Hypercholesterolemia accelerates bone loss in postmenopausal women.

OBJECTIVES: To clarify the effect of lipid profiles on postmenopausal bone loss using a longitudinal method and to determine whether cytokines are involved in bone loss. METHODS: The subjects were Japanese residents participating in the Iwaki Health Promotion Projects. Women with one or more of the following factors were excluded: a history of surgical menopause, current or past users of bisphosphonates or current user of other drugs known to influence bone and lipid metabolism, and current medication for diabetes or hypertension. Consequently, 99 postmenopausal women (61.2 ± 7.7 years old) and 85 premenopausal women (41.2 ± 8.6 years old) were selected for this study. The osteo-sono-assessment index (OSI) of the left calcaneal bone was obtained twice at 1-year intervals and the annual percentage change in OSI was calculated. Serum total cholesterol, high and low density lipoprotein cholesterol, triglycerides, homocysteine and cytokines such as adipocytokines, interleukins and tumor necrosis factor-α were measured. Postmenopausal women were grouped into three groups according to their basal cholesterol level, and the relationship between basal cholesterol level and annual change in OSI was studied. RESULTS: The annual percentage change in OSI in postmenopausal women with a serum total cholesterol level ≥240 mg/dl was significantly higher compared to those with a normal total cholesterol level, suggesting that hypercholesterolemia accelerates postmenopausal bone loss. No significant differences were seen in any of the cytokines that presumably cause bone resorption. CONCLUSION: These results showed that hypercholesterolemia has an inverse effect on bone loss independent of cytokines presumed to mediate bone loss.

Hypothermic preservation of rat hearts using antifreeze glycoprotein.

Antifreeze proteins are an effective additive for low-temperature preservation of solid organs. Here, we compared static hypothermic preservation with and without antifreeze glycoprotein (AFGP), followed by nonfreezing cryopreservation of rat hearts. The heart was surgically extracted and immersed in one of the cardioplegia solutions after cardiac arrest. Control rat hearts (n=6) were immersed in University of Wisconsin (UW) solution whereas AFGP-treated hearts (AFGP group) (n=6) were immersed in UW solution containing 500 ?g/ml AFGP. After static hypothermic preservation, a Langendorff apparatus was used to reperfuse the coronary arteries with oxygenated Krebs-Henseleit solution. After 30, 60, 90, and 120 min, the heart rate (HR), coronary flow (CF), cardiac contractile force (max dP/dt), and cardiac diastolic force (min dP/dt) were measured. Tissue water content (TWC) and tissue adenosine triphosphate (ATP) levels in the reperfused preserved hearts were also assessed. All the parameters were compared between the control and AFGP groups. Compared with the control group, the AFGP group had significantly (p<0.05) higher values of the following parameters: HR at 60, 90, and 120 min; CF at all four time points; max dP/dt at 90 min; min dP/dt at 90 and 120 min; and tissue ATP levels at 120 min. TWC did not differ significantly between the groups. The higher HR, CF, max dP/dt, min dP/dt, and tissue ATP levels in the AFGP compared with those in control hearts suggested that AFGP conferred superior hemodynamic and metabolic functions. Thus, AFGP might be a useful additive for the static/nonfreezing hypothermic preservation of hearts.

Serum high-molecular weight adiponectin is related to early postprandial glycemic increases and gastric emptying in patients with type 2 diabetes mellitus.

