Jun Saito

Pituitary adenylate cyclase activating polypeptide (PACAP) stimulates mitogen-activated protein kinase (MAPK) in cultured rat astrocytes

Astrocytes, a subtype of glial cells, have been demonstrated to have an abundant number of receptors for pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide of the VIP/secretin family which stimulates cAMP accumulation 1000 times more potent than VIP in astrocytes. PACAP is reported to stimulate the proliferation of astrocytes at low concentrations at which it does not yet stimulate the cAMP accumulation. In the present study, we examined the effect of PACAP on the activation of mitogen-activated protein kinase (MAPK), one of the important intracellular signals for the proliferation, and compared it with that of epidermal growth factor (EGF). To investigate the activation of MAPK, we focused on ERK2, one of MAPK, in cultured rat astrocytes. The activation of ERK2 was determined by immunoblotting and measurement of the activity in terms of the phosphorylating activity of immunoprecipitates with MAPK antibody on myelin basic protein. One pM of PACAP38 temporarily activated ERK2 at 10 min. In contrast, EGF activated ERK2 from 10 min to 60 min continuously. As for the dose-response effect, PACAP stimulated ERK2 at as low a concentration as 10-14 M and peaked at 10-12 M. Thereafter, its activating effect gradually decreased at 10-10 M and returned to the basal level at 10-8 M, forming a bell-shaped dose-dependency. Neither an inhibitor of PKA (H89) nor inhibitors of PKC (staurosporine and calphostin C) had any effect on the ERK2 activation induced by 1 pM PACAP38. Dibutyryl cAMP suppressed ERK2 activity in a dose-dependent manner. These data clearly demonstrated that PACAP stimulates MAPK in both a PKA- and a PKC-independent manner in cultured rat astrocytes.

Mechanisms of enhanced production of PGI2 in cultured rat vascular smooth muscle cells enriched with eicosapentaenoic acid

The present investigation was performed to clarify the effect of EPA on PGI2 production in vitro using cultured rat vascular smooth muscle cells (VSMC). To simulate in vivo conditions, a triacylglycerol (TG) emulsified form of EPA was used. An increase in EPA content was achieved without alteration of arachidonic acid concentration. These experiments clearly demonstrated that co-incubation of EPA-TG increased PGI2 production by cultured VSMC in a dose dependent fashion. Among polyunsaturated fatty acid TG examined (docosahexaenoic acid, linoleic acid, oleic acid and EPA), only EPA-TG was effective. Cyclooxygenase (COX) was activated, but neither phospholipase A2 nor PGI2 synthase activity was changed. EPA treatment did not alter the amount of COX-1 and COX-2 protein in VSMC. Addition of antioxidants, such as butylated hydroxytoluene or vitamin E, decreased MDA levels in the medium and cells and reversed the enhanced PGI2 production in EPA rich-VSMC. Therefore, the high polyunsaturation of EPA could generate low levels of lipid peroxides and thereby lead to activation of COX and an increased PGI2 production. Although EPA increased PGI2 production, only a negligible amount of PGI3 was produced by rat aortic tissues. Enhanced production of PGI2 might contribute to the anti-atherogenic effect of EPA.

Clinicopathological study of SDHB mutation-related pheochromocytoma and sympathetic paraganglioma

