Kazuharu Murakami

Corticotropin-releasing factor (CRF)-like immunoreactivity in the adrenal medulla

Bovine adrenal medulla extract prepared by acid-acetone or acid methanol extraction showed two peaks of CRF-like immunoreactivity on Sephadex G-50 chromatography. One eluted near the void volume and another (low molecular weight CRF-like immunoreactivity) eluted slightly before arginine vasopressin (AVP), while most of the immunoreactivity in bovine hypothalamus coeluted with synthetic ovine CRF. When low molecular weight CRF fractions were chromatographed by reversed phase high performance liquid chromatography, three CRF-like immunoreactive peaks appeared. The first peak appeared near TRH, the second one eluted near AVP and the last one eluted near somatostatin. These three peaks of immunoreactivity showed ACTH releasing bioactivity in rat pituitary cells cultures. Therefore, the adrenal medulla-CRF-like substances might be tissue-CRF which may play a role to stimulate ACTH release in the severe stress conditions.

The Role of Angiotensin II in the Regulation of ACTH Secretiona

1. In rats and in at least some other species, IV and IVT AII stimulate ACTH secretion. 2. Although AII increases ACTH secretion by a direct action on pituitary cells in vitro, it appears to act instead by stimulating CRH secretion in vivo. 3. The CRH stimulating effect of circulating AII is mediated by an action of the AII on one or more of the circumventricular organs of the brain. 4. AII administered into the cerebral ventricles and, presumably, centrally generated AII, increase CRH secretion by acting on AII receptors inside the blood-brain barrier as well as in the circumventricular organs. 5. A possible role for the renin-angiotensin system in mediating the increase in ACTH secretion produced by stress has been suggested, and the degree of involvement may vary from one stress to another. However, as yet we have been unable to obtain any evidence that either circulating AII or centrally generated AII plays a role in the increase in ACTH secretion produced by ether stress in rats and surgical stress in dogs.

A specific radioimmunoassay for Cortricotropin Releasing Factor (CRF) using synthetic ovine CRF

This newly developed specific radioimmunoassay for corticotropin releasing factor (CRF) had a sensitivity range of 25 pg/tube to 4 ng/tube. Intra and interassay coefficient of variation were 4.6% and 9.8%, respectively. Rat median eminence extracts showed a parallel dose response curve with synthetic ovine CRF and a significant cross reaction was not evident with other tested neuropeptides. The highest mean levels of CRF were found in the median eminence (6.61 ng/mg protein). Considerable amounts of CRF were found in the arcuate nucleus, paraventricular nucleus, dorsomedial nucleus, suprachiasmatic nucleus and ventromedial nucleus. The immunoreactive CRF of the rat medial basal hypothalamus coeluted with bioassayable CRF and with iodinated CRF on Sephadex G-75 chromatography. The results indicate that rat hypothalamus contains a CRF similar to ovine CRF.

Reduction in brain immunoreactive corticotropin-releasing factor (CRF) in spontaneously hypertensive rats

The brain CRF concentration of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) was examined by rat CRF radioimmunoassay. Anti-CRF serum was developed by immunizing rabbits with synthetic rat CRF. Synthetic rat CRF was also used as tracer and standard. The displacement of 125I-rat CRF by serially diluted extracts of male Wistar rats hypothalamus, thalamus, midbrain, pons, medulla oblongata, cerebral cortex, cerebellum and neurointermediate lobe was parallel to the displacement of synthetic rat CRF. In both WKY and SHR the highest levels of CRF immunoreactivity were shown by the hypothalamus and neuro-intermediate lobe, and considerable CRF immunoreactivity was also detected in other brain regions. The CRF immunoreactivity in the hypothalamus, neurointermediate lobe, midbrain, medulla oblongata and cerebral cortex was significantly reduced in SHR and it may suggest that CRF abnormality may be implicated in the reported abnormalities in the pituitary-adrenal axis, autonomic response and behavior of SHR.

Interaction of synthetic ovine corticotropin releasing factor and arginine vasopressin on in vitro ACTH release by the anterior pituitary of rats.

Synthetic ovine corticotropin releasing factor (CRF) at 0.03-30 ng/ml evoked a significant linear release of ACTH in pituitary monolayer cell cultures. Arginine vasopressin (AVP) at 3-30 ng/ml induced a slight ACTH release in the same culture system. CRF at 100 ng/ml greatly increased the medium ACTH with intracellular ACTH remaining relatively constant. In perifused pituitary quarters, both AVP and CRF evoked significant release of ACTH. When AVP was added in combination with synthetic ovine CRF, an additive or synergistic effect was observed on the CRF-induced ACTH release in both in vitro systems. Preincubation of cultured pituitary cells with synthetic ovine CRF for 1 h did not have a significant effect on the subsequent AVP-induced ACTH release. However, when cells were preincubated for 6 h with CRF at 1 and 100 ng/ml, the AVP-induced ACTH release was significantly increased, but preincubation with AVP suppressed both the basal and AVP-induced ACTH release. These results suggest that synthetic ovine CRF stimulates not only the release of ACTH but also the synthesis of ACTH and that AVP can evoke significant ACTH release from corticotrophs which have enough releasable ACTH or substances needed for ACTH release under the influence of CRF.