BACKGROUND: Postprandial hyperglycemia and hyperlipidemia are frequently associated with type 2 diabetes mellitus. The aim of the present study is to investigate the clinical determinants of postprandial glycemia and lipemia, especially serum high-molecular weight adiponectin. METHODS: Twenty-seven diabetic patients treated with diet alone and 13 healthy volunteers took liquid test meal containing 53 g carbohydrate and 47 g lipid, dosed with nonradioactive isotope (13)C-acetate. Venous blood and breath samples were obtained for 180 min after the meal. Gastric emptying was evaluated by peak excretion time of (13)CO(2) in the breath samples. Delayed gastric emptying was defined as peak excretion time > 2.5 h (mean + 2 SD in the healthy volunteers). RESULTS: Diabetic patients showed delayed insulin secretion, postprandial hyperglycemia and hyperlipidemia compared with control. Postprandial glycemic increases significantly correlated with enhanced gastric emptying. Serum high-molecular weight adiponectin correlated with postprandial glycemic increases at 30 and 60 min after meal (r = 0.42, p < 0.05; r = 0.37, p < 0.05, respectively). Serum high-molecular weight adiponectin also correlated with gastric emptying (versus peak excretion time r = - 0.58, p < 0.05). In addition, diabetic patients with delayed gastric emptying showed the suppressed postprandial glycemia with lower serum high-molecular weight adiponectin than those with normal gastric emptying. On the other hand, postprandial increases in serum triglyceride were not related to serum high-molecular weight adiponectin or gastric emptying, but significantly related to liver function test (serum transaminases) and body mass index. CONCLUSIONS: Early postprandial glycemic increases were related to elevated serum high-molecular weight adiponectin, which might be associated with enhanced gastric emptying.

Altered postural regulation of foot skin oxygenation and blood flow in patients with type 2 diabetes mellitus.

AIMS: Although skin oxygenation is an important factor in the development and healing of foot ulcers, its regulation was not fully understood. We studied changes in foot skin oxygenation and blood flow during postural changes in patients with type 2 diabetes mellitus. METHODS: Skin oxygenation was measured using transcutaneous oxygen pressure (TcPO(2)) and skin blood flow by laser Doppler flowmetry in 40 patients with type 2 diabetes mellitus without evidence of peripheral arterial disease and 13 healthy control subjects. RESULTS: TcPO(2) in the supine position was significantly lower in patients with type 2 diabetes mellitus compared with control, although skin blood flow was not different. In the sitting position, TcPO(2) significantly increased in control and diabetic patients. The postural change-related increase in TcPO(2) was significantly enhanced in diabetic patients. On the other hand, skin blood blow significantly decreased in the sitting position from the supine position in control subjects but remained stable in diabetic patients. Orthostatic drop in systolic blood pressure correlated negatively with TcPO(2) in the supine position while correlated positively with %change in TcPO(2) and blood flow by postural changes. CONCLUSIONS: The present study demonstrated the dissociated regulation of skin oxygenation and blood flow in response to leg dependency. Impaired postural vasoconstriction was associated with altered regulation of skin oxygenation probably due to sympathetic vascular dysfunction in diabetic patients.

Heparin inhibits the accumulation of re-esterified cholesterol in macrophages loaded with acetylated low-density lipoprotein.

Heparin enhances the endocytosis of low density lipoprotein (LDL) in macrophages via a formation of complex with LDL. The direct effect of heparin on the metabolism of cholesterol in macrophages has not been elucidated. We therefore evaluated the effects of heparin on the accumulation and reesterification of cholesterol in cultured macrophages. We used acetylated LDL (acetyl-LDL), which lacks an affinity for heparin. Rat peritoneal macrophages induced with thioglycollate were incubated with 100 micrograms of acetyl-LDL for 14 h. Heparin significantly inhibited the accumulation of total and esterified cholesterol but did not affect the binding of 125I-labeled acetyl-LDL to macrophages or its cellular degradation. Heparin at concentration above 5 micrograms/ml inhibited the incorporation of [3H]oleate into cholesteryl oleate in macrophages. Heparin significantly inhibited the acyl CoA:cholesterol acyl transferase (ACAT) activity of macrophages by 68%. Data suggest that heparin inhibits the accumulation and reesterification of cholesterol in macrophages loaded with acetyl-LDL. Heparin-like proteoglycans may thus protect the macrophages against the excessive accumulation of esterified cholesterol.

Peroxisome proliferator-activated receptor gamma1 (PPARgamma1) expresses in rat mesangial cells and PPARgamma agonists modulate its differentiation.

