Mei Ling Tsai

Gap junctional communication and vascular smooth muscle reactivity: Use of tetraethylammonium chloride

Oscillatory contractions in uterine smooth muscle are mechanistically related to gap junction complex formation. We have tested the hypothesis that agonist-induced oscillations in vascular smooth muscle are also mediated by gap junctions and that gap junctions are important for vascular smooth muscle cell communication. Total RNA from cultured Wistar-Kyoto rat (WKY) mesenteric arterial cells hybridized strongly with a cDNA probe for the message for connexin43, a monomer of the gap junction. In these same cells, the quaternary ion tetraethylammonium (TEA) (10 mM) increased Lucifer yellow dye transfer between contiguous cells, a measure of cell-to-cell communication via gap junctions, approximately 35% above basal levels. Heptanol, an established inhibitor of gap junction communication, completely blocked both basal- and TEA-stimulated dye transfer between neighboring cells. In other experiments, helical strips of superior mesenteric and tail arteries from WKY rats were mounted in tissue baths for measurement of isometric contractile force. TEA (10(-3)-10(-1) M) induced oscillatory contractions (1-5 cycle/min) in both mesenteric and tail arteries. Removal of endothelium did not affect the pattern of TEA-stimulated oscillations. Oscillations to TEA were blocked in a concentration-dependent manner in both arteries by heptanol (10(-7)-10(-3) M). Heptanol (10(-3) M) also significantly reduced (40%) acetylcholine-induced relaxation in the mesenteric artery (contracted with phenylephrine).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of somatostatin prophylaxis after pylorus-preserving pancreaticoduodenectomy: increased delayed gastric emptying and reduced plasma motilin.

Somatostatin inhibits gastroenteropancreatic exocrine secretion and is often used after pancreaticoduodenectomy to reduce pancreatic secretion to minimize tissue damage and pancreatic stump complications. Because our earlier clinical work saw a major increase in delayed gastric emptying (DGE) with somatostatin prophylaxis after pylorus-preserving pancreaticoduodenectomy (PPPD), this small-group study was designed to confirm or disprove that observation. From August 1997 to December 2000, a total of 23 post-PPPD patients were randomized to receive somatostatin prophylaxis [somatostain (+)] (n = 11) or not [somatostatin] (−) (n = 12). The incidence of DGE, scintographic solid-phase emptying results on day 14 postoperatively, and sequential fasting plasma motilin levels were compared, as motilin levels are related to both gastric motility and somatostatin levels. The somatostatin(+) group exhibited greatly increased patient complaints of DGE: 9 of 11 (82%) versus 3 of 12 (25%) in the somatostatin(−) group. Radiologic scintography showed somatostatin prophylaxis prolonged the half-time (T1/2) of solid-phase emptying: 144.5 ± 51.4 minutes for somatostatin(+) versus 89.0 ± 59.9 minutes for somatostatin(−) (p < 0.001). Comparing pre-PPPD and post-PPPD plasma motilin levels prior to somatostatin infusion, motilin decreased 80% in reaction to the surgery. For somatostatin(−) patients, motilin levels oscillated, or “rang,” postoperatively, reaching a higher level on day 3, declined to a new record minimum on day 7, and by day 21 were 50% of the original and the slope of the recovery curve was increasing well. In somatostatin(+) patients the same ringing pattern was observed but decreased with motilin levels 30% to 70% lower than in the somatostatin(−) patients. By day 21 somatostatin(+) motilin levels were recovering but still only 20% original levels, and the slope of the recovery curve was not optimistic. On postoperative day 14 the plasma motilin levels (below approximately 6 bg/ml) correlated strongly with DGE for both groups. Despite the small sample size, the results indicated that (1) somatostatin prophylaxis significantly decreases fasting plasma motilin; (2) somatostatin prophylaxis produces lingering suppression of plasma motilin; (3) PPPD surgery itself significantly reduces fasting motilin levels with recovery to 50% normal at day 21; (4) the mechanism of somatostatin-induced DGE seems related to reduced fasting plasma motilin levels.

