Selective alpha(1A)-Adrenoceptor Stimulation Induces Mueller's Smooth Muscle Contraction in an Isolated Canine Upper Eyelid Preparation

Purpose: It has been demonstrated that in patients with aponeurotic blepharoptosis, alpha(1)-adrenoceptor stimulation causes the contraction of the upper eyelid tarsal smooth muscle (Mueller's muscle) and opening of the eye. However, alpha(1)-adrenoceptor subtypes mediating the contraction of Mueller's muscle are still unclear. This study was designed to identify the alpha(1)-adrenoceptor subtypes in Mueller's muscle. Materials and Methods: A newly developed canine upper eyelid preparation was retrogradely perfused with a drug-containing Krebs-Henseleit solution through the angular vein in a temperature-controlled organ chamber. The contraction of the preparation was measured with a force-displacement transducer. Results: Phenylephrine, an alpha(1)-adrenoceptor agonist, increased the upper eyelid contractile force in a dose-dependent manner (K(0.5) = 110 nmol). Interestingly, the contraction in response to phenylephrine was persistent and hardly recovered to a base line level for more than 100 min after washout of the drug. WB4101 (100 nM), an alpha(1A)- and alpha(1D)-adrenoceptor antagonist, but not BMY7378 (100 nM), a selective alpha(1D)-adrenoceptor antagonist, competitively inhibited the phenylephrine-induced contraction. ABT-866, a selective alpha(1A)-adrenoceptor agonist, increased the upper eyelid contractile force as effectively as phenylephrine in a dose-dependent manner (K(0.5) = 190 nmol), and the contraction continued again for more than 100 min. Conclusion: These results suggest that selective alpha(1A)-adrenoceptor agonists, such as ABT-866, induce the sustained Mueller's muscle contraction and may be useful in pharmacological treatment of blepharoptosis.ArticleCURRENT EYE RESEARCH. 35(5):363-369 (2010)journal articl

The proximal C-terminus of alpha(1C) subunits is necessary for junctional membrane targeting of cardiac L-type calcium channels

In cardiac myocytes, LTCCs (L-type calcium channels) form a functional signalling complex with ryanodine receptors at the JM (junctional membrane). Although the specific localization of LTCCs to the JM is critical for excitation-contraction coupling. their targeting mechanism is unclear. Transient transfection of GFP (green fluorescent protein)-alpha(1S) or GFP-alpha(1C) but not P/Q-type calcium channel alpha(1A), in dysgenic (alpha(1S)-null) GLT myotubes results in correct targeting of these LTCCs to the JMs and restoration of action-potential-induced Ca2+ transients. To identify the sequences of alpha(1C) responsible for JM targeting, we generated a range of alpha(1C)-alpha(1A) chimaeras, deletion mutants and alanine substitution mutants and studied their targeting properties in GLT myotubes. The results revealed that amino acids L-1681 QAGLRTL(1688) and P(1693)EIRRAIS(1700), predicted to form two adjacent alpha-helices in the proximal C-terminus, are necessary for the JM targeting of alpha(1C). The efficiency of restoration of action-potential-induced Ca2+ transients in GLT myotubes was significantly decreased by mutations in the targeting motif. JM targeting was not disrupted by the distal C-terminus of alpha(1C) which binds to the second alpha-helix. Therefore we have identified a new structural motif in the C-terminus of alpha(1C) that mediates the targeting of cardiac LTCCs to JMs independently of the interaction between proximal and distal C-termini of alpha(1C).ArticleBIOCHEMICAL JOURNAL. 448:221-231 (2012)journal articl

β(2)-Adrenergic and M(2)-muscarinic receptors decrease basal t-tubular L-type Ca2+ channel activity and suppress ventricular contractility in heart failure