Thiazolidinediones, synthetic ligands of peroxisome proliferator-activated receptor gamma (PPARgamma), are reported to have direct beneficial effects on diabetic nephropathy without lowering blood glucose levels in human and rat. We hypothesized these effects of thiazolidinediones might be derived from PPARgamma activation of kidney cells, and we examined the expression of PPARgamma and the effect of PPARgamma agonists, troglitazone and 15-deoxy-delta-prostaglandin J2 (15d-PGJ2), on the proliferation and differentiation in rat mesangial cells. A single band of mRNA of PPARgamma with a predicted size was detected in reverse transcription-polymerase chain reaction products (RT-PCR) using established PCR probes of PPARgamma. PPARgamma protein in rat mesangial cells was identified as PPARgamma1 by a Western blot. In a gel mobility shift assay to determine a binding activity of PPARgamma, the nuclear protein from rat mesangial cells bound to a (32)P-labeled oligonucleotide probe, including PPAR response elements. A synthetic and a natural ligand of PPARgamma, troglitazone and 15d-PGJ2, decreased thymidine incorporation in a dose dependent manner. After 7 days incubation with troglitazone and 15d-PGJ2, alpha-smooth muscle actin expression, a marker of mesangial cell de-differentiation, was decreased significantly compared to that of control. These results indicate that PPARgamma1 is expressing in rat mesangial cells, and PPARgamma1 activation with its agonists modulates the proliferation and differentiation of cultured rat mesangial cells.

Normalization of glucose entry under the high glucose condition by phlorizin attenuates the high glucose-induced morphological and functional changes of cultured bovine retinal pericytes.

We previously reported that sodium-dependent glucose uptake is present in bovine retinal pericytes and that phlorizin normalizes its glucose consumption under high glucose conditions. To clarify the effect of phlorizin on morphological and functional change of retinal pericytes under high glucose conditions, retinal pericytes were incubated in media with 5 mM glucose, 30 mM glucose, and 30 mM glucose plus 0.2 mM phlorizin for 7 days. The diameter of cells in the concentrations of glucose more than 10 mM were significantly larger than those in 5 mM glucose and 30 mM glucose plus phlorizin. Glucose, sorbitol and fructose contents of the cells in 30 mM glucose were significantly increased compared with those in 5 mM glucose, and were normalized by phlorizin. Thymidine uptake in the concentrations of glucose more than 20 mM was significantly decreased compared with that in 5 mM glucose. Myoinositol uptake, and DNA in 30 mM glucose were significantly reduced, and were normalized with phlorizin. Myoinositol content in 30 mM glucose was the same as that in 5 mM glucose, but was significantly decreased by phlorizin. The ratios of glucose to sorbitol or fructose in 30 mM glucose were significantly decreased, compared with those in 5 mM glucose and 30 mM glucose plus phlorizin. Therefore, the cellular enlargement and decreased DNA synthesis in cultured bovine retinal pericytes with abnormal glucose metabolism under high glucose conditions are attenuated by phlorizin, independent of the cellular myoinositol content.

The role of sodium in mediating adrenocorticotropin secretion by perifused rat anterior pituitary cells.

