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Volume 64, Issue 2
Displaying 1-7 of 7 articles from this issue
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Yasumasa IWASAKI, Yutaka OISO, Kensuke TAKATSUKI, Akio TOMITA, Kunikaz ...1988 Volume 64 Issue 2 Pages 69-77
Published: February 20, 1988
Released on J-STAGE: September 24, 2012
JOURNAL FREE ACCESSHypernatremia has occasionally been observed in patients with myotonic muscular. dystrophy (MyD). To elucidate the possibility of osmoregulatory dysfunction, we investigated hypothalamo-posterior pituitary function as well as serum electrolytes in eight patients with MyD.
Blood samples were obtained early in the morning after overnight dehydration. Renal function was estimated by blood urea nitrogen, serum creatinine and creatinine clearance. Posterior pituitary function was evaluated by direct measurement of plasma vasopressin (AVP) during a 5% hypertonic saline infusion. Plasma AVP concentrations were determined by sensitive radioimmunoassay. In five patients, circulating blood volume (CBV), plasma renin activity (PRA) and serum aldosterone (S-Aldo.) were also measured.
The mean serum sodium level (143.9 ± 1.7mEq/1 : Mean±SD) was significantly higher than in the controls (139.4 ± 2.2mEq/1). A 5% hypertonic saline infusion showed a subnormal increase in AVP and diminished thirst, despite sufficient elevation of plasma osmolality, in all patients as compared with healthy adults. Renal function was intact. Biochemical evidence of dehydration, estimated by PRA, S-Aldo and CBV, was unremarkable in four of the five patients.
These findings suggest that patients with MyD have neurogenic disorders of osmoregulation in addition to previously reported endocrine abnormalities. Impaired AVP secretion in response to osmotic stimuli and reduced thirst might be responsible for such failure.View full abstractDownload PDF (909K) -
Naoki HATTORI, Yuzuru KATO, Yoshio MURAKAMI, Hiroyuki KOSHIYAMA, Tatsu ...1988 Volume 64 Issue 2 Pages 78-92
Published: February 20, 1988
Released on J-STAGE: September 24, 2012
JOURNAL FREE ACCESSA highly sensitive enzyme immunoassay (EIA) for measurement of urine hGH was set up by a modification of the method of Hashida and Ishikawa, which was a sandwich enzyme immunoassay using anti-hGH antibody coated polystyrene balls and anti-hGH antibody-peroxidase conjugate. Anti-hGH serum was obtained in rabbits by subcutaneous injections of hGH emulsified in complete Freund's adjuvant. In order to reduce non-specific binding to the solid phase, anti-hGH IgG was precipitated from rabbit serum followed by digestion to F (ab′) 2 and affinity purification. Fab′-peroxidase conjugate was produced by maleimide method.
The assay procedure was as follows.
1. 100μl of urine samples or hGH standard were incubated with anti-hGH IgG coated polystyrene balls.
2. Polystyrene balls were then incubated with Fab′-peroxidase conjugate. Polystyrene balls were carefully washed three times in saline after incubation with Fab′-peroxidase conjugate, which reduced contamination and non-specific binding.
3. Peroxidase activity bound to the balls was assayed by enzyme reaction using 3 (p-hydroxyphenyl) propionic acid as a substrate, and fluorescence intensity was measured by a spectrofluorophotometer (Shimadzu RF-540). Reducing the energy of excitation by setting the slit width of the spectrofluorophotometer at 2nm made it possible to gain stable fluorescence.
The minimum detectable quantity of hGH was 30fg/tube in the assay, so that the detection limit was 0.3pg/ml when 100μl of urine samples were used. Coefficient of intra and inter-assay variation was 6.0% and 8.6%, respectively. The recovery was 98.8± 2.8 (±SE) on average. Multiple dilution of acromegalic urine and urine after insulin injection produced dose-response curves parallel to those of the standards.
Urine hGH levels in acromegalic patients were significantly greater than those in normal subjects.
These findings indicate that sensitive EIA of urine hGH is potentially useful for evaluating the pituitary function.View full abstractDownload PDF (1131K) -
Autonomic Function and Plasma CatecholaminesNoriyuki KIKUCHI, Masataka NANNO, Hiroyuki KURAHACHI, Teruya YOSHIMI1988 Volume 64 Issue 2 Pages 93-108
Published: February 20, 1988
Released on J-STAGE: September 24, 2012
JOURNAL FREE ACCESSAutonomic neuropathy is one of the complications of diabetes. Recently, several authors reported that measuring R-R interval variation of ECG is a noninvasive and useful method for testing parasympathetic function. However, there were few reports about sympathetic function in diabetics.
