• The Korean Journal of Physiology and Pharmacology
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• 제1권5호
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• pp.485-493
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• 1997

We investigated the effect of ${\alpha}-adrenergic$ and cholinergic receptor agonists on $Ca^{2+}$ current in adult rat trigeminal ganglion neurons using whole-cell patch clamp methods. The application of acetylcholine, carbachol, and oxotremorine ($50\;{\mu}M\;each$) produced a rapid and reversible reduction of the $Ca^{2+}$ current by $17{\pm}6%,\;19{\pm}3%,\;and\;18{\pm}4%$, respectively. Atropine, a muscarinic antagonist, blocked carbachol- induced $Ca^{2+}$ current inhibition to $3{\pm}1%$. Norepinephrine ($50\;{\mu}M$) reduced $Ca^{2+}$ current by $18{\pm}2%$, while clonidine ($50\;{\mu}M$), an ${\alpha}2-adrenergic$ receptor agonist, inhibited $Ca^{2+}$ current by only $4{\pm}1%$. Yohimbine, an ${\alpha}2-adrenergic$ receptor antagonist, did not block the inhibitory effect of norepinephrine on $Ca^{2+}$ current, whereas prazosin, an ${\alpha}1-adrenergic$ receptor antagonist, attenuated the inhibitory effect of norepinephrine on $Ca^{2+}$ current to $6{\pm}1%$. This pharmacology contrasts with ${\alpha}2-adrenergic$ receptor modulation of $Ca^{2+}$ channels in rat sympathetic neurons, which is sensitive to clonidine and blocked by yohimbine. Our data suggest that the modulation of voltage dependent $Ca^{2+}$ channel by norepinephrine is mediated via an α1-adrenergic receptor. Pretreatment with pertussis toxin (250 ng/ml) for 16 h greatly reduced norepinephrine- and carbachol-induced $Ca^{2+}$ current inhibition from $17{\pm}3%\;and\;18{\pm}3%\;to\;2{\pm}1%\;and\;2{\pm}1%$, respectively. These results demonstrate that norepinephrine, through an ${\alpha}1-adrenergic$ receptor, and carbachol, through a muscarinic receptor, inhibit $Ca^{2+}$ currents in adult rat trigeminal ganglion neurons via pertussis toxin sensitive GTP-binding proteins.

• 한국어병학회지
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• 제9권1호
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• pp.41-51
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• 1996

경골어류의 혈관평활근에 대한 adrenaline성 조절기작을 규명의 일환으로 틸라피아의 배대동맥을 사용하여 Adrenergic agonist의 효과와 그 매개에 관여하는 수용체의 subtype에 대한 연구를 하였으며 그 결과는 다음과 같다. 1. Epinephrine, norepinephrine, phenylephrine, clonidine 및 methoxamine은 tilapia의 배대동맥에 대하여 농도의존적인 혈관수축효과만을 나타내었으며, 효력은 epinephrine, norepinephrine, phenylnephrine, clonidine, methoxamine의 순이었으며, 이들 수축반응은 혈관내피세포의 존재유무에 영향을 받지 않았다. 2. Epinephrine, norepinephrine, phenylephrine 및 clonidine 의 농도의존적인 혈관수축반응곡선은 선택적인 $\alpha_2$-adrenergic 수용체 길항제인 yohimbine의 농도가 증가함에 따라 오른쪽으로 평행이동 되었으며, epinephrine과 norepinephrine은 선택적인 $\alpha_1$-수용체 길항제인 prazosin의 농도가 증가함에 따라 오른쪽으로 평행이동되었다. 3. Epinephrine과 norepinephrine의 혈관수축반응은 calcium제거 생리적 완충용액에서는 각각 약 41%, 51% 소실되었며, calcium 유입차단제인 verapamil에 의해서도 거의 유사한 경향을 보였다. 이상의 실험결과들을 종합하면 Catecholamine류는 수축효과만을 나타내었으며 혈관내피세포 존재유무와는 무관하였다. 이러한 수축작용은 $\alpha_1$- 및 $\alpha_2$-adrenergic receptor가 모두 매개하였으며 voltage dependent $Ca^{2+}$ channel을 통하여 유입된 세포외액의 $Ca^{2+}$과 세포내 $Ca^{2+}$의해 일어난다고 사료된다.

• Biomolecules & Therapeutics
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• 제4권1호
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• pp.103-109
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• 1996

To investigate the effect of the third intracellular loop (i3 loop) peptide of human $\beta$$_2$-adrenergic receptor on receptor agonist binding, we expressed third intracellular loop region of human $\beta$$_2$-adrenergic receptor as glutathione S-transferase fusion protein in E. coli. DNA fragment of the receptor gene which encodes amino acid 221-274 of human $\beta$$_2$-adrenergic receptor was amplified by polymerase chain reaction and subcloned into the bacterial fusion protein expression vector pGEX-CS and expressed as a form of glutathione-S-transferase (GST) fusion protein in E. coli DH5$\alpha$. The receptor fusion protein was identified by SDS-PAGE and Western blot using monoclonal anti-GST antibody. The fusion protein expressed in this study was purified to an apparent homogeneity by glutathione Sepharose CL-4B affinity chromatography. The purified i3 loop fusion proteins at a concentration of 10 $\mu\textrm{g}$/ι caused right shift of the isoproterenol competition curve of [$^3$H]Dihydroalprenolol binding to hamster lung $\beta$$_2$-adrenergic receptor indicating lowered affinity of isoproterenol to $\beta$$_2$-adrenergic receptor possibly due to the uncoupling of receptor and G protein in the presence of the fusion protein. The uncoupling of receptor and G protein suggests that i3 loop region plays a critical role on $\beta$$_2$-adrenergic receptor G protein coupling.