We studied the role of sodium ions in mediating basal and stimulated ACTH release from perifused rat anterior pituitary cells by exposing the cells to the sodium channel opener veratridine or the Na+/K(+)-adenosine triphosphatase inhibitor ouabain to increase the intracellular Na+ concentration or, conversely, by omitting Na+ from the perifusion medium or blocking Na+ entry into the cell with tetrodotoxin, a voltage-dependent sodium channel blocker, to decrease the intracellular Na+ concentration. Neither tetrodotoxin nor Na(+)-free medium had a significant effect on 100 nM arginine vasopressin (AVP) or 10 nM ovine corticotropin-releasing hormone (CRH)-induced ACTH secretion. Veratridine increased basal ACTH secretion by 122% (41.3 +/- 2.9 vs. 18.6 +/- 0.4 pg/min; P < 0.001), the initial spike phase of the response to AVP by 65% (0.28 +/- 0.01 vs. 0.17 +/- 0.03 ng/3 min; P < 0.005), the subsequent sustained phase to AVP by 129% (0.16 +/- 0.01 vs. 0.07 +/- 0.01 ng/7 min; P < 0.005), and the total response to CRH by 70% (0.39 +/- 0.01 vs. 0.23 +/- 0.04 ng/10 min; P < 0.05). Ouabain increased basal ACTH secretion by 39% (45.7 +/- 2.8 vs. 32.9 +/- 2.1 pg/min; P < 0.05), the initial spike phase of the response to AVP by 88% (0.32 +/- 0.02 vs. 0.17 +/- 0.01 ng/3 min; P < 0.005), the sustained phase response to AVP by 67% (0.10 +/- 0.01 vs. 0.06 +/- 0.01 ng/7 min; P < 0.05), and the total integrated response to CRH by 49% (0.88 +/- 0.09 vs. 0.59 +/- 0.03 ng/10 min; P < 0.05). However, the effects of both veratridine and ouabain on basal ACTH secretion were significantly attenuated in Ca(2+)-free EGTA-containing medium, suggesting that this effect was indirect, due to membrane depolarization and consequent influx of extracellular Ca2+. Dexamethasone (100 nM) had no effect on the ACTH response to either veratridine or ouabain. We conclude that changes in the intracellular Na+ concentration and sodium channel activity are not directly involved in AVP- or CRH-induced ACTH secretion.

Urocortin expression in human pituitary gland and pituitary adenoma.

Urocortin is a recently identified neuropeptide of the CRF family in the mammalian brain, but its expression in human tissue has been little studied. In this study, we examined urocortin expression in human anterior pituitary gland and pituitary adenomas by RIA, high performance liquid chromatography, immunohistochemistry, messenger ribonucleic acid (mRNA) in situ hybridization, and reverse transcriptase-PCR. Immunoreactive urocortin concentrations in normal pituitary tissue extract were 103.25 +/- 39.05 ng/g wet wt (mean +/- SEM; n = 4), and their levels were all significantly higher than those in other portions of central nervous system of the same subjects. High performance liquid chromatography analysis of human pituitary extract demonstrated a single peak corresponding to that of the expected chromatographic mobility of synthetic human urocortin-(1-40). Urocortin-immunoreactive cells were detected in the anterior pituitary gland. Neither urocortin-immunoreactive nerve fibers nor cells were detected in the posterior lobe. Immunostaining in serial mirror tissue sections revealed that 76.55 +/- 3.06% of urocortin-immunoreactive cells expressed GH immunoreactivity, whereas 22.25 +/- 3.02% and less than 1% of urocortin-immunoreactive cells expressed PRL and ACTH, respectively. mRNA hybridization signals of urocortin were also detected in urocortin-immunopositive pituitary cells. The reverse transcriptase-PCR analysis demonstrated a 145-bp RNA band corresponding to that of the expected length of urocortin in all cases of normal pituitary glands examined (n = 3). We also immunostained urocortin in 52 cases of human anterior pituitary adenomas, including GH-producing adenomas (n = 14), ACTH-producing adenomas (n = 13), PRL-producing adenomas (n = 11), and nonfunctioning hormonally inactive adenomas (n = 14). No urocortin immunoreactivity was detected in these adenoma cells, except for one case of GH-producing adenoma and one case of nonfunctioning adenoma. We also performed mRNA in situ hybridization in 27 adenomas. No hybridization signals were detected in these adenomas, except in two cases. The results described above indicated that urocortin is synthesized in human anterior pituitary cells and may play an important role in biological features of normal pituitary gland, possibly as an autocrine or a paracrine regulator

Advanced glycosylation end products induced tissue factor expression in human monocyte-like U937 cells and increased tissue factor expression in monocytes from diabetic patients.