L-Lype Ca2+ channels (LTCC) play a crucial role in cardiac excitation-contraction coupling. We previously found that in failing ventricular myocytes of mice chronically treated with isoproterenol, basal t-tubular (TT) LTCC activity was halved by activation of protein phosphatase (PP)2A whereas basal surface sarcolemmal (SS) LTCC activity was doubled by inhibition of PP1. Interestingly, chronic treatment of these mice with pertussis toxin almost completely normalized TT and SS LTCC densities and cardiac contractility. In the present study, we therefore sought to identify the G(i/o) protein coupled receptors in cardiac myocytes (i.e. beta(2)-adrenergic, M-2-muscarinic and A(1)-adenosine receptors) that are responsible for these abnormalities in heart failure by chronically administrating mice a selective antagonist of each receptor (ICI118,551, atropine and 8-cyclopentyl-1,3-dipropilxanthine (DPCPX), respectively) with isoproterenol. Compared with mice treated with isoproterenol alone, mice treated with isoproterenol plus ICI118,551 or atropine, but not DPCPX showed significantly lower lung weight/tibial length, higher fractional shortening, lower left ventricular end-diastolic pressure and higher dP/dt(max) and dP/dt(min). In addition, ventricular myocytes of mice treated with isoproterenol plus ICI118,551 or atropine, but not DPCPX exhibited significantly higher TT and lower SS LTCC current densities than those of mice treated with isoproterenol alone due to normalization of the PP activities. These results indicate that beta(2)-adrenergic, M-2-muscarinic, but not A(1)-adenosine receptors contribute to reduced ventricular contractility at least partially by decreasing basal TT LTCC activity in heart failure. Therefore, antagonists of beta(2)-alrenergic and/or M-2-muscarinic receptors can be good adjuncts to beta(1)-adrenergic receptor antagonists in the treatment of heart failure.ArticleEUROPEAN JOURNAL OF PHARMACOLOGY. 724:122-131 (2014)journal articl

Two mechanistically distinct effects of dihydropyridine nifedipine on Ca(V)1.2 L-type Ca2+ channels revealed by Timothy syndrome mutation

Dihydropyridine Ca2+ channel antagonists (DHPs) block Ca(V)1.2 L-type Ca2+ channels (LTCCs) by stabilizing their voltage-dependent inactivation (VDI); however, it is still not clear how DHPs allosterically interact with the kinetically distinct (fast and slow) VDI. Thus, we analyzed the effect of a prototypical DHP, nifedipine on LTCCs with or without the Timothy syndrome mutation that resides in the I-II linker (LI-II) of Ca(V)1.2 subunits and impairs VDI. Whole-cell Ba2+ currents mediated by rabbit Ca(V)1.2 with or without the Timothy mutation (G436R) (analogous to the human G406R mutation) were analyzed in the presence and absence of nifedipine. In the absence of nifedipine, the mutation significantly impaired fast closed-and open-state VDI (CSI and OSI) at -40 and 0 mV, respectively, but did not affect channels' kinetics at -100 mV. Nifedipine equipotently blocked these channels at -80 mV. In wild-type LTCCs, nifedipine promoted fast CSI and OSI at -40 and 0 mV and promoted or stabilized slow CSI at -40 and -100 mV, respectively. In LTCCs with the mutation, nifedipine resumed the impaired fast CSI and OSI at -40 and 0 mV, respectively, and had the same effect on slow CSI as in wild-type LTCCs. Therefore, nifedipine has two mechanistically distinct effects on LTCCs: the promotion of fast CSI/OSI caused by LI-II at potentials positive to the sub-threshold potential and the promotion or stabilization of slow CSI caused by different mechanisms at potentials negative to the subthreshold potential.ArticleEUROPEAN JOURNAL OF PHARMACOLOGY. 685(1-3):15-23 (2012)journal articl

Thrombin Activates Ca2+-permeating Nonselective Cation Channels through Protein Kinase C in Human Umbilical Vein Endothelial Cells

We analyzed Ca-permeating nonselective cation channels (NSCs)mediating thrombin-induced contraction of human umbilical vein endothelial cells (HUVECs). A Ca chelater, BAPTA-AM (10μM), significantly inhibited the thrombin-induced contraction of HUVECs.Thrombin induced inward currents at -60 mV in the presence of intracellular MgATP. Removal of extracellular Caﾌﾞｸﾞsignificantly decreased the currents. A selective phospholipase C inhibitor, U73122 (1μM) but not its inactive analogue, U73343 (1μM) almost completely inhibited the currents. Neither a selective inhibitor of Caﾌﾞｸﾞ-ATPase of endoplasmic reticulum, thapsigargin (1μM)nor a diacylglycerol analogue, 1-oleoyl-2-acetyl-glycerol (30μM)activated the currents. However, a selective protein kinase C inhibitor, bisindolylmaleimide I (500 nM) significantly inhibited the currents.The thrombin-induced currents were significantly inhibited by SKF96365 (50μM)but not by La(1mM), ruthenium red (10μM) or flufenamic acid (100μM). As assessed with RT-PCR, HUVECs expressed transient receptor potential(TRP)M4,7,TRPV1,2,4,TRPC1,4 and 6 subunits of NSCs.These results indicate that thrombin activates Ca-permeating NSCs containing TRPC4 through protein kinase C in HUVECs. Thus,drugs specifically inhibiting TRPC4-containing channels might be effective to control fatal diseases such as sepsis where thrombin mediates the vicious cycle between inflammation and coagulation.Article信州医学雑誌 59(1): 13-26(2011)departmental bulletin pape

