Endothelin receptor antagonist reverses decreased NO system in the kidney in vivo during exercise

Vascular endothelial cells produce endothelin (ET)-1, a potent vasoconstrictor peptide, and nitric oxide (NO), a potent vasodilator substance. There are interactions between ET-1 and NO. Exercise results in a marked decrease in renal blood flow. We previously reported that exercise causes an increase of ET-1 production in the kidney, whereas production of NO in the kidney is decreased. Furthermore, we recently revealed that the magnitude of decrease in blood flow to the kidney during exercise was significantly attenuated by the administration of the endothelin-A (ET(A)) receptor antagonist, strongly suggesting that endogenously increased ET-1 participates in the decrease of blood flow in the kidney during exercise. Because it was demonstrated that ET-1 depresses NO synthase (NOS) activity of cultured cells in vitro, we hypothesized that an increase of ET-1 production in kidney during exercise contributes to a decrease of NO production in kidney in vivo. We studied whether administration of the ET(A) receptor antagonist attenuates the decreases of NOS activity and NO production in the kidney during exercise. Rats performed treadmill running for 30 min after pretreatment with an ET(A) receptor antagonist (TA-0201, 0.5 mg/kg; TA-0201-treated exercise group) or vehicle (vehicle-treated exercise group). Control rats remained at rest (vehicle-treated sedentary group). Blood flow in the kidney was decreased by this exercise, but the magnitude of the decrease after pretreatment with TA-0201 was significantly smaller than that after pretreatment with vehicle. NOS activity in kidney was significantly lower in the vehicle-treated exercise group than in the vehicle-treated sedentary group, whereas that in the TA-0201-treated exercise group was significantly higher than that in the vehicle-treated exercise group. Expressions of endothelial NOS protein and NOx, the stable end product of NO, i.e., nitrite/nitrate, concentration in the kidney were significantly lower in the vehicle-treated exercise group than in the vehicle-treated sedentary group, whereas those in the TA-0201-treated exercise group were significantly higher than those in the vehicle-treated exercise group. The data suggest that increased ET-1 production in the kidney during exercise contributes to the decreases of NOS activity and NO production. Therefore, the present study provides a possibility that the exercise-induced increase in production of ET-1 in the kidney causes a decrease in blood flow in the kidney through two pathways, i.e., vasoconstrictive action and the action of attenuating NO production.     

Involvement of BNIP1 in apoptosis and endoplasmic reticulum membrane fusion

BNIP1, a member of the BH3-only protein family, was first discovered as one of the proteins that are capable of interacting with the antiapoptotic adenovirus E1B 19-kDa protein. Here we disclose a totally unexpected finding that BNIP1 is a component of the complex comprising syntaxin 18, an endoplasmic reticulum (ER)-located soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE). Functional analysis revealed that BNIP1 participates in the formation of the ER network structure, but not in membrane trafficking between the ER and Golgi. Notably, a highly conserved leucine residue in the BH3 domain of BNIP1 plays an important role not only in the induction of apoptosis but also in the binding of alpha-SNAP, an adaptor that serves as a link between the chaperone ATPase NSF and SNAREs. This predicts that alpha-SNAP may suppress apoptosis by competing with antiapoptotic proteins for the BH3 domain of BNIP1. Indeed, overexpression of alpha-SNAP markedly delayed staurosporine-induced apoptosis. Our results shed light on possible crosstalk between apparently independent cellular events, apoptosis and ER membrane fusion.     

Involvement of ATP,increase of intracellular calcium and the early expression of <Emphasis Type="Italic">c-fos</Emphasis> in the repair of rat fetal articular cartilage

To compare the potential of adult and fetal animals to repair articular cartilage, we investigated the early process after creating superficial defects in the femoral knee cartilage in rat models. In fetuses at 19 days of gestation, both chondrocytes and the extracellular matrix responded notably by 48 h after artificial injury. Staining patterns with safranin O revealed that, by 1 h after injury, some components of the extracellular matrix around the wound were modified, and the change spread from the limited region to the entire knee cartilage within 24 h. The chondrocytes in the area surrounding the wound transiently expressed increased level of c-fos from 1 h to 6 h. The wound remained 1 day after birth, i.e., 72 h after injury, but was completely repaired 10 days after birth. In contrast, neither visible responses nor transient c-fos expression was observed in 12-week-old adult articular cartilage 48 h after injury. We also examined the relationships between the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the induction of c-fos expression in the cartilage. Applications of ATP or Ca²⁺ ionophore A23187, both of which increase [Ca²⁺]_i, induced immediate expression of c-fos in primary cultured chondrocytes: 1 M ATP elicited an increase of [Ca²⁺]_i in chondrocytes in fetal cartilage slices, but 1 mM was required in adult cartilage slices. Our findings show the presence of a signaling pathway that is apparently active in the repair of fetal but not adult articular cartilage and that involves the intercellular transfer of ATP, increase of [Ca²⁺]_i, and expression of c-fos in cartilage.This study was supported in part by Health Sciences Research Grants for Research on Human Genome, Tissue Engineering and Food Biotechnology to M.O. from the Japanese Ministry of Health, Labor and Welfare     

Generation of pluripotent stem cells from neonatal mouse testis

Although germline cells can form multipotential embryonic stem (ES)/embryonic germ (EG) cells, these cells can be derived only from embryonic tissues, and such multipotent cells have not been available from neonatal gonads. Here we report the successful establishment of ES-like cells from neonatal mouse testis. These ES-like cells were phenotypically similar to ES/EG cells except in their genomic imprinting pattern. They differentiated into various types of somatic cells in vitro under conditions used to induce the differentiation of ES cells and produced teratomas after inoculation into mice. Furthermore, these ES-like cells formed germline chimeras when injected into blastocysts. Thus, the capacity to form multipotent cells persists in neonatal testis. The ability to derive multipotential stem cells from the neonatal testis has important implications for germ cell biology and opens the possibility of using these cells for biotechnology and medicine.     