Tissue factor (TF) plays a central role in the initial activation of the extrinsic coagulation pathway and is thought to be involved in the development of atherosclerosis and thrombosis. The effect of advanced glycosylation end products (AGEs) on TF expression and its mechanism were assessed by flow cytometric analysis. Human macrophage-like U937 cells, which were shown to contain mRNA encoding the receptors of advanced glycosylation end products (RAGE), expressed TF in a dose-dependent manner on incubation with AGE-albumin. AGE-albumin-induced TF expression was completely inhibited by the anti-oxidant agents, catalase and probucol. TF expression in peripheral monocytes from normal volunteers was also increased by AGE-albumin. Finally, TF expression in monocytes from individuals with diabetes mellitus, in whom the concentration of circulating AGEs is reported to be increased, was higher than that in monocytes from normal controls. These results suggest that AGE-induced TF expression in macrophages/monocytes is mediated by oxidant stress. AGEs may promote thrombosis and the development of atherosclerosis by inducing TF expression in monocytes in patients with diabetes mellitus.

Hypercoagulable state under low-intensity warfarin anticoagulation assessed with hemostatic markers in cardiac disorders.

The hemostatic condition under low-intensity anticoagulation in cardiac disorders is not fully elucidated. The aim of this study was to ascertain whether hemostatic molecular markers are a useful assessment for anticoagulation to detect the hypercoagulable state. A hematologic study was performed in 75 outpatients, without thromboembolic episodes, treated with low-intensity anticoagulation (average international normalized ratio [INR] 1.72) because of potential cardiac sources of arterial emboli, and in 40 age-matched control subjects. The average level of thrombin-antithrombin III complex (TAT) was significantly lower in patients than in control subjects (p = 0.005), and the mean value of D-dimer was not statistically different between patients and control subjects. Although TAT correlated moderately with D-dimer (r = 0.45, p = 0.0001), INR did not correlate with TAT or D-dimer. Elevated TAT > 3.0 ng/ml and/or D-dimer S 150 ng/ml were observed in 15 patients (20.0%), whereas the remaining 60 patients (80.0%) had no obvious increase in the level of TAT or D-dimer at overall INR. Antithrombin III activity did not correlate significantly with INR, but protein C activity and free protein S antigen showed a significant negative relation to INR (r = 0.82, r = 0.62, respectively, p = 0.0001). Low-intensity anticoagulation was sufficient to reduce coagulation and subsequent fibrinolytic activation in cardiac disorders, but may not be sufficient in some patients with elevated TAT or D-dimer concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulatory effect of calcitonin gene-related peptide on adrenocorticotropin release from rat anterior pituitary cells.

In the present study, we examined the direct regulatory effect of rat calcitonin gene-related peptide (CGRP) on adrenocorticotropin (ACTH) release from rat cultured anterior pituitary cells. CGRP significantly increased ACTH release at concentrations of 10(-8)-10(-11) M. The ACTH release was gradually increased by CGRP concentrations lower than 10(-10) M, and was decreased at concentrations higher than 10(-9) M, presenting a bell-shaped dose-response curve. As well as having an additive effect on corticotropin-releasing factor-induced ACTH release, CGRP stimulated the accumulation of intracellular cAMP. The CGRP-induced ACTH release was inhibited by a protein kinase A inhibitor, suggesting that its stimulatory effect on the ACTH release was mediated via an adenylate-cyclase-protein kinase system. CGRP-like immunoreactive nerve fibers have been reported to innervate the anterior pituitary, so that the stimulatory effect of CGRP on the ACTH release suggests that this peptide may be involved in neural regulation of hormone secretion in the anterior pituitary.

Functional reorganization of adult cat somatosensory cortex is dependent on NMDA receptors.

We studied effects of selective blockade of N-methyl-D-aspartate (NMDA) receptors in the primary somatosensory cortex (SI) of the adult cat on reorganization of cortical maps after selective deafferentation. About two weeks after hindlimb deafferentation, cortical loci representing the 'trunk' seemingly expanded and 'migrated' into areas originally devoted to the 'hindlimb'. Selective blockade of NMDA receptors of the SI cells by continuous infusion of 2-amino-5-phosphonovalerate (APV) into the cortex disrupted such change. The APV treatment also depressed responses of SI cells to natural sensory stimulation, but left no long-lasting side effects. The results suggest that the processes of map reorganization take place within the SI and involve mechanisms dependent on NMDA-receptor-mediated activity.

Elevated circulating levels of a decidual protein, placental protein 12, in preeclampsia.

