• Archives of Pharmacal Research
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• v.18 no.2
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• pp.90-99
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• 1995

The effect of protein kinase C inhibitors, sturosporine and 1-(5-isoquinolinyl sulfonyl)-2-methyl piperazine(H7) on in vitro differentiation of erythroid progenitor cells which were isolated from spleens of mice infected with the anemia-inducing strain of Friend virus were examined. Erythropoietin-mediated differentitation of erythroid progenitor cells, as determined by the incorporation of $^{59}Fe$ into protoporphyrin, was inhibited by staurosporine and H7 in a concentration -dependent manner. Scatchard analysis of the $^3H-phorbol-12$, 13-dibutyrate binding to erythroid progenitor cells revealed that at the high affinity sites the dissociation constant was 22nM and the maximum number of $^3H-phorbol-12$, 13-dibutyrate binding to erythroid progenitor cells revealed that at the high affinity sites the dissociation constant was 22nM and the maximum number of $^3H-phorbol-12$, 13-dibutyrate binding sites per cell was approximately $3.7\times10^5$. Cytosonic protein kinase C was isolated from erthroid progenitor cells and then purified by sequential column chromatogrphy. Two isoforms of protein kinase C were found. Photoaffinity labeling of the purified protein kinase C samples with $^3H-phorbol-12$12-myristate 13-acetate followed by analysis of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and autofluorography showed radiolabeled 82-KDa pepticles. Rediolabeling of the 82-KDa peptides with $^3H-phorbol-12$myristate 13-acete was almost completely blocked by excess unlabeled phorbol 12-myristate 13-acetate was almost 12-muristate 13-acetate-promoted phosphorylation with the puyrified protein kinase C samples showed that the phosphorylation of 82-KDa peptides was increased as the concentration of phorbol 12-myristate 13-acetate was increased from $10^{-8}M{\;}to{\;}10^{-4}$M. In light of the findings that erythroid progenitor cells possessed an abundance of protein kinase C and that stauroporine and H7 inhibited erythroid differentiation, it seemed likely that protein kinase C would play a role in the erythroid progenitor cell development.

• Biomolecules & Therapeutics
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• v.3 no.1
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• pp.21-27
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• 1995

The only compounds with antagonistic activity via AT$_1$receptor, one of two subtypes of angiotensin II (AII) receptor, have been demonstrated to block the vasoconstriction effects of AII and thereby provide therapeutic potential. This initiated the search for compounds with high specific affinity to AT$_1$receptor and their effective screening methods. The radioligand binding assay for the AII receptor is regarded as the primary method for the evaluation of AT$_1$receptor antagonists for their activity. In this paper, we characterized the liver AT$_1$receptor and describe the efficient method of the radioligand binding assay using rat liver as a source of AT$_1$receptor. Equilibrium binding studies with rat adrenal cortex, adrenal medulla, liver and bovine adrenal showed that the specific bindings of [$^3$H] AII were saturable in all tissues and the Scatchard plots of those data were linear, suggesting a single population of binding sites. Hill slopes were very near to the unity in all tissues. Kinetic studies of [$^3$H) AII binding in rat liver homogenates yielded two association rate constants, 4.10$\times$10$^{7}$ M$^{-1}$ min$^{-1}$ and 4.02$\times$10$^{9}$ M$^{-1}$ min$^{-1}$ , with a single dissociation rate constant, 7.07$\times$10$^{-3}$ min-$^{-1}$ , possibly due to the partial dissociation phenomenon. The rank order of inhibition potencies of [$^3$H] AII binding in rat liver was AII>Sarile>Losartan>PD 123177. Rat liver homogenates revealed to have very high density of homogeneous population of the AT$_1$receptor subtype, as the specifically bound [$^3$H] AII was not inhibited by PD 123177, the nonpeptide antagonist of AT$_2$. The results of this study demonstrated that the liver homogenates from rats could be the best receptor preparation for the AT$_1$receptor binding assay and provide an efficient system for the screening of newly synthesized candidate compounds of AT$_1$receptor antagonist.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.10 no.10
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• pp.2997-3003
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• 2009

The pharmacological profile of KR-31081, a nonpeptide $AT_1$ selective angiotensin receptor antagonist, was investigated by receptor binding studies, functional in vitro assays with rabbit aorta. KR-31081 inhibited the specific binding of $[^{125}I]\;[Sar^1,\;Ile^8]$-angiotensin II to human recombinant $AT_1$ receptor with an 8.6-fold greater potency than losartan ($IC_{50}$: 1.43 and 12.3 nM, respectively), but it did not inhibit the binding of [$^{125}I$] CGP 42112A to human recombinant $AT_2$ receptor ($IC_{50}$: higher than $10{\mu}M$ for both). The Hill coefficient for the competition curve of KR-31081 against $AT_1$ receptor was not significantly different from unity (0.99). Scatchard analysis showed that KR-31081 interacted with human recombinant $AT_1$ receptor in a competitive manner, as with losartan. In functional studies with rabbit aorta, KR-31081 competitively inhibited the contractile response to angiotensin II ($pK_B$ values: 8.66) with 20-70% decrease in the maximum contractile responses, unlike losartan that showed competitive antagonism without any change in the maximum contractile responses to angiotensin II ($pA_2$ values: 7.59). These results suggest that KR-31081 is a highly potent $AT_1$ selective angiotensin II receptor antagonist with a mode of insurmountable antagonism to be developed as the exploratory potential of this compound.

• The Korean Journal of Nuclear Medicine
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• v.32 no.6
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• pp.516-524
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• 1998

Purpose: Radiolabeled CEA79.4 antibody has a possibility to be used in radioimmunoscintigraphy or radioimmunotherapy of cancer. We investigated the in vitro properties and biodistribution of CEA79.4 antibody labeled with Re-188 or Tc-99m. Materials and Methods: CEA79.4 was reduced by 2-mercaptoethanol to produce-SH residue, and was labeled with Re-188 or Tc-99m. For direct labeling of Tc-99m, methylene-diphosphonate was used as transchelating agent. CEA79.4 in 50 mM Acetate Buffered Saline (ABS, pH 5.3) was labeled with Re-188, using stannous tartrate as reducing agent. In order to measure immunoreactivity and the affinity constant of radiolabeled antibody, cell binding assay and Scatchard analysis using human colon cancer cells SNU-C4, were performed. Biodistribution study of labeled CEA79.4 was carried out at 1, 14 and 24 hr in ICR mice. Results: Labeling efficiencies of Tc-99m and Re-188 labeled antibodies were $92.4{\pm}5.9%$ and $84.7{\pm}4.6%$, respectively, In vitro stability of Tc-99m-CEA79.4 in human serum was higher than Re-188-CEA79.4. Immunoreactivity and affinity constant of Tc-99m-CEA79.4 were 59.2% and $6.59{\times}10^9\;M^{-1}$, respectively, while those of Re-188-CEA79.4 were 41.6% and $4.2{\times}10^9\;M^{-1}$, respectively. After 24 hr of administrations of Re-188 and Tc-99m labeled antibody, the remaining antibodies in blood were 6.32 and 9.35% ID/g respectively. The biodistribution of each labeled antibody in other organs was similar because they did not accumulate in non-targeted organs. Conclusion: In vitro properties and biodistribution of Re-188-CEA79.4 were similar to those of Tc-99m-CEA79.4. It appears that Re-188-CEA79.4 can be used as a suitable agent for radioimmunotheraphy.