Pheochromocytoma (PCC) and paraganglioma (PGL) are genetically and phenotypically heterogeneous catecholamine-producing neoplasms. They can occur sporadically or as a part of hereditary disease. Approximately 30% of PCC/PGL are believed to be caused by germline mutations (Welander et al. 2011). Of these, succinate dehydrogenase subunit B (SDHB) gene mutation is considered a high-risk factor for malignancy. Loss of heterozygosity at the SDHB locus (1p36)wasobserved inall tumorswithSDHBmutation, and Gimenez-Roqueplo et al. (2003) strongly suggested that SDHB is a tumor suppressorgene. Subsequently, loss of SDHB protein immunoreactivity in SDHB-mutated PCC/PGL (SDHB–PCC/PGL) was reported with 100% sensitivity and 84% specificity (van Nederveen et al. 2009). Thus, SDHB immunohistochemistry can be used to screen SDHB– PCC/PGL using paraffin-embedded pathological materials. SDHB mutation is the only established factor that indicates future metastasis. Therefore, it is important to analyze the histological characteristics of SDHB–PCC/PGL. It is generally accepted that it is difficult to distinguish histological differences between benign and malignant PCC/PGL. The current consensus is that a long-term follow-up is required after the surgery to screen for recurrence or metastasis in all PCC/PGL patients, regardless whether hereditary or sporadic in origin. Kimura et al. (2014) proposed a histological grading system called the Grading of Adrenal PCC and PGL (GAPP) classification for predicting metastasis. GAPP is composed of six factors: histological pattern, cellularity, presence or absence of comedo-type necrosis, vascular or capsular invasion, Ki67labeling index (%), and elevated catecholamine type. Each factor was assigned a point and the number of points was summated. Tumor scores of 0–2, 3–6, and 7–10 were classified into well differentiated (WD), moderately

Clinical and Steroidogenic Characteristics of Aldosterone-Producing Adenomas With ATPase or CACNA1D Gene Mutations

OBJECT This comparative study clarified the clinical characteristics and in vitro steroidogenic activities of aldosterone-producing adenomas (APAs) harboring ATPase or CACNA1D gene mutations. DESIGN AND PATIENTS Genetic testing was performed on 159 unilateral APAs. Somatic ATPase and CACNA1D gene mutations were analyzed in 42 APA tissues without KCNJ5 gene mutations. RESULTS ATP1A1, ATP2B3, and CACNA1D mutations were detected in one, four, and four patients, respectively. Compared with patients without KCNJ5, ATPase, or CACNA1D mutations (wild type), ATPase mutations tended to have more severe hyperaldosteronism and smaller tumors; those with CACNA1D mutations had clinical characteristics and tumor sizes similar to those with wild-type genes. APAs with ATPase mutations were composed mainly of compact eosinophilic tumor cells, whereas CACNA1D mutations resulted in predominantly clear tumor cells. Aldosterone production in APA cells with ATP2B3 mutations were more responsive to dibutyryl cAMP, whereas those with CACNA1D mutations were more responsive to adrenocorticotropic hormone than the wild-type cells. CONCLUSION APAs with ATPase mutations demonstrated a potentially severe primary aldosteronism phenotype, whereas those with CACNA1D mutations displayed characteristics similar to wild-type APAs. The status of stimulated aldosterone production was also different according to the cell types, suggesting that the regulatory effects of adrenocorticotropic hormone on aldosterone synthesis could possibly vary according to the intracellular signaling involved in hormone production.

Somatic KCNJ5 mutation occurring early in adrenal development may cause a novel form of juvenile primary aldosteronism.

We report a case of non-familial juvenile primary aldosteronism (PA). Super-selective adrenal venous sampling identified less aldosterone production in the right inferior adrenal segment than others. Bilateral adrenalectomy sparing the segment normalized blood pressure and improved PA. Both adrenals had similar histologies, consisting of a normal adrenal cortex and aldosterone synthase-positive hyperplasia/adenoma. An aldosterone-driving KCNJ5 mutation was detected in the lesions, but not in the histologically normal cortex. After taking into account that the two adrenal glands displayed a similar histological profile, as well as the fact that hyperplastic lesions in both glands exhibited a common KCNJ5 mutation, we conclude that the specific mutation may have occurred at an adrenal precursor mesodermal cell, at an early stage of development; its daughter cells were mixed with non-mutant cells and dispersed into both adrenal glands, resulting into a form of the condition known as genetic mosaicism.

Increased plasma serotonin metabolite 5-hydroxyindole acetic acid concentrations are associated with impaired systolic and late diastolic forward flows during cardiac cycle and elevated resistive index at popliteal artery and renal insufficiency in type 2 diabetic patients with microalbuminuria.

