Expression of neuromedins S and U and their receptors in the hypothalamus and endocrine glands of the rat

Neuromedin S (NMS) and neuromedin U (NMU) are regulatory peptides that share the C-terminal amino-acid sequence and act via common G protein-coupled receptors called NMUR1 and NMUR2. Semiquantitative real time-PCR showed that in the rat hypothalamus and testis NMS gene expression was markedly higher than that of the NMU gene, while the reverse occurred in the anterior pituitary and thyroid gland. Low expression of both genes was detected in the thymus, adrenal gland and ovary, whereas in the pancreatic islets only the expression of NMU mRNA was detected. In the rat hypothalamus the expression of the NMUR2 gene was strikingly higher than that of the NMUR1 gene; in contrast, in the testis and ovary the very low expression of NMUR2 contrasted with the relatively high expression of the NMUR1 gene. In the other glands examined only expression of the NMUR1 gene was found. The marked differences in the level of expression of NMU, NMS and their receptors in the hypothalamus and endocrine glands of the rat suggest that in this species such neuromedins may play different roles in the functional regulation of neuroendocrine axes.     

Human pheochromocytomas express orexin receptor type 2 gene and display an in vitro secretory response to orexins A and B

Orexins A and B are hypothalamic peptides, that act through two receptor subtypes, called OX1-R and OX2-R. OX1-R selectively binds orexin A, whereas OX2-R is nonselective for both orexins. High levels of OX1-R mRNA and low levels of OX2-R mRNA have been previously detected in the human adrenal cortex and medulla. Here we demonstrated by RT-PCR the expression of the OX2-R, but not the OX1-R, gene in 10 benign secreting pheochromocytomas. Both orexins A and B stimulated catecholamine secretion from pheochromocytoma slices; the maximal effective concentration was 10(-8) mol/liter. Orexins A and B (10(-8) mol/liter) increased IP3, but not cAMP production, by tumor slices, and the effect was blocked by the PLC inhibitor U-73122. The catecholamine response to 10(-8) mol/liter orexins A and B was abolished by either U-73122 or the PKC antagonist calphostin C and was unaffected by the adenylate cyclase inhibitor SQ-22536 and the PKA inhibitor H-89. Collectively, these findings suggest that orexins stimulate catecholamine secretion from human pheochromocytomas, acting through OX2-R coupled to the PLC-PKC signaling pathway.     

Preproorexin and orexin receptors are expressed in cortisol-secreting adrenocortical adenomas, and orexins stimulate in vitro cortisol secretion and growth of tumor cells

Orexins A and B are hypothalamic peptides that originate from the proteolytic cleavage of preproorexin and act through two subtypes of receptors, named OX1-R and OX2-R. OX1-R almost exclusively binds orexin-A, whereas OX2-R is nonselective for both orexins. We previously found that orexin-A, via the OX1-R, stimulates cortisol secretion from dispersed human adrenocortical cells. In this study, we demonstrate that six of eight cortisol-secreting adenomas expressed preproorexin mRNA, and seven of 10 adenomas contained measurable amounts of orexin-A but not orexin-B. Normal adrenal cortexes neither expressed preproorexin nor contained orexins. All adenomas expressed OX1-R and OX2-R mRNAs, and real-time PCR showed that the expression of both receptors was up-regulated in adenomas, compared with normal adrenal cortex. Orexin-A concentration-dependently raised basal cortisol secretion from freshly dispersed normal and adenomatous cells, minimal and maximal effective concentrations being 10(-10) and 10(-8) m, and the peptide efficacy (percent increase elicited by 10(-8) m orexin-A) was significantly higher in adenomas than in the normal adrenal cortex. Orexin-B was ineffective, thereby indicating that orexin secretagogue action is mediated by the OX1-R. In contrast, both orexins (10(-8) m) raised the proliferative activity of cultured normal and adenomatous cells, suggesting that this effect is mediated by OX2-R or both receptor subtypes. Collectively, our findings allow us to conclude that the orexin system is overexpressed in cortisol-secreting adenomas and suggest that orexin-A may act as an autocrine-paracrine regulator of the secretory activity and growth of some of these adrenal tumors.     