Protein kinase Cepsilon and the antiadrenergic action of adenosine in rat ventricular myocytes

Adenosine-induced antiadrenergic effects in the heart are mediated by adenosine A(1) receptors (A(1)R). The role of PKCepsilon in the antiadrenergic action of adenosine was explored with adult rat ventricular myocytes in which PKCepsilon was overexpressed. Myocytes were transfected with a pEGFP-N1 vector in the presence or absence of a PKCepsilon construct and compared with normal myocytes. The extent of myocyte shortening elicited by electrical stimulation of quiescent normal and transfected myocytes was recorded with video imaging. PKCepsilon was found localized primarily in transverse tubules. The A(1)R agonist chlorocyclopentyladenosine (CCPA) at 1 microM rendered an enhanced localization of PKCepsilon in the t-tubular system. The beta-adrenergic agonist isoproterenol (Iso; 0.4 microM) elicited a 29-36% increase in myocyte shortening in all three groups. Although CCPA significantly reduced the Iso-produced increase in shortening in all three groups, the reduction caused by CCPA was greatest with PKCepsilon overexpression. The CCPA reduction of the Iso-elicited shortening was eliminated in the presence of a PKCepsilon inhibitory peptide. These results suggest that the translocation of PKCepsilon to the t-tubular system plays an important role in A(1)R-mediated antiadrenergic actions in the heart.     

Angiotensin II receptor blockade corrects altered expression of gap junctions in vascular endothelial cells from hypertensive rats

Blockade of the renin-angiotensin system improves the impaired endothelium-dependent relaxations associated with hypertension and aging, partly through amelioration of endothelium-derived hyperpolarizing factor (EDHF)-mediated responses. Although the nature of EDHF is still controversial, recent studies have suggested the involvement of gap junctions in EDHF-mediated responses. Gap junctions consist of connexins (Cx), and we therefore tested whether the expression of Cx in vascular endothelial cells would be altered by hypertension and antihypertensive treatment. Spontaneously hypertensive rats (SHR) were treated with either the angiotensin II type 1 receptor antagonist candesartan or the combination of hydralazine and hydrochlorothiazide for 3 mo from 5 to 8 mo of age. Confocal laser scanning microscopy after immunofluorescent labeling with antibodies against Cx37, Cx40, and Cx43 revealed that the expression of Cx37 and Cx40 in endothelial cells of the mesenteric artery was significantly lower in SHR than in WKY. Treatment with candesartan, but not the combination of hydralazine and hydrochlorothiazide, significantly increased the expression of Cx37 and Cx40, although blood pressure decreased similarly. On the other hand, the expression of Cx43, though scarce and heterogeneous, was increased in SHR compared with WKY, and candesartan treatment lowered the expression of Cx43. These findings suggest that renin-angiotensin system blockade corrects the decreased expression of Cx37 and Cx40 in arterial endothelial cells of hypertensive rats, partly independently of blood pressure, whereas the expression of Cx43 changed in the opposite direction. It remains to be clarified whether these changes in Cx37 and Cx40 are related to endothelial function, particularly that attributable to EDHF.     

Novel<Emphasis Type="Italic"> SBDS</Emphasis> mutations caused by gene conversion in Japanese patients with Shwachman-Diamond syndrome

Shwachman-Diamond syndrome (SDS; OMIM 260400) is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency, bone marrow dysfunction and metaphyseal chondrodysplasia. SDS is caused by mutations in SBDS, an uncharacterized gene. A previous study in SDS patients largely of European ancestry found that most SBDS mutations occurred within a ~240-bp region of exon 2 and resulted from gene conversion due to recombination with a pseudogene, SBDSP. It is unknown, however, whether these findings are applicable to other ethnic groups. To address this question, we examined SBDS mutations in six Japanese families with SDS by direct sequencing. We identified compound heterozygous mutations in four families: two were recurrent (96–97insA, 258+2T>C), and three were novel [292–295delAAAG, (183–184TA>CT +201A>G), (141C>T+183–184TA>CT+201A>G)] mutations. Most of these mutations also appear to result from gene conversion, but the conversion events occurred at various sites between intron 1 and exon 3. Thus, gene conversion mutations in SBDS are common to different ethnic groups, but they are not confined to a limited region of the gene.Y. Makita, M. Masuno, H. Ohashi, G. Nishimura, S. Ikegawa are members of the Japanese Skeletal Dysplasia Consortium     

Ca2+-induced activation of ATPase activity of myosin Va is accompanied with a large conformational change

We succeeded in expressing the recombinant full-length myosin Va (M5Full) and studied its regulation mechanism. The actin-activated ATPase activity of M5Full was significantly activated by Ca(2+), whereas the truncated myosin Va without C-terminal globular domain is not regulated by Ca(2+) and constitutively active. Sedimentation analysis showed that the sedimentation coefficient of M5Full undergoes a Ca(2+)-induced conformational transition from 14S to 11S. Electron microscopy revealed that at low ionic strength, M5Full showed an extended conformation in high Ca(2+) while it formed a folded shape in the presence of EGTA, in which the tail domain was folded back towards the head-neck region. Furthermore, we found that the motor domain of myosin Va folds back to the neck domain in Ca(2+) while the head-neck domain is more extended in EGTA. It is thought that the association of the motor domain to the neck inhibits the binding of the tail to the neck thus destabilizing a folded conformation in Ca(2+). This conformational transition is closely correlated to the actin-activated ATPase activity. These results suggest that the tail and neck domain play a role in the Ca(2+) dependent regulation of myosin Va.