In order to evaluate sympathetic function in diabetics quantitatively, we studied the responses of plasma norepinephrine (NE), epinephrine (E) and related factors after 60 min bed rest and sequentially during 10 min of upright posture and 5 min handgrip while still upright. We also studied the responses of NE and E during 5 min smoking in supine position. Subjects were divided into four age-matched groups. These were 15 normal subjects (Group I), 20 diabetics without complications (Group II), 20 diabetics with peripheral neuropathy but no autonomic symptoms (Group III) and 15 diabetics with autonomic symptoms (Group IV). We also studied R-R interval variation (CV : Coefficient of Variation) as a parameter of parasympathetic function and compared this with sympathetic function.
Upon standing, blood pressure (BP) dropped precipitously in Group IV, whereas no significant changes were observed in the other three groups. Heart rate (HR) increased in Groups I and II, but not in Groups III and IV. During handgrip, BP and HR did not change significantly in all groups. Basal NE levels in Group IV were significantly smaller than those in Group I. NE responses to both standing and handgrip stimuli were markedly reduced in Group IV and, even in Group III, increments were significantly smaller than those in Groups I and II. Basal E levels did not differ, and significant changes were not observed after standing and handgrip in all groups. Both plasma renin activity (PRA) and plasma aldosterone concentrations (PAC) in Groups III and IV were lower than those in Groups I and II at rest and standing.
After smoking, both BP and HR increased significantly in Groups I, II and III, whereas no changes were observed in Group IV. Both NE and E responses were markedly reduced in Group IV and, even in Group III, responses were significantly smaller than those in Groups I and II.
CV in Groups III and IV were significantly smaller than those in Groups I and II. In diabetics, CV was strongly correlated with NE increments after standing (r=0.78, p<0.01). Also, CV was correlated with both NE and E increments after smoking (r=0.71 (NE), r=0.82 (E), p<0.01). CV was weakly correlated with PRA increments after standing (r=0.44, p<0.05).
These results suggested : 1) Measuring NE and E after standing, handgrip and smoking is a sensitive, specific and useful method in evaluating sympathetic neuropathy in diabetics. 2) The sympathoadrenomedullary function was already impaired before autonomic symptoms appear. 3) There is parallel dysfunction between the sympathetic and parasympathetic nervous systems.View full abstractDownload PDF (1451K) -
Masayuki AOKI, Shohei KAGAWA, Takehira YAMAMURA, Akira MATSUOKA, Jyoji ...1988 Volume 64 Issue 2 Pages 109-118
Published: February 20, 1988
Released on J-STAGE: September 24, 2012
JOURNAL FREE ACCESSIn the present study, the culture system for preparing monolayer islet cells of the neonatal rat was applied and modified for use with the pancreas of the adult rat. In this procedure, whole pancreatic tissues were enzymatically dispersed and then cultured for 30 days in TCM 199 medium with either 5.5mM glucose or 5.5mM glucose plus 1mM 2-deoxy-2-fluoroglucose. Under culture conditions without 2-deoxy-2-fluoroglucose, the responsiveness of B cells was totally abolished by day 20 of culture. The addition of 2-deoxy-2-fluoroglucose destroyed fibroblasts selectively in a period of 20 days, yielding the monolayers mostly consisting of islet cells, and the morphological characteristics were well preserved at the end of the culture study period.
After culture for 20 days in medium with 2-deoxy-2-fluoroglucose, insulin secretion was raised in a dose-dependent fashion due to the increasing concentrations of glucose, leucine and 2-ketoisocaproate. The dose-response curve for insulin secretion evoked by glucose was sigmoid with a Km of 7mM glucose, and the secretion threshold was observed at a concentration of between 2.8 and 5.5mM glucose. The ratios of the maximum level to the basal were 6, 5 and 3 respectively for glucose, leucine and 2-ketoisocaproate. The secretory competence was preserved in the B cells on day 30 as well.
