Arachidonic acid and docosahexaenoic acid supplementation increases coronary flow velocity reserve in Japanese elderly individuals.

BACKGROUND: Arachidonic acid (ARA) and docosahexaenoic acid (DHA) are important components of phospholipids and cell membranes. There has, however, been no clinical report on the direct effects of ARA and DHA on coronary circulation. OBJECTIVE: To evaluate the effects of ARA and DHA on coronary circulation using the measurement of coronary flow velocity reserve (CFVR) by transthoracic Doppler echocardiography (TTDE). METHODS: A double-blind, placebo-matched study of 28 Japanese elderly individuals (19 men, mean age 65 years) conducted to compare the effects of polyunsaturated fatty acids (PUFA; ARA 240 mg/day, DHA 240 mg/day) and placebo on CFVR. Coronary flow velocity (CFV) of the left anterior descending coronary artery was measured at rest and during hyperaemia by TTDE to determine CFVR. RESULTS: There were no significant differences in CFV at rest or during hyperaemia in CFVR at baseline in the two groups (PUFA versus placebo 17 (7 SD) versus 16 (6), 62 (20) versus 59 (12), and 3.85 (1.04) versus 3.98 (0.83) cm/s, respectively). After three months' supplementation, CFV during hyperaemia was significantly higher in the PUFA than in the placebo group (73 (19) versus 64 (12) cm/s, p<0.01) although no significant difference was found between the two groups in CFV at rest (17 (7) versus 16 (4) cm/s). CFVR thus significantly increased after PUFA consumption (3.85 (1.04) versus 4.46 (0.95), p = 0.0023). CONCLUSION: Three months' supplementation of PUFA increased CFVR in Japanese elderly individuals, which suggests beneficial effects of PUFA on the coronary microcirculation.

Quantitative RT-PCR for inhibin/activin subunits: measurements of rat hypothalamic and ovarian inhibin/activin subunit mRNAs during the estrous cycle.

Inhibins (alpha-beta(A) and alpha-beta(B)) and activins (beta(A)-beta(A), beta(A)-beta(B) and beta(B)-beta(B)) were originally isolated from ovarian follicular fluids as FSH secretion modifiers. Inhibin/activin subunits, alpha, beta(A) and beta(B), are widely distributed in several tissues, including gonads and brain, and inhibins and activins have been reported to be involved in ovarian or hypothalamic functions. In this study, we established and employed a competitive RT-PCR assay system for rat inhibin/activin subunits by capillary electrophoresis to determine rat hypothalamic and ovarian inhibin/activin subunit mRNA levels during the estrous cycle. Linearity of standards for alpha, beta(A), and beta(B) subunit assays were between 0.01-0.3 amol, 0.003-0.09 amol and 0.002-0.02 amol of each fragment DNA as a standard, respectively. Hypothalamic beta(A) subunit mRNA during the estrous morning (1000 h) tended to be increased compared with that of the proestrous evening (1700 h), although they were not significantly different. Ovarian alpha subunit mRNA levels tended to be increased during the proestrous morning (1000 h) and were significantly increased in the proestrous evening (1700 h), compared with diestrus and estrus (P < 0.05). Ovarian beta(A) subunit mRNA was also significantly higher in the proestrous evening, compared with diestrus and estrus (P < 0.05), but in the case of beta(B) subunit mRNA there was no difference among diestrus, proestrus and estrus. We thus established a sensitive competitive RT-PCR system for the measurement of inhibin/activin alpha, beta(A) and beta(B) subunits, and this assay system would be helpful for the study of inhibin/activin action in brain and other tissues where these factors are expressed at low levels.

Immunolocalization of type I or type II activin receptors in the rat brain.

We have studied immunolocalization of activin receptors in the central nervous system using polyclonal antibodies (IgG) to type I (50-55 kDa, ActRI), type II (70-75 kDa, ActRII) or a subtype of type II known as type IIB (ActRIIB) receptors of activin. A total of 7 antisera to rat activin receptors was generated, i.e. 3 kinds of antisera to the extracellular domain (ActRI(81-89), ActRII(91-100), or ActRIIB(90-99)) and 4 antisera to the kinase domain (ActRI(323-333), ActRII(307-319), ActRII(407-420) or ActRIIB(306-319)). The region of aa 407-420 of ActRII is identical with that of ActRIIB. At first, we characterized these antibodies by Western blot analysis using ovarian proteins fractionated by preparative SDS-PAGE. All antibodies to ActRII and ActRIIB specifically reacted with 75 kDa-proteins which could also bind to activin-A. Anti-ActRII(91-100) antibody also reacted with 62 kDa-proteins which were capable of binding with activin-A. Although no positive reactions to anti-ActRI(81-89) antibody were seen in ovarian proteins, a positive reaction was detected at 52 kDa only when the proteins were deglycosylated. By use of these antibodies, immunolocalization of activin receptors was examined in the rat brain. The patterns of expression of activin type I and type II receptors were different. Positive reactions to anti-ActRII(91-100) antibody were detected in neurons of the cerebral cortex, hippocampus, medial amygdala and thalamus. In the hypothalamus, some neurons of the supraoptic nucleus were weakly stained, and widely scattered neurons of the lateral hypothalamic area were moderately stained. On the contrary, the most intense reactions to anti-ActRI(81-89) antibody were detected in neurons of the lateral hypothalamic area. In addition, many neurons of the cerebral cortex were also stained, but neurons of the hippocampus and the amygdala were not stained. These results suggest that activin may have physiological roles not only for hypothalamic neuroendocrinological and feeding-related systems as suggested previously but may also have functions in cortical and limbic pathways as a neuromodulator or for maintenance of neurons.

Suppressed bone induction by follistatin in spontaneously hypercholesterolemic rat bone.

Bone inducing activity in demineralized bone matrix (DBM) of young spontaneously hypercholesterolemic (SHC) rats has been shown to be lower than that of aged SHC rats. This study examined the involvement of bone follistatin, an activin-binding protein, in bone induction. Immunoreactive follistatin was higher in DBM from 10-week-old SHC rats (DBM-10wk) than in DBM from 6-month-old SHC rats (DBM-6mo). When DBM without follistatin supplement was implanted, the C-propeptide of type II procollagen and calcium contents on day 12 in implants of DBM-6mo were 68% and 40% higher than those of DBM-10wk, respectively. In contrast, follistatin supplement to DBM decreased C-propeptide of type II procollagen and calcium contents in implants of both DBM-10wk and DBM-6mo, and the levels of these parameters were comparable between DBM-10wk and DBM-6mo, indicating reduced formation of cartilage and bone. These findings suggest that 1) follistatin content in bone matrix decreases with advancing age in SHC rats, and 2) the follistatin interferes with endochondral bone formation. We demonstrate that the lower bone induction of DBM from young SHC rats was partly due to the abundance of follistatin in bone matrix.