Epithelial-mesenchymal transition contributes to SWCNT-induced pulmonary fibrosis

Abstract Previous studies suggest that single-walled carbon nanotube (SWCNT) exposure causes pulmonary fibrosis. We investigated the contribution of epithelial-mesenchymal transition (EMT) during SWCNT-induced pulmonary fibrosis. C57BL6 female mice were intratracheally instilled with SWCNT at 80 μg/mouse for up to 56 days. SWCNT exposure caused pulmonary epithelial and mesenchymal injury, followed by granulomatous and fibrotic changes. Immunofluorescence staining demonstrated the increasing occurrence of epithelial-derived fibroblasts up to 42 days post-exposure. Flow cytometry analysis revealed that 42.60% of N-cadherin (N-cad)-positive fibroblasts were derived from pulmonary epithelial cells, and, in separate experiments, 30.68% of SPC positive cells were stained for N-cad at 42 days. These epithelial-derived fibroblasts were functional in collagen production. With the progression of fibrosis, there were increases in the number of hyperplastic epithelial cells stained positively for TGF-β/p-Smad2 or β-catenin. Therefore, EMT contributes significantly to fibroblast expansion. Aberrant activations of TGF-β/p-Smad2 and β-catenin are postulated to induce EMT during SWCNT-induced pathogenic fibrosis.

Detection of Total and A1c-Glycosylated Hemoglobin in Human Whole Blood Using Sandwich Immunoassays on Polydimethylsiloxane-Based Antibody Microarrays

The percentage of glycosylated hemoglobin A1c (%GHbA1c) in human whole blood indicates the average plasma glucose concentration over a prolonged period of time and is used to diagnose diabetes. However, detecting GHbA1c in the whole blood using immunoassays has limited detection sensitivity due to its low percentage in total hemoglobin (tHb) and interference from various glycan moieties in the sample. We have developed a sandwich immunoassay using an antibody microarray on a polydimethylsiloxane (PDMS) substrate modified with fluorinated compounds to detect tHb and glycosylated hemoglobin A1c (GHbA1c) in human whole blood without sample pretreatment. A polyclonal antibody against hemoglobin (Hb) immobilized on PDMS is used as a common capture probe to enrich all forms of Hb followed by detection via monoclonal anti-Hb and specific monoclonal anti-GHbA1c antibodies for tHb and GHbA1c detection, respectively. This method prevents the use of glycan binding molecules and dramatically reduces the background interference, yielding a detection limit of 3.58 ng/mL for tHb and 0.20 ng/mL for GHbA1c. The fluorinated modification on PDMS is superior to the glass substrate and eliminates the need for the blocking step which is required in commercial enzyme linked immunosorbent assay (ELISA) kits. Moreover, the detection sensitivity for GHbA1c is 4-5 orders of magnitude higher, but the required sample amount is 25 times less than the commercial method. On the basis of patient sample data, a good linear correlation between %GHbA1c values determined by our method and the certified high performance liquid chromatography (HPLC) standard method is shown with R(2) > 0.98, indicating the great promise of the developed method for clinical applications.

Quantitative shot-gun proteomics and MS-based activity assay for revealing gender differences in enzyme contents for rat liver microsome

Liver microsomes are subcellular fractions that contain many metabolizing enzymes for drugs and endogeneous compounds. Some of these enzymes are regulated by sex hormonal control and exhibit sex-dependent expression pattern and metabolizing speed. Studying these enzymes, however, are complicated by the presence of isoforms such as cytochrome P450 (CYP450), which families share more than 50% amino acid identities. In this study, we applied quantitative shot-gun proteomics approach coupled with stable-isotope dimethyl labeling, two-dimensional reversed-phase peptide separation and tandem mass spectrometry (MS) to explore the gender-dependent expression of rat liver microsomal proteins. A total of 391 proteins were identified and quantified by this approach, and 56% of quantified proteins were enzymes. Although shot-gun approach is rarely used for identifying protein isoforms, we identified 53 isoforms by at least one unique peptide including 21 isoforms of CYP450s. Moreover, by quantitative and statistics assessment, we were able to classify them into 28 male dominant enzymes including CYP2C12 CYP2C11, CYP2C13, CYP2B3, CYP2C11, CYP2C70 and CYP3A2 which are known to be male specific, 21 female dominant enzymes including CYP2A1, CYP2C7, CYP2C12, CYP2D26, alcohol dehydrogenase 1, carboxylesterase 3, glutathione S-transferase, liver carboxylesterase 4, UDP-glucuronosyltransferase 2B1, and glyceraldehyde-3-phosphate dehydrogenase which are known to be female specific; and 125 sex-independent enzymes. However, most of the sex specificities revealed from this study, such as the male specificity of CYP2D1, were novel and not yet reported. We then conducted a mass spectrometry-multiple reaction mode (MS-MRM) based enzyme activity method to determine the catalyzing rate of CYP2D1 in male and female liver microsomes using carteolol as its specific substrate. The reaction rate catalyzed by CYP2D1 in female rats was determined to differ significantly with the rate in male rats. Moreover, the ratio (female/male) of reaction rate (0.68) was found to correlate with their relative protein abundance (0.72). This study revealed novel sex dependences of many rat liver enzymes and also demonstrated a unique MS-based analytical platform that could identify novel iso-enzymes and further quantify their abundance and enzyme activity.

