Nitric oxide (NO) is synthesized from l-arginine by the Ca(2+)/calmodulin-sensitive endothelial NO synthase (NOS) isoform (eNOS). The present study assesses the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) in endothelium-dependent relaxation and NO synthesis. The effects of three CaMK II inhibitors were investigated in endothelium-intact aortic rings of normotensive rats. NO synthesis was assessed by a NO sensor and chemiluminescence in culture medium of cultured porcine aortic endothelial cells stimulated with the Ca(2+) ionophore A23187 and thapsigargin. Rat aortic endothelial NOS activity was measured by the conversion of l-[(3)H]arginine to l-[(3)H]citrulline. Three CaMK II inhibitors, polypeptide 281-302, KN-93, and lavendustin C, attenuated the endothelium-dependent relaxation of endothelium-intact rat aortic rings in response to acetylcholine, A23187, and thapsigargin. None of the CaMK II inhibitors affected the relaxation induced by NO donors. In a porcine aortic endothelial cell line, KN-93 decreased NO synthesis and caused a rightward shift of the concentration-response curves to A23187 and thapsigargin. In rat aortic endothelial cells, KN-93 significantly decreased bradykinin-induced eNOS activity. These results suggest that CaMK II was involved in NO synthesis as a result of Ca(2+)-dependent activation of eNOS.
"Thus, it is tempting to speculate that OA activates Octβ2R and OAMB in the epithelium positioned between the visceral muscle and lumen for dual physiological processes, i.e. oviduct muscle relaxation and fluid secretion to the lumen (Figure 7). CaMKII activated by OAMB together with Octβ2R may act on nitric oxide synthase (NOS) to release NO, which in turn travels to the muscle for relaxation in a similar mechanism known in other systems –. This is supported by the observation that NOS knockdown in the oviduct epithelium significantly reduces ovulation (our unpublished data). "
[Show abstract][Hide abstract] ABSTRACT: Oviposition is induced upon mating in most insects. Ovulation is a primary step in oviposition, representing an important target to control insect pests and vectors, but limited information is available on the underlying mechanism. Here we report that the beta adrenergic-like octopamine receptor Octβ2R serves as a key signaling molecule for ovulation and recruits protein kinase A and Ca2+/calmodulin-sensitive kinase II as downstream effectors for this activity. We found that the octβ2r homozygous mutant females are sterile. They displayed normal courtship, copulation, sperm storage and post-mating rejection behavior but were unable to lay eggs. We have previously shown that octopamine neurons in the abdominal ganglion innervate the oviduct epithelium. Consistently, restored expression of Octβ2R in oviduct epithelial cells was sufficient to reinstate ovulation and full fecundity in the octβ2r mutant females, demonstrating that the oviduct epithelium is a major site of Octβ2R's function in oviposition. We also found that overexpression of the protein kinase A catalytic subunit or Ca2+/calmodulin-sensitive protein kinase II led to partial rescue of octβ2r's sterility. This suggests that Octβ2R activates cAMP as well as additional effectors including Ca2+/calmodulin-sensitive protein kinase II for oviposition. All three known beta adrenergic-like octopamine receptors stimulate cAMP production in vitro. Octβ1R, when ectopically expressed in the octβ2r's oviduct epithelium, fully reinstated ovulation and fecundity. Ectopically expressed Octβ3R, on the other hand, partly restored ovulation and fecundity while OAMB-K3 and OAMB-AS that increase Ca2+ levels yielded partial rescue of ovulation but not fecundity deficit. These observations suggest that Octβ2R have distinct signaling capacities in vivo and activate multiple signaling pathways to induce egg laying. The findings reported here narrow the knowledge gap and offer insight into novel strategies for insect control.
PLoS ONE 08/2014; 9(8):e104441. DOI:10.1371/journal.pone.0104441 · 3.23 Impact Factor
"Because the phosphorylation of eNOS-Ser 1179 is mediated by several protein kinases, including Akt, AMPK, PKA and CaMKII     , we attempted to identify a kinase responsible for FIR radiation-stimulated eNOS-Ser 1179 phosphorylation. As shown in Fig. 2, we found that H-89 and KN-93 inhibited eNOS-Ser 1179 phosphorylation induced by FIR radiation, suggesting the involvement of PKA and CaMKII in the stimulatory effect of FIR radiation. "
[Show abstract][Hide abstract] ABSTRACT: Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser(1179)) in a time-dependent manner (up to 40 min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca(2+) levels. Treatment with KN-93, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. This study suggests that FIR radiation increases NO production via increasing CaMKII-mediated eNOS-Ser(1179) phosphorylation but TRPV channels may not be involved in this pathway. Our results may provide the molecular mechanism by which FIR radiation improves endothelial function.
Biochemical and Biophysical Research Communications 06/2013; 436(4). DOI:10.1016/j.bbrc.2013.06.003 · 2.30 Impact Factor
"Indeed, the activity of eNOS is regulated by both a calcium/calmodulin dependent fashion59 and AKT dependent eNOS phosphorylation in Ser 1177 8, 60–63. AKT is primarily activated in response to stimulation of transmembrane receptors with intrinsic tyrosine kinase activity or indirectly coupled to tyrosine kinases or to seven transmembrane G protein-coupled receptor11, 61, 64. "
[Show abstract][Hide abstract] ABSTRACT: There is considerable evidence that many disease are associated with endothelial dysfunction and reduced nitric oxide production such as hypertension, obesity, dyslipidemias, diabetes, heart failure, atherosclerosis. Notably these conditions are also characterized by alteration in the adrenergic tone. Whether these two mechanisms are just epiphenomenal each other or there is a functional link, it is still to be established. A starting ground to establish this issue is that vascular endothelium plays an important role in the function of cardiovascular system and that adrenergic receptors on endothelial cells contribute to the regulation of vasomotor tone. The aim of this excerpt is to review current knowledge on the physiology of endothelial adrenergic receptors to contribute to the basis for newer and better approaches to endothelial dysfunction in the setup of cardiovascular conditions.
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