フクマク ハシュ オ トモナッタ コウド シンコウ イガン ニ タイスル TS 1 ニヨル ジュツゼン カガク リョウホウ ノ ユウヨウセイ

TS -１ is an oral anticancer agent developed by utilizing biochemical modulation. We used TS -１in neoadjuvant chemotherapy for a patient with highly advanced gastric cancer that was accompanied by peritoneal dissemination. This enabled us to resect tumor. This patient was a６０-year-old woman. Fluoroscopic upper gastrointestinal series revealed a circumferential, type４lesion extending from the middle of the corpus to the antrum. This was diagnosed by endoscopy as poorly differentiated adenocarcinoma. CT showed ascites, thickening of the gastric wall, and direct infiltration into the head of the pancreas. In endoscopy of the large bowel, a strawberry jelly-like elevation was detected at the ileum. This was diagnosed as poorly differentiated adenocarcinoma, and considered a metastatic lesion produced by dissemination. Chest CT showed a single metastasis in the upper lobe of the right lung. We gave her３cycles of combined TS-１and low-dose CDDP for neoadjuvant chemotherapy. On laparotomy, we found that there was no ascites, and miliary scars were present at several sites near the ascending colon. The antrum of the stomach firmly adhered to the head of the pancreas, and scarred. We judged that the tumor was resectable, and performed distal gastrectomy（D２）plus ileocecal resection（D２）． In histopathological examination, poorly differentiated adenocarcinoma was detected only on a part of the muscular layer in the lesser curvature and posterior wall of the corpus, and marked fibrosis was observed in the submucosal layer. The effect of chemotherapy was histologically evaluated as grade２. The tumor was diagnosed as poorly differentiated adenocarcinoma（por）, with muscularis propria（mp）, lymph invasion２（ly２），vein invasion ０（v０）and degree of lymph node metastasis２（+）［n２（+）］. Tubular adenocarcinoma was detected in a part of the submucosal layer of the ileum. The patient was alive with cancer as of２７months after operation

チョメイナ ノウホウ ケイセイ オ トモナッタ カンサイボウ ガン ノ 1ジケンレイ

It is said that in hepatocellular carcinoma, necrosis is liable to occur in the center and the percentage of cystic formation is comparatively low, but as we have experienced one case of hepatocellular carcinoma associated with marked cystic formation, we reported it. The patient was a female aged７０years. Because general fatigue and anorexia occurred, Abdominal CT test was conducted and hepatic mass was pointed out. Contrast-CT test showed a tumor of６cm in diameter in the medial segment of the left lobe of the liver. As the border was stained with arterial phase, the center was not imaged and the tumor was diagnosed as vascular proliferating type hepatic tumor associated with marked cystic change. MRI test showed that the border of the tumor was lobular and part thereof was of septal structure. The tumor was diagnosed as hepatocellular carcinoma associated with bleeding in tumor, hepatic cystadenocarcinoma or hepatic sarcoma. Abdominal angiography showed not only marked vascular proliferation and tumor stain in the area of the middle hepatic artery but also early venous return, and the middle hepatic vein was clearly imaged. The tumor size was ５×５×４cm in size and was in contact with the middle hepatic vein but it did not invade the vein. Left hepatic lobectomy was performed. Histopathologically the center of the tumor was cystic with colliquative necrosis. The tumor had the trabecular structure and the tumor cell was consisted of clear cell. The tumor was thus diagnosed as poorly differentiated hepatocellular carcinoma. Postoperative course was good and the patient was discharged from our hospital on the１９th day after operation. At the moment when１９ months have passed since the operation, the patient is alive without any recurrence of carcinoma