Coupling factor 6-induced activation of ecto-F1F(o) complex induces insulin resistance, mild glucose intolerance and elevated blood pressure in mice.

AIMS/HYPOTHESIS: Despite advances in pharmacological treatments, diabetes with hypertension continues to be a major public health problem with high morbidity and mortality rates. We recently identified a circulating peptide coupling factor 6 (CF6), which binds to the plasma membrane ATP synthase (ecto-F(1)F(o) complex), resulting in intracellular acidosis. We investigated whether overexpression of CF6 contributes to diabetes and hypertension by intracellular acidosis. METHODS: Transgenic mice overexpressing CF6 (also known as ATP5J) were generated, and physiological, biochemical and molecular biology studies were performed. RESULTS: CF6 overexpression elicited a sustained decrease in intracellular pH in tissues (aorta, kidney, skeletal muscle and liver, with the exception of adipose tissue) that express its receptor, the ß-subunit of ecto-F(1)F(o) complex. Consistent with the receptor distribution, phospho-insulin receptor ß, phosphoinositide 3-kinase activity and the phospho-Akt1:total Akt1 ratio were all decreased in the skeletal muscle and the liver in transgenic compared with wild-type mice, resulting in a decrease of plasma membrane-bound GLUT4 and an increase in hepatic glucose production. Under a high-sucrose diet, transgenic mice had insulin resistance and mild glucose intolerance; under a high-salt diet, they had elevated blood pressure with increased renal RAS-related C3 botulinum substrate 1 (RAC1)-GTP, which is an activator of mineralocorticoid receptor. CONCLUSIONS/INTERPRETATION: Through its action on the ß-subunit of ecto-F(1)F(o) complex, which results in intracellular acidosis, CF6 plays a crucial role in the development of insulin resistance and hypertension. This finding might advance our understanding of the mechanisms underlying diabetes and hypertension, possibly also providing a novel therapeutic target against cardiovascular disease.

Multiple (18)F-FDG, PET-CT for postoperative monitoring of breast cancer patients.

BACKGROUND: Positron emission tomography (PET)-computed tomography (CT) may be useful in the post-treatment follow-up of breast cancer patients. PURPOSE: To assess the usefulness of (18)F-fluorodeoxyglucose (FDG) PET-CT (PET-CT) for postoperative monitoring of breast cancer patients. MATERIAL AND METHODS: One hundred twenty-nine PET-CT studies performed on 55 female postoperative breast cancer patients (median age 56 years, range 36-86 years) were analyzed. The median interval between the PET-CT studies was 6 months (range 1-15 months). In order to determine the usefulness of serial PET-CT examinations in the postoperative follow-up of breast cancer patients, the PET-CT findings were compared with the physical findings, findings obtained by other imaging modalities, and the (18)F-FDG-PET (PET) findings. RESULTS: The PET findings were negative in 4 metastatic bone lesions with a positive bone scan. The PET findings were also negative in 6 of 9 osteogenic bone metastases and one of 64 osteolytic bone lesions. There were 5 cases with false-positive of PET, which were determined to be areas of soft-tissue hyperactivity. All false-positive/-negative findings were corrected by the addition of CT. CONCLUSION: The results of this study lend support to the clinical role of PET-CT in the postoperative follow-up/monitoring of breast cancer patients.

Improved shoulder contour following forequarter amputation with an osteomyocutaneous free flap from the amputated extremity: two cases.

To cover a large soft-tissue defect and to reconstruct the shoulder contour after forequarter amputation, we used an osteomyocutaneous free flap incorporating an elbow joint from the amputated extremity in two patients. These flaps were well vascularised and reliable. They provided excellent coverage of large soft-tissue defects and they maintained shoulder contours. This procedure is useful for reconstruction after extended forequarter amputation and chest wall resection.

Effect of itraconazole on the pharmacokinetics and pharmacodynamics of a single oral dose of brotizolam.

