• Korean Journal of Veterinary Research
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• v.33 no.4
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• pp.617-622
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• 1993

The preliminary studies on the localization of adrenoceptors were performed on smooth muscle strips of bovine esophageal groove. The mechanical activity of the muscle strip was recorded isometrically in vitro.w In the bottom circular muscle strips. the excitatory ${\alpha}-adrenergic$ responses were not blocked by tetrodotoxin$(2.1{\times}10^{-6}M)$ and denervation which was carried by cold storage of strips for 48 hrs in Tyrode's solution at $5-6{^{\circ}C}$ without oxygen supply. These excitatory ${\alpha}-adrenergic$ responses were partially blocked by atropine. In the lip longitudinal muscle strips, the inhibitory${\beta}-adrenergic$ responses were not blocked by pretreatment of tetrodotoxin and atropine. The results suggest that ${\beta}-adrenergic$ receptors mediating relaxations are located on the postsynaptic smooth muscle cells, whereas ${\beta}-adrenergic$ receptors mediating contractions are located both in the smooth muscle cells and in the cholinergic neurones.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.61-61
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• 1993

사람의 혈액의 백혈구로부터 Polymerase Chain Reaction 방법으로 $\beta$$_2$-adrenergic receptor 유전자를 증폭하였다. 이 증폭된 유전자를 pbluescript KS(＋)에 cloning하였으며, 제한효소지도 작성과 부분적인 염기배열 확인으로 증폭된 유전자가 사람의 $\beta$$_2$-adrenergic receptor 유전자임을 확인하였다. $\beta$$_2$-adrenergic receptor 유전자를 N말단의 아미노산이 21개 제거되도록 결손시킨후 yeast의 pSec5의 Killer toxin signal sequence의 뒤에 in-frame으로 연결하였다. 현재 효모에서의 발현양상 및 발현된 단백질의 활성을 조사중이다.

• Biomolecules & Therapeutics
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• v.1 no.2
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• pp.188-195
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• 1993

In order to understand the machanism of action and regulation of ${\beta}$-adrenergic receptor in terms of molecular level, the purification of receptor protein has a fundamental importance. Moreover, species differences among avian, amphibian and mammalian ${\beta}$-adrenergic receptors make it more important to purify mammalian ${\beta}$-adrenergic receptor. Because ${\beta}$-adrenergic receptor is an integral membrane protein, it must be solubilized from the membrane for the purification. The purpose of the present study was to solubilize and characterize the mammalian $\beta$-adrenergic receptor from guinea pig lung in quantities by more efficient and practical method eventually to purify receptor. Guinea pig lung membrane preparation was solubilized by sequential treatment of buffers containing low and high concentration of digitonin which are 0.2 and 1.2% respectively. About 50% of the total receptor pool was released by this double extraction procedure. The $\beta$-adrenoceptors in the digitonin extract were identified using the ${\beta}$-adrenergic antagonist, (-)-[$^3H$]-dihydroalprenolol ([$^3H$]DHA). The solubilized receptor retained all of the essential characteristics of membrane-bound receptor, namely saturability; stereoselectivity; high affinity to ${\beta}$-adrenergic drugs. For the measurement of soluble receptor activity, Sephadex G-50 chromatography method has been widely used. Inspite of its accuracy and wide acceptance, this technique employed troublesome column work which required long time to assay the activity of receptor. We employed another methods to measure receptor activity. When using 0.5% polyethylenimine pretreated GF/B glass fiber filter, filtration technique could be used to measure soluble receptor activity. This technique enabled us to reduce the total amount of time to assay by a factor of 4 as well as to detect soluble receptor. In the present study, we could establish more efficient and practical solubilization method of mammalian $\beta$-adrenergic receptor. The rapidity and high yield of this solubilization scheme, together with the favorable recovery of the receptor activity, are significant steps toward the ultimate purification of the mammalian $\beta$-adrenergic receptor. The result of this study together with more convenient purification method could provide large amount of purified receptor with ease for various research purposes.

• The Korean Journal of Pain
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• v.12 no.2
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• pp.177-182
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• 1999