Calmodulin inhibitors decrease the CRF-and AVP-induced ACTH release in vitro: interaction of calcium-calmodulin and the cyclic AMP system.

The effect of N-(6-aminohexyl)-5-chloro-naphthalene-1-sulfomide (W-7) and trifluoperazine (TFP) was examined on ACTH release from cultured rat anterior pituitary cells and pituitary halves. These drugs significantly inhibited the ACTH release induced by synthetic ovine corticotropin-releasing factor (CRF) in a dose-related manner. In pituitary halves, arginine vasopressin (AVP) at 10 and 100 ng/ml showed almost the same ACTH-releasing activity as CRF at the same concentrations. W-7 and TFP inhibited the CRF-and AVP-induced ACTH release from pituitary halves. The cyclic AMP levels in the pituitary halves were significantly increased by CRF, but not AVP. Although W-7 inhibited CRF-induced ACTH release, it did not have an effect in cyclic AMP accumulation. These results suggest that CRF exerts ACTH-releasing activity through both the calcium-calmodulin system and cyclic AMP system and that AVP stimulates ACTH release mainly through the calcium-calmodulin system.

Effect of proteinase inhibitor camostat mesilate on nephrotic syndrome with diabetic nephropathy.

The effect of serine protease inhibitor Camostat Mesilate on nephrotic syndrome with diabetic nephropathy was evaluated. Eight patients with nephrotic syndrome associated with diabetic nephropathy were orally administered 600 mg of Camostat Mesilate per day. Three patients showed a reduction of urinary protein excretion promptly at 4 weeks. Serum protein and degree of edema improved significantly at 4 weeks. Camostat Mesilate had no effect on renal function assessed by creatinine clearance. During the observation period there were no significant changes in blood pressure, level of blood sugar, or HbA1c. Camostat Mesilate would be beneficial for the treatment of diabetic nephropathy.

Characterization of Rat Hypothalamic Corticotropin-Releasing Factor by Reversed-Phase, High-Performance Liquid Chromatography with Synthetic Ovine Corticotropin-Releasing Factor as a Marker

Reversed-phase, high-performance liquid chromatography (HPLC) was carried out to characterize rat hypothalamic corticotropin-releasing factor (CRF) using synthetic ovine CRF as a marker. Samples were injected onto a stainless steel column (4 X 250 mm) packed with Hitachi gel 3053. The column was eluted using a gradient elution of increasing acetonitrile concentration, in a mixture of NaCl-HCl at a flow rate of 1.0 ml/min, monitoring the column effluent at 220 nm with an UV detector. Fractions eluted every 1-2 min were collected and lyophilized for subsequent CRF bioassay and radioimmunoassay. When various neuropeptide mixtures including synthetic ovine CRF were injected onto the column, synthetic ovine CRF was separated from the other neuropeptides with a gradient of 0-60% acetonitrile in 0.1 M NaCl-0.01 N HCl or 0-08% acetonitrile in 0.05 M NaCl-0.01 N HCl. The median eminence extracts showed two main peaks of CRF bioactivity on HPLC. One (small CRF) coeluted with arginine vasopressin and oxytocin markers, and the other (big CRF) appeared near the position of synthetic CRF and was divided into two peaks. One coeluted with synthetic ovine CRF and the second eluted after synthetic CRF, showing high CRF activity. Three or four peaks of CRF immunoreactivity appeared on HPLC and the main peak appeared after synthetic ovine CRF marker. Our results suggest that rat CRF is different from ovine CRF, and the total lipophilicity of amino acid residues of rat CRF may be higher than that of ovine CRF.

Corticotrophin‐Releasing Factor Antagonist [alpha helical CRF(9–41)] Blocks Central Noradrenaline‐lnduced ACTH Secretion

Plasma ACTH increased after an intra‐third ventricular administration of noradrenaline (NA). An iv corticotrophin‐releasing factor (CRF) antagonist [alpha‐helical CRF(9–41)] injection did not affect ACTH secretion by itself, whereas it significantly reduced NA‐induced ACTH secretion. These results suggest that NA centrally stimulated ACTH secretion and that endogenous CRF is involved in this ACTH secretion.

Myeloma kidney associated with acute renal failure after pneumonia.

骨髄腫腎に肺炎を契機に発症した急性腎不全の1例を経験し, 治療により血液透析より離脱し得たので報告する.症例は74歳, 男性. 肺炎に続発して無尿となり補液や利尿剤などの投与を受けた. しかし急性腎不全がさらに進行したため血液透析を開始した. 骨髄穿刺, 血清および尿中免疫電気泳動などで多発性骨髄腫と診断した. 腎機能の回復が遅れていたため腎生検を施行し骨髄腫腎と診断した. そこで化学療法 (MP療法) を開始したところ, 腎機能が次第に回復し血液透析より離脱し得た.多発性骨髄腫の急性腎不全合併例には, 脱水, 感染症, その治療に対する抗生剤, 高カルシウム血症, 造影剤などの誘因に対する処置や, 血液透析や血漿交換などの適切な血液浄化法の選択, および多発性骨髄腫に対する化学療法など, 積極的な治療が重要であると考えられた.