AIMS: To assess the effect of itraconazole, a potent inhibitor of cytochrome P450 (CYP)3A4, on the single oral dose pharmacokinetics and pharmacodynamics of brotizolam. METHODS: In this randomized, double-blind, cross-over trial 10 healthy male subjects received either itraconazole 200 mg or matched placebo once daily for 4 days. On day 4, a single 0.5 mg dose of brotizolam was administered orally. Plasma concentrations of brotizolam were followed up to 24 h, together with assessment of psychomotor function measured by the digit symbol substitution test (DSST), visual analogue scales and UKU side-effect rating scale. RESULTS: Itraconazole significantly (P < 0.001) decreased the apparent oral clearance (CL/F) (16.47 +/- 4.3 vs 3.91 +/- 2.1), increased the area under the concentration-time curves (AUC) from 0 h to 24 h (28.37 +/- 10.8 vs 68.71 +/- 24.1 ng ml h(-1)), and prolonged the elimination half-life (4.56 +/- 1.4 vs 23.27 +/- 10.3 h) of brotizolam. The AUC(0,24 h) of the DSST (P < 0.001) and the item 'sleepiness' of UKU (P < 0.05) were significantly decreased. CONCLUSIONS: Itraconazole increases plasma concentrations of brotizolam probably via its inhibitory effect on CYP3A4 brotizolam metabolism.

Ochronotic spondylarthropathy: two case reports of progressive destructive changes in the hip.

Abstract We report two cases of ochronotic spondylarthropathy who presented with spinal involvement followed by progressive destructive changes in the hip joint, which led to total hip replacement with a satisfactory outcome. Pathological examination revealed severe deterioration of the affected hip with unique cartilage degeneration associated with active inflammation in the synovium and bone marrow. These features were also evaluated by magnetic resonance imaging in one case which presented with rapidly destructive changes in the hip. Spinal involvement may contribute to progressive destructive hip arthropathy in ochronosis.

Mitochondrial coupling factor 6 is present on the surface of human vascular endothelial cells and is released by shear stress.

BACKGROUND: We showed that mitochondrial coupling factor 6 (CF6), an endogenous inhibitor of prostacyclin synthesis, is present in the systemic circulation as a pressor substance in rats. We investigated the possibility of vascular endothelial cells as a source of circulating CF6. METHODS AND RESULTS: We used 2 cultured endothelial cell lines, human umbilical vein endothelial cells (HUVECs) and ECV 304 cells (transformed HUVECs), for this study. Immunofluorescence microscopy of both ECV 304 and HUVECs confirmed the surface-associated immunoreactivity of anti-CF6 antibody on the plasma membrane. The concentration of CF6 in the medium increased gradually with time in both ECV 304 and HUVECs in static conditions. Exposure of ECV 304 and HUVECs to a fluid shear stress enhanced the release of CF6: In ECV 304, the concentration of CF6 in the medium (ng. well(-1). 6 hours(-1)) was 2.1+/-0.8 at baseline, 4.3+/-0.8 after shear at 15 dynes/cm(2), and 57.7+/-8.4 after shear at 25 dynes/cm(2). CF6 contents in the cell homogenate and mitochondria were both significantly increased after exposure of ECV 304 to 6-hour shear at 15 dynes/cm(2), whereas they were unchanged after shear stress at 25 dynes/cm(2). The ratio of CF6 to GAPDH mRNA was enhanced significantly, by 1.8+/-0.2-fold, after 6-hour shear stress at 25 dynes/cm(2). Flow cytometry analysis revealed that the surface-associated CF6 was significantly increased in a 3-hour static condition after the previous exposure of the cells to shear stress for 3 hours. CONCLUSIONS: Vascular endothelial cells are a source of CF6, and shear stress regulates the release of the surface-associated CF6.

Expression of pyrimidine nucleoside phosphorylase (PyNPase) in colorectal cancer.