Addition of epinephrine or clonidine inhibited the glucose-induced insulin secretion dose-dependently. At a concentration of 10-7M, both drugs produced an 80% drop in insulin secretion evoked by glucose. The inhibitory effect of epinephrine or clonidine was reversed by 3 × 10-5M yohimbine or 10-5M phentolamine, whereas 10-5M propranolol had little or no effect and alpha adrenergic blockade of prazosin (5 × 10-5M) was weak as compared to that of yohimbine. At a high concentration (10-5M) of phenylephrine, a marked drop of insulin secretion was observed.
In summary, the present culture system facilitates the establishment of monolayers of adult rat pancreas that consist mostly of islet cells. In addition, it is certain that the response of the B cells in 2-deoxy-2-fluoroglucose to nutrient secretagogues and the adrenergic modulation of insulin secretion are well preserved for a long-term culture period of 30 days. These preparations may provide a useful tool not only for the in vitro study of the B-cell function, but also for use in implantation resource.View full abstractDownload PDF (1333K) -
Masateru HORIMOTO, Mitsushige NISHIKAWA1988 Volume 64 Issue 2 Pages 119-127
Published: February 20, 1988
Released on J-STAGE: September 24, 2012
JOURNAL FREE ACCESSSerum thyroid hormone and TSH concentrations were determined in patients with nonthyroidal illnesses (NTI) including 26 patients with chronic renal failure on hemodialysis, 15 patients with end-stage malignancy and 21 normal controls. Serum TSH levels were measured by a highly sensitive immunoradiometric assay Kit (RIA-gnost hTSH) and free-T4 levels were determined not only by both back-titration method and T4 derivative method, but also by magnesium precipitation method using equilibrium dialysis. TSH levels in normal controls ranged from 0.6 to 2.4μU/ml. In 40 of 41 patients with NTI, serum T3 levels were less than 100ng/dl, and 37 patients had diminished T3 levels, as compared with those in normal controls. Moreover, serum T4 levels were also diminished in 19 patients with NTI, and T4 levels related significantly to serum TBG levels in all patients with NTI (r=+0.76, p<;0.01), suggesting that the diminution of T4 levels in patients with NTI can be explained partly by decreased TBG levels. However, it was remarkable that free T4 levels in serum, determined by RIA kits, were decreased in approximately half of the patients with NTI. Free T4 levels by the magnesium precipitation method were still lower in these patients when compared with those in normal controls. Serum TSH levels in patients with NTI showed a wide scattering from below to above the normal range, and the mean value was not significantly different from that in normal controls. No significant relations were found between TSH levels and T4, T3, or free T4 levels in patients with NTI. TSH increments at 30 minutes after TRH infusion in the patients were significantly lower than those in normal controls. The mean value of serum cortisol concentrations in patients with NTI was not significantly different from that in normal controls, and there was no correlation between serum cortisol concentrations and TSH increments at 30 minutes after TRH infusion. Moreover, values for free T4/log TSH, which could be used as the index of the thyroid response to TSH, were significantly lower than those in normal controls. The present findings suggest that the responsiveness of the thyroid to TSH as well as that of the pituitary to TRH in patients with NTI is deteriorated, and these may be associated with the diminution of free T4 in patients with NTI.View full abstractDownload PDF (859K) -
Mitsutoshi IWASHITA, Yoshihiko TAKEDA, Shoichi SAKAMOTO1988 Volume 64 Issue 2 Pages 128-137
Published: February 20, 1988
Released on J-STAGE: September 24, 2012
JOURNAL FREE ACCESSGnRH stimulates LH release from pituitary cells, and this process is calcium dependent. On the other hand, phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA), a potent activator of calcium- and phospholipid-dependent protein kinase (protein kinase C), stimulates luteinizing hormone (LH) release from rat pituitary cells.
To investigate the involvement of the calcium dependent process in LH release by TPA, the effects of calcium channel antagonists, verapamil and nifedipine, on TPA-mediated LH release were compared with those of a GnRH superagonist, [D-Ala6] des-Gly10-GnRH N-ethyramide (GnRHa) in cultured pituitary cells. Furthermore, pituitary cells saturated with 45 Ca2+ were stimulated by GnRHa or TPA and calcium mobilization after the stimuli were monitored.
The pituitary cells from adult male rats were dispersed by tripsin and cultured for 3 days. Cultured pituitary cells were incubated with GnRHa or TPA in the presence of increasing concentrations of verapamil or nifedipine for 3hrs, and LH released into medium was measured by RIA for rat LH. For 45 Ca2+ experiment, 3 day-cultured pituitary cells were saturated with 45 Ca2+ (106 cells/1μCi/100μl) and incubated with secretgogues for the indicated times. Incubations were terminated by filtration, and the radioactivity on the filter was measured by a beta-counter.