Although lower extremity arterial disease is frequently accompanied by diabetes mellitus, the association of circulating biomarkers with flow components during the cardiac cycle in lower-leg arteries has yet to be fully elucidated. We enrolled 165 type 2 diabetic patients with normal ankle-brachial index (ABI 1.0-1.4), comprising 106 normoalbuminuric and 59 microalbuminuric patients, and 40 age-matched nondiabetic subjects consecutively admitted to our hospital. Serum high sensitivity C-reactive protein (hsCRP) level and plasma von Willebrand factor ristocetin cofactor activity (VWF) and vasoconstrictor serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA) concentrations were measured. An automatic device was used to measure ABI and brachial-ankle pulse wave velocity (baPWV). Flow components during the cardiac cycle, total flow volume, and resistive index at popliteal artery were evaluated using gated magnetic resonance imaging. Although estimated glomerular filtration rate (eGFR), early diastolic flow reversal, heart rate, and ABI were similar between the groups, diabetic patients had higher log hsCRP (p<0.001), VWF (p<0.001), 5-HIAA (p=0.002), resistive index (p<0.001) and baPWV (p<0.001) and lower systolic (p=0.026) and late diastolic (p<0.001) forward flows and total flow volume (p<0.001) than nondiabetic subjects. Multivariate analyses demonstrated that 5-HIAA in microalbuminuric patients showed higher associations with systolic and late diastolic forward flows during the cardiac cycle, total flow volume and resistive index at popliteal artery, and eGFR compared to normoalbuminuric patients. In microalbuminuric patients, 5-HIAA was a significant independent determinant among these factors. Thus, increased plasma 5-HIAA levels are involved in the pathogenesis of impaired blood flow in lower extremities and renal insufficiency in diabetic patients with microalbuminuria.

Evaluation of Cortisol Production in Aldosterone-Producing Adenoma

Aldosterone-producing adenoma (APA) is sometimes accompanied with subclinical hypercortisolism. We investigated the ability of cortisol production in APA, both clinically and pathologically. A retrospective cohort study was conducted at Yokohama Rosai Hospital from 2009 to 2016. Thirty patients with APA and serum cortisol levels during the 1 mg dexamethasone suppression test (F-DST)<3.0 μg/dl were included. We evaluated the 1) difference between pre-adrenalectomy F-DST (pre-F-DST) and post-adrenalectomy F-DST (ΔF-DST), 2) correlation between ∆F-DST and pre-F-DST, tumour size determined by CT, and type of adrenalectomy (total or partial), and 3) relationship between the ratio of F-DST divided by tumour size (ΔF-DST/pre-F-DST/mm) and immunoreactivity of CYP17A1, CYP11B1, and CYP11B2. The median [interquartile range] age was 48 [38-58] years. We found a significant decrease in F-DST after adrenalectomy [before: 1.4 (1.1-1.8); after: 0.9 (0.6-1.2); p<0.001]. Additionally, a significant correlation was found for ΔF-DST and both pre-F-DST (Spearman, ρ=-0.68, p<0.001) and tumour size (ρ=-0.51, p 0.005). No significant difference was found in ΔF-DST between total and partial adrenalectomy. CYP17A1 and CYP11B1 were positive in 21 (100%) and 17 (81%) adenomas, respectively. CYP17A1 immunoreactivity in the tumour was significantly related with ΔF-DST/pre-F-DST/mm (p 0.049). F-DST significantly decreased after adrenalectomy, and most of the adenomas were immunohistochemically positive for CYP17A1 and CYP11B1 as well as CYP11B2. We should consider the possibility of autonomous cortisol production as well as hyperaldosteronism in the evaluation and treatment of APA patients.

Elevated plasma B-type natriuretic peptide concentration and resistive index, but not decreased aortic distensibility, associate with impaired blood flow at popliteal artery in type 2 diabetic patients