BACKGROUND: We investigated the expression of PyNPase both in cancer cells and in stroma cells to clarify the correlation between PyNPase expression and the prognosis of patients with colorectal cancer. METHODS: Using immunohistochemical staining with an anti-PyNPase antibody, the PyNPase expression in tissues from 114 patients with stage II or III colorectal cancer was examined. From the correlation between PyNPase expression and the clinicopathological findings, the prognosis for survival was analyzed. RESULTS: The expression of PyNPase was classified as negative or positive on the basis of staining. No relationship between PyNPase expression and any of the clinicopathological findings was identified. However, a relationship was observed regarding positive staining between cancer cells and stroma cells. The prognosis of patients with positive staining in both cancer cells and stroma was worse than that of other patients. In multivariate analyses, expression in cancer cells was the strongest predictor of prognosis. CONCLUSIONS: PyNPase expression appears to be a relevant factor for predicting the prognosis of colorectal cancer.

Mitochondrial coupling factor 6 as a potent endogenous vasoconstrictor.

We demonstrated recently that coupling factor 6, an essential component of the energy-transducing stalk of mitochondrial ATP synthase, suppresses the synthesis of prostacyclin in vascular endothelial cells. Here, we tested the hypothesis that coupling factor 6 is present on the cell surface and is involved in the regulation of systemic circulation. This peptide is present on the surface of CRL-2222 vascular endothelial cells and is released by these cells into the medium. In vivo, the peptide circulates in the vascular system of the rat, and its gene expression and plasma concentration are higher in spontaneously hypertensive rats (SHRs) than in normotensive controls. Elevation of blood pressure with norepinephrine did not affect the plasma concentration of coupling factor 6. Intravenous injection of recombinant peptide increased blood pressure, apparently by suppressing prostacyclin synthesis, whereas a specific Ab to coupling factor 6 decreased systemic blood pressure concomitantly with an increase in plasma prostacyclin. Interestingly, the antibody's hypotensive effect could be abolished by treating with the cyclooxygenase inhibitor indomethacin. These findings indicate that mitochondrial coupling factor 6 functions as a potent endogenous vasoconstrictor in the fashion of a circulating hormone and may suggest a new mechanism for hypertension.

Cross talk between prostacyclin and nitric oxide under shear in smooth muscle cell: role in monocyte adhesion.

We tested the hypothesis that at sites of vascular damage, vessel homeostasis is maintained through the cross talk of shear-induced production of prostacyclin and nitric oxide (NO) in vascular smooth muscle cells (VSMC). Confluent A7r5 cells derived from rat aortic VSMC and mesenteric VSMC were exposed to shear stress at 15 dyn/cm(2) for 90 min with the use of a cone-plate device, and productions of prostacyclin and NO were examined. Shear stress increased cumulative production of prostacyclin by 3- to 3.5-fold and that of NO by 6- to 7.5-fold. Western blot analysis showed that inducible NO synthase protein was expressed after shear stress in both types of VSMC. Inhibition of NO synthase enhanced the shear-induced production of prostacyclin from 40 to 60%. Shear-induced production of NO was suppressed by 70% after treatment with 10(-4) M of indomethacin. A7r5 cells adhesiveness for monocytes was suppressed by 50% after shear stress. This suppression was abolished by pretreatment with 10(-4) M of indomethacin, whereas inhibition of NO synthase only minimally inhibited it. We conclude that there is a cross talk of shear-induced production of prostacyclin and NO in VSMC. At sites of vascular damage, prostacyclin synthesis may prevent monocyte adhesiveness for VSMC through the concomitant enhancement of NO production.

Expression of glycoconjugates bearing the Lewis X epitope during neural differentiation of P19 EC cells.

The Lewis X (Le(x)) bearing glycolipids were noticeably increased in amounts during the course of neural differentiation of P19 EC cells induced by retinoic acid (RA, all-trans form). Applying neoglycolipid technology and in situ TLC-LSIMS, the oligosaccharide chains of these scarce Le(x) bearing glycolipids were partially characterized after released by endoglycoceramidase and subsequent conversion into neoglycolipids. In order to understand the enzymatic basis for the expression of Le(x) bearing glycolipids, we measured glycolipid, glycoprotein and oligosaccharide fucosyltransferase (Fuc-T) activities using appropriate substrates in P19 EC cells with or without RA treatment. All three Fuc-Ts were increased after RA treatment and the highest activity was in the differentiated neural cells. We then investigated the two possible Fuc-T genes that might be responsible for these changes using RT-PCR analysis. Mouse Fuc-TIX (mFuc-TIX) transcript was detected in all cell types but it was only strongly expressed in RA-induced aggregates and neural cells. In the case of mouse Fuc-TIV (mFuc-TIV) gene, its transcript was only detectable in RA-induced aggregates and not found in either undifferentiated or RA-induced neural cells. These results strongly support that RA induces only a transient expression of the mFuc-TIV gene in cell aggregates but a more persistent expression of the mFuc-TIX gene at the transcription level throughout neural cell differentiation. The mFuc-TIX gene is probably the main cause for the increased expression of Le(x) glycoconjugates during neural differentiation of P19 EC cells.