Bradykinin and matrix metalloproteinases are involved the structural alterations of rat small resistance arteries with inhibition of ACE and NEP

BACKGROUND AND AIM: Increased vascular resistance is a hallmark of hypertension and involves structural alterations, which may entail smooth muscle cell hypertrophy or hyperplasia, or qualitative or quantitative changes in extracellular matrix (ECM) proteins. Since the renin-angiotensin-aldosterone system modulates these changes, we investigated the effects of 8 weeks of treatment with an angiotensin-converting enzyme (ACE) inhibitor, ramipril (RAM), or a dual ACE and neutral endopeptidase (NEP) inhibitor, MDL-100240 (MDL), on mesenteric small artery structure and ECM proteins in mRen2-transgenic rats (TGRs), an animal model of hypertension with severe cardiovascular damage. MATERIALS AND METHODS: Thirty-five 5-week-old rats were included in the study: six TGRs received RAM; five TGRs RAM + the bradykinin receptor inhibitor, icatibant; six TGRs, MDL; and five TGRs MDL + icatibant, while eight TGRs and five normotensive Sprague-Dawley controls were kept untreated. Mesenteric small arteries were dissected and mounted on a micromyograph. The media-to-lumen ratio (M/L) was then calculated. Vascular metalloproteinase (MMP) content was evaluated by zymography. RESULTS: In untreated TGRs severe hypertension was associated with inward eutrophic remodelling of small arteries. Both RAM and MDL prevented the increase in blood pressure and M/L and decreased MMPs. Icatibant blunted the effect of MDL on BP, M/L and MMPs. CONCLUSIONS: Changes in collagenase activity induced by ramipril and MDL are associated with prevention of small artery structural alterations in TGRs. Furthermore, MDL-induced enhancement of bradykinin could play a role in both the prevention of vascular structural alterations and in the stimulation of MMPs.     

Effects of prolonged cyclosporine-A treatment on the morphology and function of rat adrenal cortex

The effects of prolonged (30 day) treatment with daily therapeutical doses of cyclosporine A (CSA) (20 mg/kg) on the function and morphology of adrenal cortex were studied in adult male rats. CSA-treated animals developed a notable hypertension, along with a striking rise in PRA, which was not coupled with significant changes in the plasma concentrations of aldosterone and corticosterone (hyperreninemic hypoaldosteronism). Morphometry showed that zona glomerulosa (ZG) and zona fasciculata, and their parenchymal cells were atrophic. Isolated capsular (ZG) and inner (zona fasciculata/reticularis) cells displayed reduced basal and stimulated secretory responses. However, while the response of ZG cells to angiotensin II was almost completely suppressed (96%), basal steroid secretion of isolated cells, as well as the aldosterone and corticosterone response of ZG cells to potassium and ACTH, and corticosterone production of inner cells in response to ACTH were decreased by only about 30-40%. The hypothesis is advanced that CSA exerts a dual effect on rat adrenal cortex: 1) a general inhibitory effect on the growth and steroidogenic capacity of adrenocortical cells, which manifests itself only after very prolonged treatment and may be caused by an impairment of protein synthesis; and 2) an acute effect involving the specific blockade of the angiotensin-II-induced stimulation of the secretory activity of ZG cells.     

Anomalous ABO inheritance explained by ovum transplantation

BACKGROUND: Modern fertilization techniques can lead to unexpected ABO phenotypes in newborn infants and can raise questions as to maternity, paternity, and infant misidentification. Ovum transplantation can result in an infant with an ABO phenotype that is unexpected, given the birth mother's ABO type. STUDY DESIGN AND METHODS: A group AB, Rh- positive female infant was born to a group O, Rh-positive woman as a result of ovum transplantation. The case report is provided. RESULTS: The birth mother typed group O, Rh-positive both before and after delivery. The infant typed group AB, Rh-positive on cord blood and heelstick specimens. CONCLUSION: Ovum transplantation can result in newborns whose ABO phenotypes are unexpected, in relation to the birth mother's ABO type. To ensure patient privacy, such fertilization techniques may not be clearly documented in the delivery room chart. A complete obstetric history helps prevent repeat phlebotomies, expensive and unnecessary typing studies, and concern of the clinical staff with possible sample or infant misidentification.     