The induction of vascular endothelial growth factor by ultrafine carbon black contributes to the increase of alveolar-capillary permeability

Ultrafine carbon black (ufCB) can cause proinflammatory response and increase alveolar-capillary permeability. However, the mechanism underlying the increased permeability is not well characterized. Vascular endothelial growth factor (VEGF) is originally recognized as a vascular permeability factor. Oxidative stress generated by hydrogen peroxide (H2O2) stimulates VEGF gene expression. The purpose of this study was to explore the role of VEGF in ufCB-induced alveolar-capillary permeability. Intratracheal instillation of 200 μg ufCB in mice caused a significant and sustained increase of total proteins in bronchoalveolar lavage (BAL) fluid, with the maximal increase at 21 hr postinstillation. The influx of neutrophils did not significantly increase until 16 hr. It reached the highest level at 21 hr and returned to the basal level by 42 hr. Tumor necrosis factor-α was significantly elevated only at 4 hr. ufCB induced significant increases of VEGF in BAL fluid throughout the study period, with the peak increase at 16 hr. The nonsecreted isoform VEGF188 was not altered after 16 hr of exposure to ufCB. Moreover, there was a strong correlation between VEGF and total proteins in BAL fluid (R2 = 0.7352, p < 0.01). In vivo study supported the role of reactive oxygen species (ROSs) in ufCB-induced VEGF release and protein leakage. The involvement of ROSs was strengthened by the fact that interventions with N-acetyl-cysteine prevented ufCB-induced generation of ROSs and VEGF in vitro. Our study for the first time demonstrates that ufCB induces the production of VEGF, which is associated with the increase of alveolar-capillary permeability. The induction of VEGF by ufCB acts through an ROS-dependent pathway.

Reconsideration of Delayed Gastric Emptying in Pancreaticoduodenectomy

Literature reports indicate that the incidence of delayed gastric emptying (DGE) is higher after pylorus-preserving pancreaticoduodenectomy (PPPD) than after conventional pancreaticoduodenectomy (CPD), but DGE is traditionally diagnosed from patient-reported subjective sensations. Our clinical radiological experience suggests higher rates for physiological DGE post-CPD. We therefore sought to quantify rates of subjective DGE (sDGE, based on patient complaint) verses objective DGE (oDGE, based on scintography) post-CPD and post-PPPD. Contractile motility of post-PPPD residual stomach was also studied. For 21 PPPD and 33 CPD patients between October 1997 and June 2000, sDGE and oDGE data were collected preoperatively, on postoperative day 14, and 6 months postoperatively, with cholescintography for pylorus ring competency on postoperative day 14. The incidence of sDGE was higher for PPPD (42%) than for CPD (15%) at 14 days, with zero sDGE for both at 6 months. The incidence oDGE was higher for CPD (91%) than for PPPD (76%) at 14 days, with a 6-month incidence of 4.7% in PPPD but ~33% for CPD. Solid-phase emptying in PPPD showed that residual stomach retained partial gastric emptying function at 14 days but not at 6 months. Cholescintography showed abnormal pylorus closure function in 2 of 21 PPPD patients but was not related to DGE. Literature reports of higher DGE incidence post-PPPD are true only for subjective symptoms. Radiological measurement of oDGE shows that both CPD and PPPD manifest ~80% incidence of DGE in the early postoperative period. At 6 months, ~33% of CPD show persistent oDGE. We concluded that (1) the concept of DGE should distinguish between subjective and objective symptoms; (2) loss of distal stomach mechanoreceptors in CPD reduces patients sensation of oDGE, producing “silent” DGE;(3) both CPD and PPPD have high and approximately equal rates oDGE;(4) the previously unnoticed silent oDGE in CPD may contribute to the higher rates of ulceration and related morbidity in association with CPD