[Exercise training improves activities of daily living in elderly patients with congestive heart failure].

Exercise training which is one of the multidisciplinary interventions for elderly patients with congestive heart failure, plays an important role for improving the quality of life and reducing the re-admission rate of these patients. We assessed the validity of exercise training for the improvement of patient's skeletal muscle functions and activities of daily living along with monitoring cardiac functions. Exercise training programs were performed in 12 patients with congestive heart failure (New York Heart Association class III or IV), including 5 with valvular disease, 4 with dilated cardiomyopathy and 3 with ischemic cardiomyopathy (mean 79 +/- 9 years). All patients were admitted because of exacerbation of congestive heart failure and were treated conventionally. The exercise training program was started after stabilization of their cardiac condition. The medication was not changed during the training period. After exercise training programs, the cardio-thoracic ratio decreased from 63.8 +/- 7.9% to 60.1 +/- 6.9% (p < 0.01), ejection fraction on echocardiography increased from 47.4 +/- 18.2% to 56.0 +/- 17.5% (p < 0.01), and brain natriuretic peptide decreased from 404.8 +/- 267.5 pg/ml to 313.6 +/- 239.5 pg/ml (p < 0.05). The quadriceps muscle power increased from 0.77 +/- 0.36 Nm/kg to 0.97 +/- 0.41 Nm/kg (p < 0.01). The maximum walking distance on flat surface increased from 149 +/- 164 m to 456 +/- 394 m (p < 0.05). In most patients, the activities of daily living, especially mobility, improved. Appropriate exercise training for the elderly patients with congestive heart failure improves activities of daily living and also reduces the amount of required care by the patients.

Enhanced phospholipase C activity in the cultured skin fibroblast obtained from patients with coronary spastic angina: possible role for enhanced vasoconstrictor response.

OBJECTIVES: We measured phospholipase C (PLC) activity in the cultured skin fibroblasts obtained from patients with and without coronary spasm and examined its correlation with coronary artery vasomotility. BACKGROUND: Coronary artery vasomotility is enhanced in coronary spastic angina (CSA), but no information is available for the intracellular signaling. In spontaneously hypertensive rats, PLC activity in the skin fibroblasts has been shown to be enhanced. METHODS: Skin fibroblasts obtained from 24 patients with CSA-14 with organic coronary artery disease (CAD) and 12 control subjects--were cultured by the explant method. Activity of PLC was determined by incubating the membrane fraction with 3H-phosphatidyl inositol bisphosphate and by quantifying 3H-inositol trisphosphate. In patients with CSA and control subjects, the relations between PLC activity and coronary artery basal tone and constrictor response to intracoronary acetylcholine (ACh) were examined. RESULTS: Activity of PLC (pmol/protein [mg] per min) was 1.74+/-0.19 in patients with CSA; 0.90+/-0.12 in patients with CAD; and 0.65+/-0.07 in control subjects (p<0.001, patients with CSA vs. patients with CAD and control subjects; p = NS, patients with CAD vs. control subjects). According to the Lineweaver-Burk plot, Michaelis constant (micromol/liter) of PLC was 28+/-4 in patients with CSA; 49+/-14 in patients with CAD; and 56+/-10 in control subjects (p<0.05, patients with CSA vs. control subjects), whereas the maximal velocity was not different between the three groups. There were significant positive correlations between PLC activity and both basal tone (p = 0.0108) and response to ACh (p = 0.0053). Western blot analysis using membrane fraction demonstrated that 89% of PLC isoenzymes detected was of the delta1 isoform. CONCLUSIONS: Because the PLC activity measured was genetically defined and was positively correlated with coronary artery vasomotility, enhanced PLC activity may be involved in the pathogenesis of coronary spasm.