Cytokines in inflammatory malignant fibrous histiocytoma presenting with leukemoid reaction

Inflammatory malignant fibrous histiocytomas (IMFH) are rare tumors and are frequently associated with leukocytosis. In rare cases, leukemoid reactions were attributed to tumor production of unidentified hematopoietic factors. In this study, we used immunohistochemical techniques to show cytokine immunoreactivity in the malignant cells of two cases of IMFH presenting with leukemoid reactions and compared them with two malignant fibrous histocytomas, noninflammatory type. All four tumors stained positively for stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), interleukin-2 (IL-2), IL-4, IL-5, interferon-alpha (IFN-alpha), and insulin-like growth factor-I. Other cytokines detected only in the two IMFH included IL-6, IL-7, IL-8, IFN- gamma, and keratinocyte growth factor. Granulocyte-macrophage-CSF, IL- 3, and transforming growth factor-beta staining was present in one of the two IMFH tumors and was not present in the noninflammatory tumors. The immunohistochemical staining was localized to the malignant cells, suggesting deregulated cytokine expression consistent with their monocytic/histocytic origin. Expression of certain cytokines in the IMFH may account for the local inflammatory infiltrate, tumor fibrosis, and the aggressive nature of the malignant cells. We also detected elevated serum levels of SCF, G-CSF, IL-6, and tumor necrosis factor in one or both of the IMFH patients. These latter observations may explain the bone marrow hypercellularity and other paraneoplastic symptoms, including fever, malaise, and weight loss, observed in both patients. Different cytokines present in the two IMFH tumors appear to be responsible for the eosinophilic leukemoid reaction observed in one case and for the granulocytic leukemoid reaction observed in the other patient. They may also be responsible for expansion of the tumor-cell population, fibroblast proliferation, and enhanced secretion of extracellular collagen.     

Effects of prolactin administration on the zona glomerulosa of the rat adrenal cortex: stereology and plasma hormone concentrations

The effects of short and prolonged treatments with prolactin on the morphology and hormone secretion of the rat adrenal zona glomerulosa were investigated by coupled morphometric and radioimmunological techniques. Short-term prolactin administration did not alter plasma aldosterone concentration or zona glomerulosa morphology. Conversely, chronic prolactin treatment caused both a notable hypertrophy of zona glomerulosa cells and a significant rise in the blood level of aldosterone. The possibility is discussed that prolactin may be involved in the control of the growth and steroidogenic capacity of the rat adrenal zona glomerulosa.     

Proof-of-concept study on the suitability of 13C-urea as a marker substance for assessment of in vivo behaviour of oral colon-targeted dosage forms

Background and purpose:

¹³C-urea may be a suitable marker to assess the in vivo fate of colon-targeted dosage forms given by mouth. We postulated that release in the colon (urease-rich segment) of ¹³C-urea from colon-targeted capsules would lead to fermentation of ¹³C-urea by bacterial ureases into ¹³CO₂. Subsequent absorption into the blood and circulation would lead to detectable ¹³C (as ¹³CO₂) in breath. If, however, release of ¹³C-urea occurred in the small intestine (urease-poor segment), we expected detectable ¹³C (as ¹³C-urea) in blood but no breath ¹³C (as ¹³CO₂). The differential kinetics of ¹³C-urea could thus potentially describe both release kinetics and indicate the gastrointestinal segment of release.

Experimental approach:

The in vivo study consisted of three experiments, during which the same group of four volunteers participated.

Key results:

The kinetic model was internally valid. The appearance of ¹³C-in breath CO₂ (F_fermented) and the appearance of ¹³C in blood as ¹³C-urea (F_{not fermented}) show a high inverse correlation (Pearson''s r=−0.981, P= 0.06). The total recovery of ¹³C (F_fermented+F_{not fermented}) averaged 99%, indicating complete recovery of the administered ¹³C via breath and blood. ¹³CO₂ exhalation was observed in all subjects. This indicates that ¹³C-urea was available in urease-rich segments, such as the caecum or colon.

Conclusions and implications:

In this proof-of-concept study, ¹³C-urea was able to provide information on both the release kinetics of a colon-targeted oral dosage form and the gastrointestinal segment where it was released.