Background: Clonidine, an ${\alpha}_2$ adrenergic agonist blocks nerve conduction. However, in our previous experiment we found that adrenaline neither blocks nerve conduction by itself nor augment nerve conduction blockade by lidocaine near clinical concentrations. Possible explanations are: 1) there may be antagonism between some of adrenergic receptors, 2) clonidine may block nerve conduction via non-adrenergic mechanism. The purpose of this study is to obtain dose-response curves of several different forms of adrenergic receptor agonist to see the relative potencies of each adrenergic receptors to block nerve conduction. Methods: Recordings of compound action potentials of A-fiber components (A-CAPs) were obtained from isolated sciatic nerves of adult male Sprague-Dawley rats. Nerve sheath of the sciatic nerve was removed and desheathed nerve bundle was mounted on a recording chamber. Single pulse stimuli (0.5 msec, supramaximal stimuli) were repeatedly applied (2Hz) to one end of the nerve and recordings of A-CAPs were made on the other end of the nerve. Dose-response curves of epinephrine, phenylephrine, isoproterenol, clonidine were obtained. Results: $ED_{50}$ of each adrenergic agonist was: $4.51\times10^{-2}$ M for epinephrine; phenylephrine, $7.74\times10^{-2}$ M; isoproterenol, $9.61\times10^{-2}$ M; clonidine, $1.57\times10^{-3}$ M. Conclusion: This study showed that only clonidine, ${\alpha}_2$ adrenergic agonist, showed some nerve blocking action while other adrenergic agonists showed similar poor degree of nerve blockade. This data suggest that non-effectiveness of epinephrine in blocking nerve conduction is not from the antagonism between adrenergic receptors.

• Biomolecules & Therapeutics
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• v.4 no.1
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• pp.97-102
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• 1996

To investigate whether human $\beta$$_2$-adrenergic receptor devoid of the C-terminal two transmembrane helices retain its ligand binding activity and specificity, 5'780-bp DNA fragment of the receptor gene which encodes amino acid 1-260 of human $\beta$$_2$-adrenergic receptor was subcloned into the bacterial fusion protein expression vector and expressed as a form of glutathione-S-transferase (GST) fusion protein in E. coli DH5$\alpha$. The receptor fusion protein was expressed as a membrane bound form which was verified by SDS-PAGE and Western blot. The fusion protein expressed in this study specifically bound $\beta$-adrenergic receptor ligand [$^3$H] Dihydroalprenolol. In saturation ligand binding assay, the $K_{d}$ value was 7.6 nM which was similar to that of intact $\beta$$_2$-adrenergic receptor in normal animal tissue ( $K_{d}$=1~2 nM) and the $B_{max}$ value was 266 fmol/mg membrane protein. In competition binding assay, the order of binding affinity of various adrenergic receptor agonists to the fusion protein was isoproterenol》epinephrine norepinephrine, which was similar to that of intact receptor in normal animal tissue. These results suggest that N-terminal five transmembrane helices of the $\beta$$_2$-adrenergic receptor be sufficient to determine the ligand binding activity and specificity, irrespective of the presence or absence of the C-terminal two transmembrane helices.s.s.s.

• Biomolecules & Therapeutics
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• v.5 no.4
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• pp.344-350
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• 1997

The purpose of the present study was to produce and characterize a monoclonal antibody against human ${\beta}_2$-adrenergic receptor. Male BALB/c mice were immunized with glutathione S-transferase (GST) fusion protein of the C-terminal portion of the human ${\beta}_2$-adrenergic receptor which was expressed in E.Coli. The immunized splenocytes were fused with myeloma SP2/0-Agl4 cells. The resulting hybridomas were screened for the production of a monoclonal antibody which can recognize human ${\beta}_2$-adrenergic receptor, and then subcloned by limiting dilution. The resulting monoclonal antibody was named as mAb$\beta$CO2. The mono-clonal antibody $\beta$CO2 was determined as IgM subtype and then purified by anti-mouse IgM-agarose affinity chromatography. The results of ELISA, Western blot, and immunocytochemistry showed that mAb$\beta$CO2 recognized human ${\beta}_2$-adrenergic receptor in the ${\beta}_2$-adrenergic receptor-GST fusion protein and human spider-moid carcinoma cell line A431 with highly specific immunoreactivity, The monoclonal antibody $\beta$CO2 may provide useful tools for the study of the $\beta$-adrenergic receptor of human and other species including rats.

• Biomolecules & Therapeutics
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• v.8 no.2
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• pp.125-131
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• 2000

Idazoxan, $\alpha$$_2$-adrenergic antagonist, produced antidiuretic action by administration into the vein and diuretic action only in ipsilateral kidney by injection into a renal artery in dog. These studies were performed for investigation of mechanism on the renal action induced by idazoxan. Antiduretic action by idazoxan given into vein and diuretic action only in ipsilateral kidney by idazoxan injected into a renal artery were blocked entirely by renal denervation. Antidiuretic action of idazoxan given into the vein was weakened by UK 14,304, $\alpha$$_2$-adrenergic agonist, pretreated into the vein. Above results suggest that antidiuretic action of idazoxan given into the vein is caused by blocking of $\alpha$$_2$-adrenergic receptor, diuretic action only in ipsilateral kidney of idazoxan injected into a renal artery by blocking of $\alpha$$_2$-adrenergic receptor in the kidney.

