Activation of cAMP-dependent Protein Kinase in Epidermis by the Compounds which Increase Epidermal cAMP

Pig epidermal slices were incubated with various compounds which increased epidermal cAMP (adenosine 3',5'-monophosphate), and the change in cAMP-dependent protein kinase activity ratio was studied by the method of Cherrington et al (J Biol Chem 251:5209–5218, 1976) with modification.Epinephrine (5 × 10−5 m), histamine (10−4 m) and adenosine (10−3 m), potent agonists of epidermal adenyl cyclase, fully activated the protein kinase (PK) during an incubation of 30 to 45 seconds, that was much shorter than that required for maximal cAMP accumulation under the same conditions (5 min). With such a brief stimulus, the epidermal cAMP-PK system did not become refractory and responded to repeated stimuli. Prostaglandin E2 (PGE2) and isobuthylmethylxanthine (IBMX) and ethanol only partially activated the enzyme. Prostaglandin F2α. (PGF2α) and theophylline which were much less effective in increasing epidermal cAMP, activated the enzyme to the same extent as PGE2 and IBMX respectively.These results suggest that protein kinase activation takes place in response to a cAMP increase in small locus of the cell. Such an increase in cAMP can be very small or even not measurable when measured as total cAMP in the tissue homogenate. Also, increases above this level may not be physiologic.It is concluded that measurement of cAMP-dependent protein kinase activity ratio is a more direct and more sensitive way to study the effect of compounds which act through cAMP mediated mechanism

Phosphorylation of Pig Epidermal Soluble Protein by Endogenous cAMP-Dependent Protein Kinase

The distribution of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase and its substrate proteins was analyzed using soluble and particulate fractions of pig epidermal homogenates. When histone was used as a substrate for this enzyme reaction, protein kinase activity was distributed almost equally between the soluble and particulate fractions. However, the effect of exogenously added cAMP was confined almost exclusively to the soluble enzyme. Endogenous protein phosphorylation in the absence of exogenous histone was higher in the particulate fraction than in the soluble fraction, but the stimulating effect of cAMP was observed only in the soluble fraction. These results indicate that cAMP-dependent protein kinase is predominantly localized in the soluble fraction and phosphorylates soluble epidermal proteins. The particulate fraction contains protein kinase which is cAMP-independent and phosphorylates particulate-bound proteins as well as histone. Based on these observations, the soluble fraction was incubated with [γ-32P]-ATP in the presence or absence of cAMP, and phosphorylated protein was analyzed by SDS disc- or slab-gel electrophoresis followed by autoradiography. Among many proteins whose phosphorylation was slightly increased by cAMP, a protein with Mr ∼45,000 was found which was markedly phosphorylated in the presence of cAMP. Although this protein corresponds to one of the richest proteins in the epidermal soluble fraction, an important physiologic role for this phosphorylation has not been clarified

Cyclic AMP-Dependent Protein Kinase Isozymes of Pig Skin and Human Skin from Normal and Psoriatic Subjects

Cyclic AMP-dependent protein kinase isozymes of pig and human skin (epidermis) were separated by DEAE- cellulose column chromatography after micromodification for small biopsy samples. Clear-cut separations of type I and type II isozymes, winch were of about equal amounts, could be obtained only when the ischemia effect was avoided by in vivo freezing of skin and homogenization for less than 10 s. Intradermal injections of epinephrine caused dose-dependent activation of type I isozyme, but not of type 11. Injections of other skin adenylate cyclase stimulators such as histamine, adenosine, and prostaglandin E2 elevated the local cyclic AMP levels to not more than 5 pmol/mg protein and also stimulated only the type I isozyme. Incubation of keratome-sliced pig skin under various conditions caused both activation by dissociation and inactivation by dissociation of the subunits, which appeared to be dependent on the cyclic AMP content. Epinephrine added to the incubation medium led to complete activation of both type I and type II isozymes (the intraepidermal cyclic AMP contents ranged from 20–50 pmol/mg protein). The isozymes of normal skin and involved skin of psoriatics showed identical peaks of type I and type II Isozymes of equal amounts. The data indicate that protein kinase in the involved skin is not in an activated (by cyclic AMP) state