Responses of cerebral circulation produced by adrenoceptor agonists and antagonists in rats.

The effects of intravenous administration of adrenoceptor agonists and antagonists on relative cerebral blood flow, cerebral perfusion pressure, intracranial pressure, mean arterial blood pressure and heart rate were assessed in rats under urethane anesthesia. Administration of phenylephrine (a preferential alpha 1-adrenoceptor agonist), adrenaline (a mixed alpha/beta-adrenoceptor agonist), noradrenaline (a mixed alpha/beta-adrenoceptor agonist) raised mean arterial pressure, cerebral perfusion pressure, cerebral blood flow and intracranial pressure, but lowered heart rate. On the other hand, administration of isoproterenol (a beta-adrenoceptor agonist), phentolamine (an alpha-adrenoceptor antagonist) or propranolol (a beta-adrenoceptor antagonist) lowered mean arterial pressure, cerebral perfusion pressure or cerebral blood flow. In addition, phentolamine raised both intracranial pressure and heart rate, whereas propranolol lowered both intracranial pressure and heart rate. However, isoproterenol produced a decrease in heart rate, without affecting intracranial pressure. There was no significant difference between the groups of animals for PCO2, PO2 or pH throughout the studies. The results suggest that adrenoceptor agonists or antagonists act through breakthrough of autoregulation, with acute hypertension or hypotension, to enhance or to reduce cerebral blood flow in rats.

Hypertension and tachycardia produced by inhibition of reuptake of 5-Hydroxytryptamine by fluoxetine in the rat

In rats anaesthetized with urethane, increasing the activity of 5-hydroxytryptamine receptors or the level of cuntional serotonin in the brain with the inhibitors of the reuptake of serotonin, fluoxetine, produced both hypertension and tachycardia. The hypertension induced by fluoxetine was significantly inhibited by pretreatment of the animals with ketanserin (a serotonin receptor antagonist), by bilateral vagotomy, spinal transection or bilateral adrenalectomy. On the other hand, the tachycardia induced by fluoxetine was significantly inhibited by pretreatment with ketanserin or bilateral vagotomy, but not by spinal transection or adrenalectomy. The data indicate that fluoxetine acts through serotonin receptors in the central nervous system by influencing autonomic outflow to induce both hypertension and tachycardia.

Participation of a bulbospinal serotonergic pathway in the rat brain in clonidine-induced hypotension and bradycardia.

The effects of microinjection of clonidine (1-10 micrograms in 1 microliter) into a region adjacent to the ventrolateral surface of the medulla oblongata on cardiovascular function were assessed in urethane-anesthetized rats. Intramedullary administration of clonidine, but not saline vehicle, caused a dose-dependent decrease in both the mean arterial pressure and the heart rate. The clonidine-induced hypotension was antagonized by prior spinal transection, but not bilateral vagotomy. On the other hand, the clonidine-induced bradycardia was antagonized by prior bilateral vagotomy, but not spinal transection. Furthermore, selective destruction of the spinal 5-HT nerves, produced by bilateral spinal injection of 5,7-dihydroxytryptamine, reduced the magnitude of the vasodepressor or the bradycardiac responses to clonidine microinjected into the area near the ventrolateral surface of the medulla oblongata in rats. The data indicate that a bulbospinal serotonergic pathway is involved in development of clonidine-induced hypotension and bradycardia. The induced hypotension is brought about by a decrease in sympathetic efferent activity, whereas the induced bradycardia was due to an increase in vagal efferent activity.