and 20 metastatic tumors. Cholangiocarcinoma and metastatic tumors were more susceptible to this treatment than HCC. Of the three types of HCC, higher intratumor temperatures were achieved in the diffuse type than in the nodular or massive types. The minimum tumor temperature of HCC stayed below 40 degrees C in 46% of cases, especially in larger tumors. The local response rates (complete remission plus partial remission/all) were 28% and 11% for HCC and non-HCC, respectively, for thermochemotherapy; 86% and 33%, for thermoradiotherapy; and 33% and 89%, for thermotherapy with embolization. No apparent relationship was observed between the intratumor temperatures and local response rate.

[Therapeutic effectiveness of vitamin D3 in patients with myelodysplastic syndromes, leukemias and myeloproliferative disorders].

We tried to treat 13 patients with myelodysplastic syndromes (MDS), leukemias and myeloproliferative disorders, with alfacalcidol for their hematological improvement. Eight of them had MDS, 2 acute leukemia (M3, M4), 1 chronic myelogenous leukemia and 2 primary myelofibrosis. All patients were untreated except for 3 patients (PASA, RAEB, AML-M4) who had been treated with mepitiostane, prednisolone and BH.AC-AMP regimen, respectively, prior to alfacalcidol therapy. All patients received alfacalcidol orally for at least one month. The dosage of alfacalcidol ranged from 0.25 to 10 micrograms/day, and the medicine was administrated intermittently when the dosage exceeded 6 micrograms/day to prevent hypercalcemia. The therapeutic effectiveness of alfacalcidol was evaluated according to a criteria by Koeffler (Cancer Treat Rep 69: 1399, 1985) with minor modifications. Three patients (PASA, RAEB, CMML) showed partial response, 3 (RAEB, RAEB in T, AML-M4) minor response and rest of the patients did not respond. The hematological improvement of 6 responders was transient (from 1 to 2 months), however, one patient (PASA) is still responding to alfacalcidol therapy (0.25 microgram/day) for over 12 months. The dysplastic features of hemopoietic cells in the bone marrow showed no noticeable change during the hematological improvement in these responders, suggesting the improvement was obtained as a result of alteration in the proliferation or differentiation of neoplastic clone. None of 13 patients developed hypercalcemia. One patient (AML-M4) became excitable on high dose alfacalcidol (10 micrograms/day). In conclusion, alfacalcidol therapy is effective in some patients with MDS or leukemias and appears worthy especially in the clinical state in which chemotherapy is not indicated.

Galanin stimulates the release of vasoactive intestinal polypeptide from perifused hypothalamic fragments in vitro and from periventricular structures into the cerebrospinal fluid in vivo in the rat.

Intracerebroventricular injection of galanin (2 micrograms/rat) raised plasma prolactin (PRL) levels in the rat, which was accompanied by an increase in immunoreactive vasoactive intestinal polypeptide (VIP) in the cerebrospinal fluid (CSF). Immunoreactive VIP release from superfused rat hypothalamic fragments in vitro was dose-relatedly stimulated by galanin (10(-7) and 10(-8) M). PRL release from superfused rat anterior pituitary cells was stimulated by TRH (10(-8) M) but not affected by galanin (10(-7) to 10(-5) M). These findings indicate that central galanin has a stimulating role in the release of hypothalamic VIP, which results in pituitary PRL secretion in the rat.

Central galanin stimulates pituitary prolactin secretion in rats: possible involvement of hypothalamic vasoactive intestinal polypeptide.

The effect of galanin, a newly identified neuropeptide, on pituitary prolactin (PRL) secretion was examined in the rat. Intracerebroventricular (i.c.v.) injection of all 5 doses of galanin (0.4, 1, 2, 5 and 10 micrograms/rat) raised plasma PRL levels in urethane-anesthetized rats. Galanin injection (2 micrograms/rat, i.c.v.) also increased plasma PRL levels in conscious rats. The intermediate dose of galanin (2 micrograms/rat, i.c.v.) produced a greater response in plasma PRL levels than either smaller or larger doses of galanin. Intravenous injection of galanin did not affect plasma PRL levels. Passive immunization with specific anti-vasoactive intestinal polypeptide (VIP) rabbit serum suppressed plasma PRL response to galanin (2 micrograms/rat, i.c.v.) in anesthetized rats. These findings indicate that central galanin has a stimulatory role in pituitary PRL secretion via the hypothalamus in the rat and that VIP may be involved in rat PRL release induced by galanin.

