Matanobu Abe

Role of activin A in murine mast cells: modulation of cell growth, differentiation, and migration

Activins, members of the transforming growth factor‐β (TGF‐β) superfamily, are potent growth and differentiation factors. Our previous studies revealed that activin A, a homodimer of inhibin/activin βA, was induced in mast cells and peritoneal macrophages in response to their activation. In the present study, we examined the roles of activin A in murine bone marrow‐derived, cultured mast cell progenitors (BMCMCs), which expressed gene transcripts for molecules involved in activin signaling, suggesting that BMCMCs could be target cells of activin A. Treatment of activin A inhibited 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide uptake into BMCMCs in a dose‐dependent manner. The IC50 concentration was 2.1 nM, which was less potent than 185 pM TGF‐β1. Activin A treatment caused morphological changes toward the differentiated cells at 2 nM and up‐regulated mRNA of mouse mast cell protease‐1 (mMCP‐1), a marker enzyme of mature mucosal mast cells, at 1 nM. Activin A also showed activity in inducing migration of BMCMCs; the optimal concentration for maximal migration was 10 pM, which was much lower than the concentrations to inhibit cell growth and to activate the mMCP‐1 gene. Taking the present results together with our previous results, it is suggested that activin A secreted from activated immune cells recruits mast cell progenitors to sites of inflammation and that with increasing activin A concentration, the progenitors differentiate into mature mast cells. Thus, activin A may positively regulate the functions of mast cells as effector cells of the immune system.

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.

Degranulation in RBL-2H3 cells: regulation by calmodulin pathway

Involvement of the calmodulin pathway in Ca2+‐induced degranulation was evaluated in RBL‐2H3 mast cells. Pretreatment of RBL‐2H3 cells with a calmodulin antagonist, W‐13, blocked ionomycin‐dependent release of β‐hexosaminidase into the supernatant, although W‐13 treatment alone slightly but significantly increased the release. Ca2+/calmodulin activates various protein kinases and phosphatases including myosin‐light chain kinase (MLCK), calmodulin‐dependent protein kinases (CaMKs), and calcineurin. When RBL‐2H3 cells were pretreated with a MLCK inhibitor, ML‐7, or a CaMKs inhibitor, KN‐93, the ionomycin‐dependent release of β‐hexosaminidase into the supernatant was inhibited. In addition, pretreatment with calcineurin inhibitors, cyclosporin A and FR901725, resulted in blockage of the ionomycin‐dependent release of β‐hexosaminidase into the supernatant. Our results indicate that Ca2+/calmodulin, activated calmodulin, is indispensable for Ca2+‐induced degranulation, and that within the calmodulin pathways, at least MLCK, CaMKs and calcineurin positively regulate the release of granules initiated by increasing cytosolic Ca2+concentrations in RBL‐2H3 cells.

Relation between diet and protozoal population in the rumen.

I. Four feeding trials were made to investigate relations between diet and protozoal population in the rumen. 2. When a ration containing no concentrate was used, the number of entodiniomorphs decreased rapidly. The number of entodinioniorphs increased with the amount of concentrate. Rice straw, which was used as a sole source of roughage, was not always necessary for protozoa to survive in the rumen. 3. The type of diet affected the holotrich population to a much smaller extent than the entodiniomorph population.

Calcium-regulated expression of activin A in RBL-2H3 mast cells.

The present study examined the regulatory expression of activin A, a potent growth and differentiation factor, in rat basophilic leukemia (RBL-2H3) mast cells. Treatment of RBL-2H3 cells sensitized with anti-dinitrophenyl IgE with multivalent dinitrophenyl led to a clear increase in RT-PCR products of inhibin/activin beta(A). The steady-state mRNA of inhibin/activin beta(A) was also induced by increasing cytosolic Ca(2+) concentration with ionomycin, which required de novo protein synthesis, and was regulated at the transcriptional level. Pretreatment of RBL-2H3 cells with antagonists or inhibitors for the calmodulin pathway blocked ionomycin-dependent inhibin/activin beta(A) transcription and mRNA induction, suggesting the involvement of calmodulin-dependent kinase (CaMK) and calcineurin. The ionomycin-dependent inhibin/activin beta(A) induction was also partially blocked by preincubation with c-Jun NH(2)-terminal kinase (JNK) and p38 kinase inhibitors, but not with MEK1 inhibitor. These results suggest that inhibin/activin beta(A) gene activation is achieved by the JNK and p38 kinase activation through the calmodulin pathway in mast cells.

Effects of nipple or bucket feeding of milk-substitute on rumen by-pass and on rate of passage in calves.