• The Korean Journal of Physiology and Pharmacology
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• 제2권6호
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• pp.753-761
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• 1998

Preconditioning of a heart with small doses of catecholamines induces a tolerance against the subsequent lethal ischemia. The present study was performed to find a specific receptor pathway involved with the catecholamine preconditioning and to test if adenosine plays a role in this cardioprotective effect. Isolated rat hearts, pretreated with small doses of ${\alpha}-\;or\;{\beta}-adrenergic$ agonists/antagonists, were subjected to 20 minutes ischemia and 20 minutes reperfusion by Langendorff perfusion method. Cardiac mechanical functions, lactate dehydrogenase and adenosine release from the hearts were measured before and after the drug treatments and ischemia. In another series of experiments, adenosine $A_1\;or\;A_2$ receptor blockers were treated prior to administration of adrenergic agonists. Pretreatments of a ${\beta}-agonist,\;isoproterenol(10^{-9}{\sim}10^{-7}\;M)$ markedly improved the post-ischemic mechanical function and reduced the lactate dehydrogenase release. Similar cardioprotective effect was observed with an ?-agonist, phenylephrine pretreatment, but much higher $concentration(10^{-4}\;M)$ was needed to achieve the same degree of cardioprotection. The cardioprotective effects of isoproterenol and phenylephrine pretreatments were blocked by a ${\beta}_1-adrenergic$ receptor antagonist, atenolol, but not by an ${\alpha}_1-antagonist,$ prazosin. Adenosine release from the heart was increased by isoproterenol, and the increase was also blocked by atenolol, but not by prazosin. A selective $A_1-adenosine$ receptor antagonist, 1,3-dipropyl-8-cyclopentyl xanthine (DPCPX) blocked the cardioprotection by isoproterenol pretreatment. These results suggest that catecholamine pretreatment protects rat myocardium against ischemia and reperfusion injury by mediation of ${\beta}_1-adrenergic$ receptor pathway, and that adenosine is involved in this cardioprotective effect.

• Biomolecules & Therapeutics
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• 제4권1호
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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.

• The Korean Journal of Pain
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• 제12권2호
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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.

• 한국어류학회지
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• 제12권1호
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• pp.38-45
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• 2000

어종에 따라 혈관의 긴장도 조절은 다양한 신경전달물질에 의하여 조절되고 있다. 그러나 아직 대부분의 어종에서 자율신경계 신경전달물질 및 혈관긴장도 조절인자들의 기능에 대하여 명확하게 규명되어 있지 않다. 본 연구는 아직 연구되지 않은 분야인 이스라엘잉어에서의 자율신경계 신경전달물질들의 혈관긴장도 조절에서의 역할을 규명하고자 적출복대동맥을 이용하여 시험하였다. 이 적출혈관에서 아세틸콜린 (ACh)은 정상혈관과 미리 일정수준 수축시킨 혈관 모두에서 수축을 유발하였으며 수축작용은 무스카린성 길항제인 아트로핀에 의해 거의 완벽하게 차단되었다. 여러 가지 아드레날린성 수용체를 동시에 흥분시키는 내인성 물질인 에피네프린 (Epi)은 혈관의 조건에 상관없이 이완반응을 유발하였다. 그러나 유사한 내인성물질인 노르에피네프린 (NE)은 정상혈관에서는 미약한 수축율, 미리수축된 혈관에서는 이완작용을 유발하였다. 한편 ${\alpha}_1$ 아드레날린성 수용체 흥분제인 페닐에프린은 수축을, $\beta$수용체 홍분제인 이소프로테레놀은 이완을 각각 유발하였으며 ${\alpha}_2$수용체 흥분제인 클로니딘은 아무런 반응을 유발하지 않았다. Epi, NE 및 이소프로테레놀에 의해 유발된 혈관이완 반응은 $\beta$ 아드레날린성 수용체 길항제인 프로프라놀롤에 의해 유의하게 억제되었다. 따라서 살아있는 상태의 이스라엘 잉어에서는 ACh는 주로 무스카린성 수용체 활성화에 의한 혈관을 수축하는 기능을, Epi과 NE는 $\beta$수용체 흥분에 의한 이완작용을 각각 발휘하는 것으로 판단된다.