A case of Klinefelter's syndrome associated with hypothalamic-pituitary dysfunction caused by an intracranial germ cell tumor.

A 17 year-old boy was admitted to the hospital because of thirst, polyuria (5-61/day), delayed sexual development and muscle weakness. He appeared obese, had an eunuchoidal body habitus and was excessively tall. Chromosomal analysis revealed a 47XXY karyotype. Serum cortisol was 1.3 microgram/dl, LH, 10.4 mIU/ml, FSH, 2.0 mIU/ml, and testosterone, 10 ng/dl. Endocrinological dynamic tests indicated diabetes insipidus and hypopituitarism of a hypothalamic type. Brain CT disclosed the existence of a tumor shadow around the calcified pineal body, extending towards the suprasellar region. Replacement therapy with glucocorticoid and DDAVP was started. The patient complained of a headache and plasma AFP and hCG concentrations were 868 ng/ml and 68.6 IU/ml respectively. A hCG- and AFP- producing germ cell tumor was suspected and radiation therapy with 60Co was performed. Plasma AFP and hCG were decreased with significant clinical improvement. Soon after irradiation, he started to complain of a headache and had elevated AFP and hCG levels. Right hemiparesis and unconsciousness suddenly appeared and he died of left thalamic bleeding. This is the first case of Klinefelter's syndrome associated with intracranial germ cell tumor. Plasma testosterone levels fluctuated in parallel with the change in plasma hCG levels. This shows that the Leydig cells in this patient could respond to some extent to tumor-producing hCG.

Involvement of growth hormone (GH)-releasing factor in GH secretion induced by serotoninergic mechanisms in conscious rats.

A possible role of hypothalamic GH-releasing factor (GRF) in GH secretion induced by serotoninergic stimuli was investigated in conscious freely moving rats with a passive immunization technique. Either iv injection of 5-hydroxy-L-tryptophan (1 mg/100 g BW) or intracerebroventricular injection of serotonin (10 micrograms/rat) caused an increase in plasma GH in rats treated with normal rabbit serum (0.5 ml/rat, iv) 30 min previously. Injection of rabbit antiserum specific for rat GRF (0.5 ml/rat, iv, 30 min previously) blunted the plasma GH increase induced by 5-hydroxy-L-tryptophan or serotonin in these animals. These results suggest that GH secretion induced by serotoninergic mechanisms is mediated, at least in part, by hypothalamic GRF in the rat.

Stimulation by alpha-adrenergic mechanisms of the secretion of growth hormone-releasing factor (GRF) from perifused rat hypothalamus.

The possible involvement of adrenergic mechanisms in regulating the secretion of growth hormone (GH)-releasing factor (GRF) from the rat hypothalamus was examined in vitro with a perifusion system. A high potassium concentration (56 mM) stimulated GRF release from the hypothalamus. The infusion of clonidine (10(-4) M), an alpha 2-adrenergic stimulant, resulted in an increase in the spontaneous release of GRF. In the presence of propranolol (10(-5) M), a beta-adrenergic blocking agent, clonidine (10(-5) and 10(-4) M) stimulated GRF release more prominently in a dose-related manner, whereas propranolol (10(-5) and 10(-4) M) by itself did not affect the spontaneous GRF release. The stimulatory effect of clonidine (10(-4) M) on GRF release in the presence of propranolol was inhibited by yohimbine (10(-4) M), an alpha 2-adrenergic blocking agent. These findings suggest that alpha 2-adrenergic mechanisms play a role in stimulating GRF release from the hypothalamus in rats.

Stimulation by gastrin-releasing peptide, neurotensin and DN1417, a novel TRH analog, of dopamine and norepinephrine release from perifused rat hypothalamic fragments in vitro.