[Chemoradiotherapy with low-dose cisplatin and 5-FU for advanced esophageal cancer].

We evaluated the efficacy of chemoradiotherapy (CRT) for advanced esophageal cancer, from the view point of response. The relationship between chemo-radiosensitivity and dihydropyridine dehydrogenase (DPD), thymidylate synthase (TS), and p53 was investigated immunohistochemically. Thirteen patients with inoperable advanced esophageal cancer were involved in this study. CDDP of 10 mg/m2/day and 5-FU of 335 mg/m2/day were infused intravenously (day 1-5, day 15-19). Radiation was delivered concomitantly at a total dose of 30 Gy. Expressions of p53, DPD and TS were detected using immunohistology in the biopsy samples taken before CRT from 8 patients. Partial response was observed in 8 cases, no change in 4 cases, and progressive disease in one case. The overall response rate was 62%. The reduction rate was higher in tumors positive for p53 expression than in negative ones. The same was true for DPD and TS. The Treatment effect was more precisely predicted by combination of p53, DPD and TS. CRT with low-dose CDDP + 5-FU chemotherapy was effective and combination with p53, DPD, and TS might be a predictive marker for CRT in patients with advanced esophageal cancer.

Metamorphosed fibroblasts and their relation to the histogenesis of malignant fibrous histiocytoma in experimental murine model.

Malignant fibrous histiocytoma (MFH) is a clinicopathologically established entity, but its histogenesis remains to be clarified. We have reported the existence of a specific cell type, the "fibrohistiocytoid (FH) cells", in various chronic inflammatory tissues. The FH cells are the metamorphosed fibroblasts and we have revealed the morphological resemblance between FH cells and MFH cells. In the present study we carried out some experiments to ascertain whether the FH cells have a possibility of neoplastic potential for the development of MFH in mice. A total of 50 female Balb/c mice treated with a chemical carcinogen, 9,10 dimethyl-1,2-benzanthracene (DMBA), were examined histopathologically from 8 to 22 weeks after the initial treatment. It was found that 1) the chemically induced tumors in the mice resembled human pleomorphic/ storiform variant of MFH and cells from the tumor were transplantable subcutaneously in the back of another mouse, 2) the tumors were composed mainly of malignant FH cells, and there were many benign FH cells and fibroblasts in granulation tissues obtained at the initial stage of the experiment, 3) all DNA histograms obtained from MFHs were aneuploid and granulation tissues were diploid, and 4) benign FH cells in the granulation tissue appeared to have higher DNA synthesis activity than typical fibroblasts on the basis of bromodeoxyuridine (BrdU) labeling and cytofluorometric studies. From these findings, we suggest that the FH cells are not only a merely morphologically changed fibroblast, but also a biologically ominous cell which may contribute to develop MFH in mice.

Therapeutic spectrum of nemonapride and its relationship with plasma concentrations of the drug and prolactin.

The therapeutic spectrum of nemonapride, a new substituted benzamide, and its relationship with plasma concentrations of the drug and prolactin were investigated by a fixed-dose study (18 mg/day for 3 weeks) in 31 patients with acutely exacerbated schizophrenia. Of 31 patients, 25 (80.6%) were responders who showed a reduction in symptoms (percentage of improvement) of 50% or more after 3 weeks. The mean values of percentage of improvement in scores on the total Brief Psychiatric Rating Scale (BPRS) and the five subscale symptoms were 71.5% for total, 73.2% for Positive, 86.0% for Excitement, 53.9% for Negative, 84.2% for Cognitive, and 67.5% for Anxiety-Depression. Responders had higher percentage of improvement in positive (84.6 +/- 17.0% vs. 25.9 +/- 15.7%; p < 0.001) and anxiety-depression (76.9 +/- 18.8% vs. 28.5 +/- 39.9%; p < 0.005) symptoms than did nonresponders after 3 weeks. The percentage of improvement in total BPRS after 2 weeks was well correlated with that after 3 weeks (Spearman rank correlation coefficient: r(s) = 0.711; p < 0.01). There was an inverted U-shaped relationship between plasma drug concentrations (nemonapride plus desmethylnemonapride) and percentage of improvement in total BPRS symptoms after a 3-week treatment (y = 46.9 + 73.9x - 44.2x2; p < 0.001). These findings suggest that nemonapride has a broad therapeutic spectrum in the treatment of acute schizophrenia. The improvements in scores for the Positive and Anxiety-Depression subscale symptoms are regarded as determinant factors for total response to nemonapride. An assessment of clinical status after 2 weeks and plasma drug monitoring may be useful for the prediction of the final outcome for patients.