I . Effects of feeding liquid milk-substitute to young calves either by nipple-pail or open-bucket o n the rumen by-pass and on the rate of passage were studied. z. Sixteen Holstein calves, aged I week initially, were used in three experiments in which calves were slaughtered after they were given liquids (milk-substituteand water) containingchromic oxide and SrCI, .6H,O as a tracer either by the nippleor bucket-feeding method, and the distribution of tracers to the rumen, abomasum and the lower alimentary tracts was examined. 3. When the liquid milk-substitute containing tracers was given by the nippleor bucket-feeding method to calves having been trained to the corresponding procedures for the preceding I week, most of the tracers was directed into the omasum and abomasum. There seemed no difference in the functioning of oesophageal groove closure between the two feeding procedures. Even when the liquid milk-substitute containing tracers was given by the nipple-or bucket-feeding method to calves which had been accustomed to different procedures for the preceding week, the majority of tracers were found in the abomasum immediately after administration, though a slightly greater proportion of the tracers entered the reticulo-rumen. 4. Continuing bucket feeding of liquid milk-substitute effected an efficient closure of the oesophageal groove at least up to 16 weeks of age. After calves were accustomed to consume liquid milk-substitute from the bucket from I to 4 weeks of age, drinking warm water from the bucket also caused efficient closure at least up to 16 weeks of age. 5 . When tracers were administered with warm water, Cr,O, and strontium, especially the latter, transferred much more rapidly to the lower gut than when they were administered with liquid milk-substitute, probably reflecting the curd formation of the milk-substitute in the abomasum. When liquid milk-substitute with tracers was fed by the bucket-feeding method, Sr transferred more rapidly to the lower gut than when the milk-substitute was fed by the nipple-feeding method, indicating that the feeding procedure of liquid milk-substitute has an apparent effect on the rate of passage.

Mechanism whereby holotrich ciliates are retained in the reticulo-rumen of cattle

The concentrations of holotrichs, reducing sugars and fermentation end-products, and the fluid dilution rates, in the cranial (CR), ventral (V) and caudal (CA) regions of the reticulo-rumen of cows were compared. Additionally, holotrich numbers in fluid and solid digesta samples taken from the dorsal (D) region were determined at different time-intervals. Holotrich numbers expressed per ml fluid at site D were almost twofold those expressed per g solid digesta, and the fluctuation of their numbers in fluid preceded that in solid digesta. Holotrich concentrations at site CR were higher before feeding than after feeding, while those at sites V and CA were lower before feeding than for a few hours after feeding. At sites V and CA, holotrich concentrations fluctuated in good agreement with reducing sugar concentrations, but the increase in the former always preceded that in the latter. The concentration of volatile fatty acids (VFA) tended to decrease from site CA to site CR, while the dilution rate was highest in site CR. The results suggest that migration of holotrichs from the reticulum to the rumen and vice versa are the cause of fluctuation in their numbers, and also the mechanism by which they are retained in the rumen of cattle.

Neonatal diarrhoea in calves given milk-substitutes differing in fat source and fed by different procedures

1. The incidence of diarrhoea, digestibilities of nutrients and the faecal bacterial flora were compared among three groups of Holstein male calves up to 3 weeks of age. Two groups of four calves were given a milk-substitute containing tallow by nipple-pail (group TN) and by open-bucket (group TB). The third group of four calves was nipple-fed a milk-substitute containing soya-bean oil (group SN). Each of the milk-substitutes contained approximately 300 g milk-protein and 100 g fat/kg dry matter (DM)., 2. Mean faecal DM contents (g/kg) were 217, 185 and 112 for groups TN, TB, and SN respectively and the corresponding pH values were 7.21, 7.00 and 6.50. The difference between groups TN and SN was statistically significant (P less than 0.05). 3. No difference was observed between groups TN and SN in the apparent digestibilities of DM, crude protein (CP, nitrogen X 6.25), diethyl ether extract (EE) and total reducing sugars. But in the group TB, the digestibility of EE was significantly lower (P less than 0.05), and that of CP tended to be, though not significantly, lower than in the other two groups. 4. Bacterial flora in faeces showed considerably wide quantitative variations among individual calves, but there was a tendency for increased viable counts of Lactobacilli in faeces of group SN. 5. The present suggested that an appreciable difference in the mechanism would exist between diarrhoea occurring when milk-substitute was offered by bucket and when highly-unsaturated vegetable oils were contained in it. Possible mechanisms were also discussed.

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.

INCREASED CARTILAGE AND BONE FORMATION IN SPONTANEOUSLY HYPERCHOLESTEROLEMIC RATS

Spontaneously hypercholesterolemic (SHC) rats are known to exhibit accelerated bone resorption. We compared endochondral bone formation induced by implantation of demineralized bone matrix (DBM) to 4-week-old SHC rats with that of age-matched Sprague-Dawley (SD) rats. When DBM prepared from adult SD rats was implanted, the cartilageous area enlarged, and C-propeptide of type II procollagen content on day 7 was higher in SHC rats. Alkaline phosphatase activity and calcium content on day 12 and tartrate-resistant acid phosphatase activity on day 19 were higher in SHC rats. These results suggest active chondrogenesis, with a subsequent increase in osteogenesis, and stimulated osteoclastic bone resorption in SHC rats. When DBM from 10-week-old SHC rats was implanted into SD or SHC rats, the levels of bone forming parameters on day 12 were reduced to one-third, suggesting inhibiting factor(s) for bone induction in bone matrix of SHC rats. In contrast, when DBM from 6-month-old SHC rats was implanted, although bone forming parameters in SD rats were comparable to the case of implantation of DBM from SD rats, the accelerated bone formation detected in SHC rats was blocked, indicating resistance to systemic bone inducing factor(s) of SHC rats in aged bone matrix. These results suggest that age-related decrease in responses to some systemic bone inducing factor may lead to the bone loss with advancing age.