• International Journal of Oral Biology
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• 제46권1호
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• pp.39-44
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• 2021

This study aimed to investigate whether neurotransmitter receptors in the nervous system were also expressed in oral keratinocytes. Expressions of various neurotransmitter receptor genes in immortalized mouse oral keratinocyte (IMOK) cells were examined by reverse transcriptase polymerase chain reaction. IMOK cells expressed calcitonin gene-related peptide (CGRP) receptor subunit genes Ramp1 and Ramp3 and glutamate receptor subunit genes Grina, Gria3, Grin1, Grin2a, and Grin2d. Moreover, IMOK cells expressed Adrb2 and Chrna5 that encode beta 2 adrenergic receptor and cholinergic receptor nicotinic alpha 5 for sympathetic and parasympathetic neurotransmitters, respectively. The expression of Bdkrb1 and Ptger4, which encode receptors for bradykinin and prostaglandin E2 involved in inflammatory responses, was also observed at low levels. Expressions of Ramp1 and Grina in the mouse gingival epithelium were also confirmed by immunohistochemistry. When the function of neurotransmitter receptors expressed on IMOK cells was tested by intracellular calcium response, CGRP, glutamate, and cholinergic receptors did not respond to their agonists, but the bradykinin receptor responded to bradykinin. Collectively, oral keratinocytes express several neurotransmitter receptors, suggesting the potential regulation of oral epithelial homeostasis by the nervous system.

• 대한약리학회지
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• 제19권1호
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• pp.85-92
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• 1983

Intestine is innervated by an interconnected plexus of both sympathetic and parasympathetic nerve fibers. Sympathetic influence causes inhibition of intestinal motility mediated by both ${\alpha}-\;and\;{\beta}-adrenergic$ receptors. The mechanism of intestinal relaxation by ${\beta}-receptors$ has been extensively studied, but the function of ${\alpha}-receptors$ in intestinal motility is still unclear. Although it is suggested that catecholamine reduces acetylcholine release and this may play an important role in ${\alpha}-receptor$ mediated intestinal relaxation, there is no definite evidences about the mechanism and site of action of ${\alpha}-receptor$ mediated relaxation. In this experiment, therefore, the effect and site of action of ${\alpha}-receptor$ agonists were investigated in the guinea pig ileum using electrical field stimulation. The results are summarized as follows : 1) Electrical field stimulation elicited tonic contraction in isolated guinea pig ileum ana this contraction was completely inhibited by the pretreatment of tetrodotoxin or atropine. 2) Norepinephrine, epinephrine and dopamine inhibited the contraction induced by electrical field stimulation but methoxamine and phenylephrine had little effects. 3) Inhibitory effects of norepinephrine and dopamine was partially blocked by yohimbine and phentolamine pretreatment. But haloperidol and propranolol pretreatment cause no effects on the electrical field stimulation induced contraction. Inhibitory effect of dopamine was completely blocked by both haloperidol and yohimbine pretreatment. 4) Inhibitory effects of norepinephrine and dopamine were little affected by the pretreatment with hexamethonium. It is suggested that electrical field stimulation causes tonic contraction of guinea pig ileum by releasing acetylcholine from postganglionic fiber, and this release is blocked by presynaptic ${\alpha}-receptor$ activation.

• The Korean Journal of Pain
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• 제22권1호
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• pp.21-27
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• 2009

Background: The neuropathic pain arising from nerve injury is difficult to treat and the therapeutic effects of opioid drugs remain debatable. Agonists acting at the ${\alpha}_2$ adrenergic and opioid receptors have analgesic properties and they act synergistically when co-administered in the spinal cord. The lack of subtype-selective pharmacological agents has previously impeded the synergistic effects that are mediated by the adrenergic receptor subtypes. Methods: We created neuropathic pain model by ligating the L5 spinal nerve in Sprague-Dawley rats (n = 18). We divided the rats into three groups (n = 6 for each group), and we administered intraperitoneal morphine (1 mg/kg, 3 mg/kg, 5 mg/kg) and then we measured the mechanical allodynia with using von-Frey filaments for 8 hours. We then injected morphine (5 mg/kg) intraperitoneally, twice a day for 2 weeks. We measured the tactile and cold allodynia in the morphine group (n = 9) and the saline group (n = 9). After 2 weeks, we decapitated the rats and harvested the spinal cords at the level of lumbar enlargement. We compared the ${\alpha}_2$ subtype mRNA expression with that of control group (n = 6) by performing real time polymerase chain reaction (RTPCR). Results: Intraperitoneal morphine reduced the neuropathic pain behavior in the dose-dependent manner. Chronic morphine administration showed an antiallodynic effect on the neuropathic pain rat model. The rats did not display tolerance or hyperalgesia. The expression of the mRNAs of the ${\alpha}_{2A}$, ${\alpha}_{2B}$, ${\alpha}_{2C}$ subtypes decreased, and morphine attenuated this effect. But we could not get statistically proven results. Conclusions: Systemic administration of morphine can attenuate allodynia during both the short-term and long-term time course. Morphine has an influence on the expression of ${\alpha}_2$ receptor subtype mRNA. Yet we need more research to determine the precise effect of morphine on the ${\alpha}_2$ subtype gene expression.