Effects of repeated ingestion of grapefruit juice on the single and multiple oral-dose pharmacokinetics and pharmacodynamics of alprazolam.

The effects of repeated ingestion of grapefruit juice, an inhibitor of cytochrome P450 3A4 (CYP3A4), on the pharmacokinetics and pharmacodynamics of both single and multiple oral doses of alprazolam, a substrate of CYP3A4, were examined. In study 1, eight healthy volunteers ingesting 600 ml/day water or grapefruit juice for 10 days took a single oral 0.8-mg dose of alprazolam on the eighth day. Plasma drug concentrations were monitored up to 48 h after alprazolam dosing together with evaluation of psychomotor function. Grapefruit juice altered neither the plasma concentrations of alprazolam at any time points, any pharmacokinetic parameters, nor the majority of psychomotor function parameters in subjects. In study 2, 11 patients with anxiety disorders receiving alprazolam (0.8-2.4 mg/day) ingested grapefruit juice (600 ml/day) for 7 days. Blood samples were collected before and during grapefruit juice ingestion and 1 week after its discontinuation together with an assessment of clinical status. Grapefruit juice altered neither the steady-state plasma concentration of alprazolam nor the clinical status in patients. The present study shows that grapefruit juice is unlikely to affect pharmacokinetics or pharmacodynamics of alprazolam due to its high bioavailability.

Ultrasound-guided core needle biopsy for breast cancer: preliminary report.

BACKGROUND: Ultrasound-guided automated percutaneous core needle biopsy (US-CNB) for breast tumors has been introduced into clinical practice, but it has not yet been used routinely. We evaluated its usefulness, especially in terms of histological accuracy. METHODS: Thirty-one consecutive patients underwent mammography followed by breast biopsy with the automated core needle biopsy device. RESULTS: Mammography was highly suggestive of malignancy or suspicious abnormalities in 17 cases whose histological findings from US-CNB specimens were invasive ductal carcinoma without exception. The other 14 cases with benign or probably benign mammography findings showed no malignancy histologically in the US-CNB specimens. In cases of malignancy, the accuracy rates of histological findings for the specimens obtained by US-CNB were 94.1% in histological type, 100% in direct infiltration, 82.4% in lymphatic infiltration, 82.4% in venous infiltration, 94.1% in histological grading and 82.4% in intraductal spread. CONCLUSION: US-CNB was useful for making reliable preoperative histopathological diagnosis and may substitute fine needle aspiration biopsy and surgical biopsy.

Study on the relationship between plasma nitrite and nitrate level and salt sensitivity in human hypertension : modulation of nitric oxide synthesis by salt intake.

BACKGROUND: High salt intake suppresses the effect of nitric oxide (NO) in the peripheral resistance vessels in animal models. We tested the hypothesis that the modulation of endogenous NO is related to salt sensitivity in human hypertension. METHODS AND RESULTS: Inpatients with essential hypertension (n=24) were maintained on a normal-salt diet (12 g/d NaCl) for 3 days, a low-salt diet (2 g), a high-salt diet (20 to 23 g), and a low-salt diet for 7 days. Normotensive subjects (n=16) were maintained on the first 2 salt diets. The hypertensive patients whose average 24-hour blood pressure was increased by >5% by salt loading were assigned to group 1 (n=8) and the others to group 2 (n=16). Nitrate plus nitrite (NO(x)) was measured by the Griess method, and asymmetrical dimethylarginine (ADMA) by high-performance liquid chromatography. The plasma NO(x) level during the normal-salt diet was lower in group 1 than in group 2 and the normotensive group. After salt loading, the plasma NO(x) level was decreased and reversed after the second salt restriction. Plasma ADMA level was increased after salt loading and decreased after salt restriction. The change in plasma NO(x) level was correlated inversely with those in blood pressure (r=-0.59, P=0.0007) and plasma ADMA level (r=-0.64, P=0.003) after salt loading and restriction. CONCLUSIONS: Modulation of NO synthesis by salt intake may be involved in a mechanism for salt sensitivity in human hypertension, presumably via the change in ADMA.