Both dopamine (DA) and norepinephrine (NE) release from perifused rat hypothalamic fragments was increased by depolarizing concentrations of potassium (K+, 20 mM and 56 mM) in a dose-related and Ca2+-dependent manner. DA and NE release induced by high K+ (20 mM) was further enhanced by gastrin-releasing peptide (GRP, 10(-5)-10(-6) M) and DN 1417 (10(-5) M), a thyrotropin-releasing hormone (TRH) analog. GRP (10(-5) M), neurotensin (NT, 10(-5) M) and DN 1417 (10(-5) M) also stimulated spontaneous release of DA and NE from the hypothalamus. These results suggest that GRP, NT and DN 1417 act at the hypothalamic catecholamine nerve terminals and stimulate the release of DA and NE in the rat.

Changes in the immunoreactivities of an opioid peptide leumorphin in the hypothalamus and anterior pituitary during the estrous cycle of the rat and their relation to sexual behavior.

Leumorphin, an opioid peptide whose functions are unknown, is found in mammalian brain and pituitary and stimulates lordosis behavior in estrogen-treated female rats. To elucidate the role of leumorphin in the physiological control of female sexual behavior, the levels of immunoreactive (ir) leumorphin as well as ir dynorphin (dynorphin A) were measured in the rat brain and pituitary during the estrous cycle. There was a clear variation of ir leumorphin in the hypothalamus and anterior pituitary during the estrous cycle. The levels of ir leumorphin in the hypothalamus and anterior pituitary on the afternoon of proestrus were significantly higher (P less than 0.01) than those on the afternoons of estrus and metestrus. The rise in the hypothalamic levels of ir leumorphin on the afternoon of proestrus was correlated with the receptivity of lordosis during the estrous cycle. Furthermore, there was a close correlation with ir dynorphin levels. These findings are in agreement with studies demonstrating a common precursor for leumorphin and dynorphin. Ir leumorphin in the hippocampus and neurointermediate pituitary did not change significantly during the estrous cycle. Because the leumorphin antiserum used recognizes rimorphin (dynorphin B) 1.78 times more than porcine leumorphin on a molar basis, high performance-gel permeation chromatography was done on pooled extracts of hypothalamus taken at proestrus and estrus. The peak in the leumorphin-like substance in the activation of sexual behavior is discussed.

Effect of 1,25-dihydroxyvitamin D3 on insulin secretion: direct or mediated?

In order to clarify the role of vitamin D (D) in regulating insulin secretion, we studied the effect of long term (10 days) and short term (3 days) supplementation with D and/or calcium (Ca) on insulin secretion from the isolated, perfused pancreas of D- and Ca-deficient rats. The influence of the nutritional state induced by D deficiency was also evaluated. The long term supplementation of either D, Ca, or both restored the body weight and improved insulin secretion induced by high glucose concentration to the same extent; thus, no significant difference in insulin secretion was found between the D-only-supplemented group and the Ca-only-supplemented group. When the insulin secretion was compared in D-deplete vs. D-replete rats given the same amount of Ca, insulin secretion was significantly higher in D-replete animals, although plasma Ca levels were also higher. In short term experiments, insulin release was significantly augmented to a similar extent in D- or Ca-replete rats as compared with D- and Ca-deficient rats, despite no significant change in body weight. In a separate experiment, the pancreas from D-deficient rats was perfused with or without 1,25-dihydroxyvitamin D3 [1,25-OH)2D3] to observe its acute effect on insulin release. The perfusion with 1,25-(OH)2D3 did not affect insulin release. This result suggests that impaired insulin secretion in D-deficient rats is caused by a decrease in Ca in the body fluid and possibly by the lack of D effect on the pancreas.

Further evidence that central neurotensin inhibits pituitary prolactin secretion by stimulating dopamine release from the hypothalamus.

Intracerebroventricular (icv) injection of neurotensin (NT) (2 micrograms/rat) suppressed prolactin (PRL) release induced by L-5-hydroxytryptophan (1 mg/100 g body wt, iv), prostaglandin E2(1 microgram/rat, icv), and FK33-824 (10 micrograms/100 g body wt, iv), a Met5-enkephalin analog, in urethane-anesthetized or conscious rats. In contrast, NT did not suppress elevated plasma PRL levels sustained by a large dose of domperidone (10 micrograms/100 g body wt, iv), a peripheral dopamine antagonist. In in vitro experiments, NT (10(-5) M) stimulated dopamine release from perifused rat hypothalamic fragments. These results suggest that central NT inhibits PRL secretion by stimulating dopamine release from the hypothalamus into hypophysical portal blood in the rat.