The serum concentration of placental protein 12 was measured by radioimmunoassay in 111 apparently healthy pregnant women at 33 to 40 weeks, and the results were compared with those of 39 women with preeclampsia either with or without proteinuria at similar stages of pregnancy. Because the placental protein 12 levels were similar between 33 and 40 weeks of pregnancy, all data were pooled for the analysis of results. The levels were generally higher in preeclamptic patients: 175 +/- 81.5 micrograms/L (mean +/- SD) for preeclampsia versus 112 +/- 38.8 micrograms/L for controls (P less than .001). Those patients with proteinuric preeclampsia had the highest levels (207 +/- 92.1 micrograms/L; P less than .001). In preeclampsia the levels were above the normal 90th percentile in 15 (38%) cases (P less than .001). Because placental protein 12 has recently been found to be synthesized by decidua and not placenta, these results suggest that decidua may be affected in preeclampsia.

Effects of monosodium glutamate-induced obesity in spontaneously hypertensive rats vs. Wistar Kyoto rats: serum leptin and blood flow to brown adipose tissue.

We compared the effects of hypothalamic obesity induced by neonatal monosodium glutamate (MSG) treatment between spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). Newborn WKY and SHR were injected intraperitoneally with 4 mg/kg body weight of MSG daily for 5 days. At 6 months of age, the obesity of SHR was more advanced than that of WKY, but at 14 months of age the severity of obesity was similar between the two strains. Hypertriglyceridemia was enhanced in MSG-treated SHR as compared with MSG-treated WKY. Systolic blood pressure measured by the tail-cuff method was consistently lower in MSG-treated SHR than in control SHR, whereas blood pressure was not affected by neonatal MSG treatment in WKY. Food restriction reduced body weight more in control SHR than in control WKY, with the former also showing enhanced ketogenesis. Neonatal MSG treatment abolished the accelerated reduction of body weight in SHR. Serum leptin concentration was markedly increased in MSG-treated obese rats, though no differences were seen between WKY and SHR in the control or MSG-treated groups. Serum leptin was closely correlated with both Lee obese index and mesenteric fat weight over the strain. Blood flow in interscapular brown adipose tissue (BAT) measured by Laser Doppler flowmetry was significantly increased in response to beta3-adrenoceptor agonist BRL26830A in both the control and MSG-treated rats. However, the response of blood flow was not affected by MSG treatment or strain difference. The present study demonstrated some strain differences in response to neonatal MSG treatment between WKY and SHR. These differences could not be explained by the difference in serum leptin level or beta3-adrenergic reactivity in BAT.

Obesity induced by neonatal monosodium glutamate treatment in spontaneously hypertensive rats: an animal model of multiple risk factors.

The present study was designed to develop an animal model of multiple risk factors, including obesity, hypertension, non-insulin-dependent diabetes mellitus, and hyperlipidemia. Hypothalamic obesity was induced by neonatal monosodium glutamate (MSG) treatment in spontaneously hypertensive rats (SHR). Female newborn SHR were treated intraperitoneally with 2 or 4 mg/kg body weight of MSG for 5 days. Obesity developed in SHR treated with 4 mg/kg of MSG but not in SHR treated with 2 mg/kg of MSG. Obese SHR had impaired glucose tolerance, hyperinsulinemia, and hypertriglyceridemia. However, the severity of hypertension was attenuated in obese SHR as compared with control SHR. The degree of obesity was closely related to the metabolic abnormalities, but inversely correlated with the blood pressure level. Macrovascular changes were investigated in obese SHR at 14 months of age. Intimal thickening was accelerated in the carotid artery of obese SHR as compared with that of nonobese SHR. Aortic contents of DNA and total cholesterol were significantly increased in obese SHR. SHR associated with MSG-induced obesity showed major manifestations of metabolic syndrome X. This animal model may be useful to study the clustering of risk factors for the development of macrovascular diseases.

Effect of urocortin and its interaction with adrenocorticotropin (ACTH) secretagogues on ACTH release.