LH release was stimulated by 0.1nM TPA, and the maximum response at 10nM TPA was 50% of the LH response to GnRHa. A23187 also stimulated LH release in relatively high concentrations (10-5- 10-4M), and no additive stimulatory effect was observed when a half-maximal dose of TPA (10-9M) was added with increasing concentrations of A23187. Verapamil partially inhibited both GnRHa- and TPA-stimulated LH release, and a similar inhibitory effect on LH release was observed when nifedipine was incubated with GnRHa or TPA, although high concentrations (10-5-10-4M) of nifedipine stimulated LH release induced by GnRHa and TPA.
GnRHa and TPA stimulated 45 Ca2+influx into the cells, and its peak was observed 15 and 30 seconds after stimulation, respectively, while GnRH antagonist did not mobilize 45 Ca2+ until 120 seconds after stimulation. These results suggest that TPA-stimulated LH release from pituitary cells involves a calcium dependent process as does GnRH-stimulated LH release.View full abstractDownload PDF (1058K) -
Hisamoto KURODA1988 Volume 64 Issue 2 Pages 138-153
Published: February 20, 1988
Released on J-STAGE: September 24, 2012
JOURNAL FREE ACCESSThe effects of phorbol esters and various growth factors on prostaglandin E2 (PGE2) synthesis by cultured porcine thyroid cells were examined. Phorbol 12-myristate, 13-acetate (PMA), phorbol 12, 13-dibutylate, epidermal growth factor (EGF) and fetal bovine serum (FBS) stimulated PGE2 production in a dose-related fashion. PMA stimulated PGE2 production over fifty-fold with a dose of 10-7M compared with controls during 4h incubation. EGF (10-7) also stimulated it about ten-fold. The ED50 values of PMA and EGF were around 1 × 10-9M and 5 × 10-10M respectively. FBS also clearly stimulated it about ten-fold with a concentration of 20%. Thyroid stimulating hormone (TSH) and inactive phorbol, however, did not stimulate PGE2 production. The release of radioactivity from [3H] arachidonic acid (A.A.) prelabeled cells was also stimulated by PMA, EGF and FBS. These results indicate that PMA, EGF and FBS are potent stimulators of PGE2 production, associated with the activity to stimulate A.A. release in thyroid cells.
Secondly, in order to elucidate some mechanism (s) of the stimulation by these factors of PGE2 production, effects of various agents such as EGTA, Ca2+-ionophore (A-23187), cycloheximide, hydrocortisone and para-bromophenacylbromide (p-BPB) were examined on PGE2 production and/or [3H] -A.A. release by thyroid cells. Chelation of Ca2+ by EGTA, treatment of cells with hydrocortisone, an inducer of lipocortin which may inhibit phospholipase A2 (PLA2) activity, or inhibition of PLA2 activity by p-BPB clearly inhibited PGE2 production and/or [3H] -A.A. release induced by PMA, EGF and FBS. Cycloheximide also blocked the PMA- or EGF-stimulated [3H] -A.A. release and PGE2 synthesis. On the other hand, Ca2+-ionophore (A-23187) potentiated PMA- or EGF-stimulation. Furthermore, the Ca2+-ionophore alone-induced stimulation was clearly inhibited by the treatment with hydrocortisone or p-BPB. These data indicate that an increase of Ca2+ in cytosol is important to PLA2 activation, and also that PLA2 may be activated by PMA, EGF and FBS in thyroid cells. The inhibition of PMA- or EGF-induced [3H] -A.A. release and PGE2 synthesis by cycloheximide suggests that both factors-induced stimulation may be sensitive to regulation by short-lived protein (s).
Finally, other growth factors such as insulin (0.1 -10μg/ml), insulin like growth factor I (IGF-1) (10-9 10-7M) and interleukin la (1 - 100ng/ml) also stimulated PGE2 production by thyroid cells in the presence or absence of EGF (10-10-10-9M). However, transferrin (0.5- 50μg/ml), somatostatin (1 - 100ng/ml), interleukin 2 (1-100ng/ml) and bombesin (10-7 10-5M) did not stimulate PGE2 production.View full abstractDownload PDF (1348K)
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