Stimulation by serotonin of immunoreactive peptide histidine isoleucine release from rat hypothalamus in vitro.

The effect of serotonin (5-HT) on the release of peptide histidine isoleucine-like immunoreactivity (PHI-LI) from rat hypothalamus was investigated in vitro with a perifusion system. A high potassium concentration (56 mM) stimulated PHI-LI release in a calcium-dependent manner. PHI-LI release was dose-relatedly stimulated by 5-HT (10(-6), 10(-5) and 10(-4) M). PHI-LI release induced by 5-HT (10(-5) M) was abolished by cyproheptadine (10(-4) M), a 5-HT antagonist. These results suggest that 5-HT has a stimulating effect on PHI release from the hypothalamus.

Involvement of peptide histidine isoleucine (PHI) in prolactin secretion induced by serotonin in rats.

The possible role of hypothalamic peptide histidine isoleucine (PHI) in prolactin (PRL) secretion induced by serotoninergic mechanisms was investigated in male rats using a passive immunization technique. Intracerebroventricular injection of serotonin (5HT, 10 micrograms/rat) raised plasma PRL levels both in urethane-anesthetized rats and in conscious rats pretreated with normal rabbit serum (0.5 ml/rat, iv, 30 min before). Plasma PRL responses to 5HT were blunted in these animals when they were pretreated with rabbit antiserum specific for PHI (0.5 ml/rat, iv, 30 min before) (mean +/- SE peak plasma PRL: anesthetized rats 271.3 +/- 38.3 ng/ml vs 150.0 +/- 12.6 ng/ml, p less than 0.01, conscious rats 54.3 +/- 6.8 ng/ml vs 30.7 +/- 4.1 ng/ml, p less than 0.025). These results suggest that hypothalamic PHI is involved, at least in part, in PRL secretion induced by central serotoninergic stimulation in the rat.

Effects of adrenalectomy and dexamethasone administration on the level of prepro-corticotropin-releasing factor messenger ribonucleic acid (mRNA) in the hypothalamus and adrenocorticotropin/beta-lipotropin precursor mRNA in the pituitary in rats.

RNA blot hybridization analysis with cloned rat CRF precursor (prepro-CRF) cDNA as a probe showed that prepro-CRF mRNA existed in rat hypothalamic and extrahypothalamic brain tissue, whereas it was undetectable in the pituitary and adrenal. To study the effect of glucocorticoid on the level of prepro-CRF mRNA in the hypothalmus and that of ACTH/beta-lipotropin (beta LPH) precursor mRNA in the pituitary, effects of adrenalectomy and dexamethasone administration were studied in rats. Adrenalectomy markedly raised mRNA coding for ACTH/beta LPH precursor in the anterior pituitary, but not in the neurointermediate pituitary lobe. Hypothalamic pre-pro-CRF mRNA increased only to 152% of the control value, 7 days after adrenalectomy. The administration of dexamethasone (200 micrograms/day for 7 days) started immediately after adrenalectomy lowered the ACTH/beta LPH precursor mRNA level in the anterior pituitary to 19% of the intact control value, whereas the level of prepro-CRF mRNA in the hypothalamus decreased only to 102%. These results suggest that glucocorticoids exert their feedback effect at the level of gene expression on both hypothalamic CRF neurons and pituitary corticotropes. Although the possibility that CRF neurons insensitive to glucocorticoid in the hypothalamus might blunt the change in the prepro-CRF mRNA could not be ruled out, it is also possible that the effect of glucocorticoids on the pituitary is dominant.

Cloning and sequence analysis of cDNA for rat corticotropin-releasing factor precursor.

DNA complementary to the rat hypothalamic mRNA coding for the corticotropin-releasing factor precursor (prepro-CRF) has been cloned by screening a cDNA library with a human genomic DNA probe. Nucleotide sequence analysis of the cloned cDNA has revealed that rat prepro-CRF consists of 187 amino acid residues including a putative signal peptide. The CRF and putative signal peptide regions are more highly conserved among rat, human and ovine prepro-CRF than is the cryptic portion.