Salt-induced exacerbation of morning surge in blood pressure in patients with essential hypertension.

The morning surge in blood pressure (BP) is related to the morning occurrence of lethal cardiovascular events. We tested the hypothesis that salt intake may be associated with the morning surge in BP in essential hypertension. Seventy-six patients were admitted and placed on a low salt diet (2 g/day) for 7 days followed by a high salt diet (20-23 g/day) for another 7 days. At the end of each salt diet, 24-h ambulatory BP and heart rate monitorings and head-up tilt (HUT) test were performed. Patients whose average mean BP (MBP) was increased by more than 10% by salt loading were assigned to the salt-sensitive (SS) group (n = 37); the remaining patients, whose MBP was increased by less than 10%, were assigned to the non-salt-sensitive (NSS) group (n = 39). The increase in ambulatory MBP during 6.30-8.00 am above the baseline (2.00-4.00 am) was significantly enhanced by salt loading in the NSS group (P < 0.05), but not in the SS group. The coefficient of variation of 24-h MBP and heart rate was increased by salt loading only in the NSS group. The significant elevation of plasma noradrenaline concentration after awakening, which was noted during the low salt diet period, was unchanged during the high salt diet period in the NSS group, but abolished in the SS group. Salt loading enhanced HUT-induced decrease in systolic BP without affecting the heart rate response only in the NSS group. We conclude that the morning surge in BP is enhanced by salt loading in the NSS type of essential hyper- tension, presumably by the excessive activation of the sympathetic nervous system. Journal of Human Hypertension (2000) 14, 57-64.

Involvement of phosphorylation of beta-subunit in cAMP-dependent activation of L-type Ca2+ channel in aortic smooth muscle-derived A7r5 cells.

We investigated the effect of intracellular cAMP on the gating kinetics of L-type Ca2+ channel in an A7r5 smooth muscle-derived cell line using the whole-cell patch-clamp technique. Application of dibutyryl cyclic AMP (db-cAMP) to the cell increased the magnitude of Ca2+ currents through L-type Ca2+ channels (I(Ca)), and shifted the current-voltage relationship (I-V curve) for I(Ca) to the left. The magnitudes of maximum I(Ca) were 14.1 +/- 0.7 before and 16.0 +/- 1.1 pA/pF after application of 1 mM db-cAMP (P < 0.05). The values of the half-activation potential (V(1/2)) of I(Ca), estimated from activation curves, were -7.0 +/- 0.8 mV before and -10.8 +/- 1.0 mV after application of db-cAMP (P < 0.05). In cells pretreated with 10 microM Rp-cAMPS (a specific inhibitor of PKA), db-cAMP affected neither the I-V curve nor the activation curve for I(Ca). In cells pretreated with the antisense oligonucleotide for the beta-subunit of L-type Ca2+ channel, db-cAMP failed to enhance I(Ca) or alter the activation curve. On the other hand, in the cells pretreated with the nonsense oligonucleotide, application of db-cAMP caused an increase in magnitude of I(Ca) and shifted the activation curve to the left. Western blot analysis revealed that the pretreatment of cells with antisense oligonucleotide but nonsense oligonucleotide reduced the expression of the beta-subunit of the L-type Ca2+ channel. We conclude that the cAMP-dependent phosphorylation of the beta-subunit potentiates the voltage dependency of the activation kinetics of the L-type Ca2+ channel in A7r5 cells.