We examined the effect of urocortin (Ucn) on the adrenocorticotropin (ACTH) release from cultured rat anterior pituitary cells and AtT 20 cells. Synthetic rat (r)Ucn was not soluble in 0.1 N HCl but soluble in alkaline solvents with diminished corticotropin-releasing activity. rUcn dissolved in 0.1 M sodium phosphate buffer as a stock solution maintained its bioactivity and had the equal corticotropin-releasing activity with rat/human corticotropin-releasing factor (r/hCRF). rUcn stimulated the adrenocorticotropin release via CRF-receptors accompanied by the additive effect with r/hCRF, the synergistic effect with arginine vasopressin and the dose-dependent inhibition of a potent CRF-receptor antagonist.

Urocortin in human placenta and maternal plasma.

Plasma immunoreactive (IR-) urocortin (Ucn) and corticotropin-releasing factor (CRF) levels in pregnant women were measured by their specific radioimmunoassays after extraction. Although plasma IR-CRF levels were increased in pregnant women as compared to men and non-pregnant women, there was no difference of plasma IR-Ucn levels among groups. Ucn mRNA was detected in cytotrophoblasts and syncytiotrophoblasts by in situ hybridization. A reverse-phase high-performance liquid chromatography (HPLC) showed the major peak of IR-Ucn in placenta and plasma that had similar chromatographic mobility to synthetic Ucn1-40. These data suggest that Ucn is produced and processed into the same form of synthetic Ucn in placenta, but not secreted into maternal blood.

Regional distribution of urocortin-like immunoreactivity and expression of urocortin mRNA in the human brain.

Regional distribution of urocortin-like immunoreactivity (UCN-LI) in the human brain was studied by radioimmunoassay and was compared with that of corticotropin-releasing hormone (CRH). In addition, the expression of UCN mRNA was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) method. UCN-LI was detected in every region of brain examined, including hypothalamus, pons, cerebral cortex, and cerebellum. The concentrations of UCN-LI in the human brain were approximately 3 pmol/g wet weight in any brain region, and no marked regional difference was noted. On the other hand, the highest concentrations of CRH-LI were found in the frontal cortex, temporal cortex, and hypothalamus and the lowest in the pons. Reverse phase high-performance liquid chromatography of the UCN-LI in the human brain extract showed two immunoreactive peaks; one peak eluting earlier and one in the position of synthetic human UCN. RT-PCR showed that UCN mRNA was expressed in every region of brain examined. These findings indicated that UCN and UCN mRNA were widely expressed in the human brain.

Urocortin and corticotropin-releasing factor receptor expression in the human colonic mucosa.

Urocortin is a newly identified member of the CRF neuropeptide family. Urocortin has been found to bind with high affinity to CRF receptors. The present study investigated urocortin and CRF receptor expression in human colonic mucosa. Non-pathologic sections of adult colorectal tissues were obtained from patients with colorectal cancer at surgery. Urocortin expression was examined using immunohistochemistry and messenger (m) RNA in situ hybridization. Isolated lamina propria mononuclear cells (LPMC) and epithelial cells were also analyzed by flow cytometry for the characterization of urocortin-positive cells, and by RT-PCR for detection of urocortin, CRF, and CRF receptor mRNA. Urocortin peptide distribution at various stages of human development (n = 35, from 11 weeks of gestation to 6 years of age) was examined by immunohistochemistry using surgical and autopsy specimens. Immunoreactive urocortin and urocortin mRNA were predominantly detected in lamina propria macrophages. Urocortin peptide expression was detected from as early as three months of age, but not before birth or in neonates. Urocortin, CRF receptor type 1 and type 2 alpha mRNA were detected in LPMC. CRF receptor type 2 beta mRNA, a minor isoform in human tissues, was also detected in LPMC, but at lower levels. Urocortin is locally synthesized in lamina propria macrophages and may act on lamina propria inflammatory cells as an autocrine/paracrine regulator of the mucosal immune system. The appearance of urocortin after birth indicates that the exposure to dietary intake and/or luminal bacteria after birth may contribute to the initiation of urocortin expression in human gastrointestinal tract mucosa.