Blood flow in lower extremity arteries is frequently impaired in diabetic patients even though they have a normal ankle-brachial index (ABI 1.0-1.4). Risk factors contributing to this lower extremity arterial disease have not been fully elucidated. We enrolled 52 type 2 diabetic patients with normal ABI and 30 age-matched nondiabetic subjects consecutively admitted to our hospital. Plasma B-type natriuretic peptide (BNP) concentrations were measured. Distensibility in ascending thoracic and abdominal aortas as well as total flow volume and resistive index at popliteal artery were evaluated by gated magnetic resonance imaging. An automatic device was used to measure ABI and brachial-ankle pulse-wave velocity (baPWV). Diabetic patients showed lower distensibility in ascending thoracic aorta (p<0.001) and total flow volume (p<0.001) and higher baPWV (p<0.001) and resistive index (p=0.005) and similar BNP and distensibility in abdominal aorta compared to nondiabetic subjects. Simple linear regression analyses revealed that distensibility in ascending thoracic (p=0.019) and abdominal (p=0.030) aortas positively as well as baPWV (p=0.020), resistive index (p<0.001) and BNP (p<0.001) negatively correlated with total flow volume. Stepwise multiple regression analysis demonstrated that increased BNP and resistive index were independent risk factors for total flow volume in diabetic patients (r(2)=0.639, p<0.001). These results indicate that increased plasma BNP levels and peripheral vascular resistance, but not decreased aortic distensibility, associate with impaired blood flow in lower extremity arteries in diabetic patients.

Multiple Sampling from the Central Veins with their Tributaries can Detect Bilateral Hyperaldosteronism with a Cortisol-Producing Adenoma in a Hypertensive Patient

A 52-year old woman was admitted to our hospital for evaluation of left adrenal incidenataloma. Endocrinological examination showed Cushing’s syndrome (CS) complicated with masked primary aldosteronism (PA). On the other hand, multiple sampling from the central veins and one or two tributaries of the adrenal veins before and after ACTH-stimulation (multiple AVS) clearly revealed bilateral hyperaldosteronism with excess cortisol secretion from the left adrenal. Thus, we diagnosed this case as CS due to left adrenal tumor with bilateral hyperaldosteronism, and left adrenalectomy was done. Immunohistochemical analysis of the removed left adrenal showed cortisol-producing adenoma and multiple aldosterone-producing cell clusters (APCCs) expressing CYP11B2 within the attached adrenal. Bilateral PA is mostly diagnosed as idiopathic hyperaldosteronism (IHA). IHA has not been examined enough pathologically. We first describe here a possible involvement of APCCs inducing hyperaldosteronism in a case of bilateral PA with a cortisol-producing-adenoma.

Aldosterone Suppression by Dexamethasone in Patients with KCNJ5-Mutated Aldosterone-producing Adenoma.

Context Aldosterone biosynthesis is regulated principally by ACTH and gene mutations as well as by angiotensin II and serum potassium. In addition, previous studies have reported the potential effects of KCNJ5 mutations in aldosterone-producing adenoma (APA) on cardiovascular diseases. However, responsiveness to ACTH in APAs according to potassium inwardly rectifying channel, subfamily J, member 5 (KCNJ5) mutations remains unknown. Objective To investigate KCNJ5 genotype-specific differences in aldosterone biosynthesis in response to ACTH stimulation. Design and Setting A cross-sectional study through retrieval of clinical records. Participants One hundred forty-one patients aged ≥20 years with APA were examined. Main Outcome Measures Associations between KCNJ5 mutations and clinical parameters reflecting the renin-angiotensin system [saline infusion test (SIT)] and ACTH pathways [dexamethasone suppression test (DST)]. Results KCNJ5 mutations were detected in 107 cases. In the crude comparison, patients with mutations in KCNJ5 had higher plasma aldosterone concentrations (PACs) both at baseline and after the SIT. PAC after the DST showed a significant inverse association with KCNJ5 genotypes after controlling for age, sex, tumor size, and PAC after the SIT. Immunohistochemical analysis of 101 cases revealed more abundant immunoreactivity of CYP11B1 and CYP17 in the KCNJ5-mutated group than in the KCNJ5 wild-type group. Conclusion This report of marked suppression of PAC by dexamethasone in patients with KCNJ5-mutated APAs indicates that such APAs respond to endogenous ACTH more readily than APAs in nonmutated cases. Further molecular and epidemiologic studies are required to validate our results and clarify the clinical effectiveness of the DST for predicting KCNJ5 mutations before adrenalectomy.