• Korean Journal of Veterinary Service
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• v.17 no.3
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• pp.255-263
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• 1994

To elucidate the action of the adrenergic nerve on the isolated uterine smooth muscle of the pig, effects of electrical transmural nerve stimulation and norepinephrine were investigated on the pretreatment of phentolamine ； non-selective ${\alpha}$-adrenoceptor blocker, propranolol ; ${\beta}$-adrenoceptor blocker and the yohimbine；${\alpha}_2$-selective adrenoceptor blocker from physiograph. 1. The relaxation response induced by norepinephrine was the concentration of $10^{-6}$ M at first and maximum response was concentration of $10^{-4}$M. 2. The relaxation response induced by norepinephrine was not effected by the pretreatment with non-selective $\alpha$-adrenoceptor blocker, phentolanune ($10^{-6}$ M) but was completely blocked by the pretreatment with ${\beta}$-adrenoceptor blocker, propranolol($10^{-6}$ M). 3. The contractile response induced by electrical transmural nerve stimulation(20V, 10Hz, 0.5msec, 20sec ) was inhibited by the pretreatment with non-selective ${\alpha}$-adrenoceptor blocker, phentolamine($10^{-6}$ M) but was not inhibited and rather increased by the pretreatment ${\beta}$-adrenoceptor blocker, propranolol($10^{-6}$ M), and was not approximately effected by the pretreatment with ${\alpha}_2$-adrenoceptor blocker, yohimbine($10^{-6}$ M). These finding suggest that it was excitatory action by ${\alpha}_1$-adrenergic nerve and inhibitory action by ${\alpha}_2$-adrenergic, ${\beta}$-adrenergic nerve on uterine smooth muscle of the pig.

• Biomolecules & Therapeutics
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• v.1 no.1
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• pp.1-8
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• 1993

It has been known that calcium antagonists also inhibit the radioligand binding to muscarinic and $\alpha$-adrenergic receptors and, in case of verapamil, these inhibitions may play a role in the effects of verapamil on the heart. In this study, the effects of nicardipine, nifedipine, nimodipine, diltiazem and verapamil on the binding of [$^3H$]dihydroalprenolol (DHA) to dog cardiac ${\beta}$-adrenergic receptors were examined. A single uniform [$^3H$]DHA binding site ($K_D/= 5nM\;and\;B_{max}=2600$ fmol/mg protein) was identified in dog cardiac sarcolemma. [$^3H$]DHA binding was not affected by the usual therapeutic concentrations of these calcium antagonists (nanomolar range) but in the "nonspecific"concentration ranges ($28-180{\mu}m$) these drugs inhibited [$^3H$]DHA binding to $\beta$-adrenergic receptors. Nicardipine, nifedipine, nimodipine and diltiazem competed for [$^3H$]DHA binding to ${\beta}$-adrenergic receptors with dissociation constants ($K_i$) of $28{\mu}m,\' 74{\mu}m, 39{\mu}m \;and \;35{\mu}m,$ respectively. Verapamil ($K_i=176.5 {\mu}m$) was less potent inhibitor than other drugs and this inhibition was noncompetitive; the maximal binding capacity ($B_{max}$) $300 {\mu}m$ verapamil without change in the apparent dissociation constant (4K_D$) for DHA. These results indicate that the inhibitory action of calcium antagonists at high concentrations on ${\beta}$-adrenergic receptors is not involved in the therapeutic effects of these drugs by the calcium channel blocking action.

• Applied Microscopy
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• v.22 no.1
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• pp.33-41
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• 1992

To clarify the exocytotic features in adrenal medullary aminergic cells, the authors observed rat adrenal medulla prepared by the TAGO method with transmission electron microscope. Rat adrenal medulla contains two types of aminergic cells, adrenergic and noradrenergic, as described. They were present as a group. In a single group both adrenergic and noradrenergic cells were present, but the same kind of cells showed the tendency forming small groups. Adrenergic cells were characterized with the granules having relatively electroluscent cores. These granules were relatively uniform in size, and the cores filled the granules with only thin halos. Noradrenergic cells were characterized with the granules of various size and forms. Most of the cores of these granules were generally more electron-dense than those of the adrenergic cells and only partly filled the granules without forming the halos. But, some granules were very similar in the shape and electron density as those of the adrenergic cells. Even empty-looking granules were present. Exocytotic figures with the classical omega figures were observed in both types of aminergic cells, but they were more frequent in adrenergic cells. These figures were mainly present along the plasma membranes toward the capillary. The excreted materials could be identified in the cleft of the omega figures. Apocrine-like secretory patterns but without cytoplasmic rims were identified in noradrenergic cells. Some vesicles, possibly formed from the cytoplsmic tubular systems were released. Some irregular lamellar structures of varying sizes were also observed. They looked like membranous structures sneaking through the plasma membranes. We could not, however, found any evidences of their involvement in exocytotic processes. These were present toward the capillaries and found only in the adrenergic cells. The authors conclude that the secretory processes in adrenal chromaffin cells may include not only the classical exocytotic processes but also the unusual direct secretions of granules or parts of cellular organelles. The membranous lamellar structures may indicate the remnants of excreted granules or functionally inactive excess membranes of the organelles removed from the cytoplasm.