In vivo dynamic analysis of BMP-2-induced ectopic bone formation

Bone morphogenetic protein (BMP)-2 plays a central role in bone-tissue engineering because of its potent bone-induction ability. However, the process of BMP-induced bone formation in vivo remains poorly elucidated. Here, we aimed to establish a method for intravital imaging of the entire process of BMP-2-induced ectopic bone formation. Using multicolor intravital imaging in transgenic mice, we visualized the spatiotemporal process of bone induction, including appearance and motility of osteoblasts and osteoclasts, angiogenesis, collagen-fiber formation, and bone-mineral deposition. Furthermore, we investigated how PTH1-34 affects BMP-2-induced bone formation, which revealed that PTH1-34 administration accelerated differentiation and increased the motility of osteoblasts, whereas it decreased morphological changes in osteoclasts. This is the first report on visualization of the entire process of BMP-2-induced bone formation using intravital imaging techniques, which, we believe, will contribute to our understanding of ectopic bone formation and provide new parameters for evaluating bone-forming activity.Hashimoto K., Kaito T., Furuya M., et al. In vivo dynamic analysis of BMP-2-induced ectopic bone formation. Scientific Reports 10, 4751 (2020); https://doi.org/10.1038/s41598-020-61825-2

Diagnosis of Myocardial Viability by Fluorodeoxyglucose Distribution at the Border Zone of a Low Uptake Region

Purpose: In cardiac 2-[F-18]fluoro-2-deoxy-D-glucose (FDG)-positron emission tomography (PET) examination, interpretation of myocardial viability in the low uptake region (LUR) has been difficult without additional perfusion imaging. We evaluated distribution patterns of FDG at the border zone of the LUR in the cardiac FDG-PET and established a novel parameter for diagnosing myocardial viability and for discriminating the LUR of normal variants. Materials and Methods: Cardiac FDG-PET was performed in patients with a myocardial ischemic event (n = 22) and in healthy volunteers (n = 22). Whether the myocardium was not a viable myocardium (not-VM) or an ischemic but viable myocardium (isch-VM) was defined by an echocardiogram under a low dose of dobutamine infusion as the gold standard. FDG images were displayed as gray scaled-bull’s eye mappings. FDG-plot profiles for LUR ( = true ischemic region in the patients or normal variant region in healthy subjects) were calculated. Maximal values of FDG change at the LUR border zone (a steepness index; Smax scale/pixel) were compared among not-VM, isch-VM, and normal myocardium. Results: Smax was significantly higher for n-VM compared to those with isch-VM or normal myocardium (ANOVA). A cut-off value of 0.30 in Smax demonstrated 100 % sensitivity and 83 % specificity for diagnosing n-VM and isch-VM. Smax less than 0.23 discriminated LUR in normal myocardium from the LUR in patients with both n-VM and isch-VM with a 94 % sensitivity and a 93 % specificity. Conclusion: Smax of the LUR in cardiac FDG-PET is a simple and useful parameter to diagnose n-VM and isch

Direct cell–cell contact between mature osteoblasts and osteoclasts dynamically controls their functions in vivo

Bone homeostasis is regulated by communication between bone-forming mature osteoblasts (mOBs) and bone-resorptive mature osteoclasts (mOCs). However, the spatial–temporal relationship and mode of interaction in vivo remain elusive. Here we show, by using an intravital imaging technique, that mOB and mOC functions are regulated via direct cell–cell contact between these cell types. The mOBs and mOCs mainly occupy discrete territories in the steady state, although direct cell–cell contact is detected in spatiotemporally limited areas. In addition, a pH-sensing fluorescence probe reveals that mOCs secrete protons for bone resorption when they are not in contact with mOBs, whereas mOCs contacting mOBs are non-resorptive, suggesting that mOBs can inhibit bone resorption by direct contact. Intermittent administration of parathyroid hormone causes bone anabolic effects, which lead to a mixed distribution of mOBs and mOCs, and increase cell–cell contact. This study reveals spatiotemporal intercellular interactions between mOBs and mOCs affecting bone homeostasis in vivo