Carrier-mediated uptake of lucifer yellow in skate and rat hepatocytes: a fluid-phase marker revisited.

Uptake of lucifer yellow (LY), a fluorescent disulfonic acid anionic dye, was studied in isolated skate (Raja erinacea) perfused livers and primary hepatocytes to evaluate its utility as a fluid-phase marker in these cells. However, our findings demonstrated that LY is transported across the plasma membrane of skate hepatocytes largely via carrier-mediated mechanisms. Isolated perfused skate livers cleared 50% of the LY from the recirculating perfusate within 1 h of addition of either 22 or 220 microM LY, with only 4.5 and 9% of the LY remaining in the perfusate after 7 h, respectively. Most of the LY was excreted into bile, resulting in high biliary LY concentrations (1 and 10 mM at the two doses, respectively), indicating concentrative transport into bile canalicular lumen. LY uptake by freshly isolated skate hepatocytes was temperature sensitive, exhibited saturation kinetics, and was inhibited by other organic anions. Uptake was mediated by both sodium-dependent [Michaelis-Menten constant (K(m)), 125 +/- 57 microM; maximal velocity (V(max)), 1.5 +/- 0.2 pmol. min(-1). mg cells(-1)] and sodium-independent (K(m), 207 +/- 55 microM; V(max), 1.7 +/- 0.2 pmol. min(-1). mg cells(-1)) mechanisms. Both of these uptake mechanisms were inhibited by various organic anions and transport inhibitors, including furosemide, bumetanide, sulfobromophthalein, rose bengal, probenecid, N-ethylmaleimide, taurocholate, and p-aminohippuric acid. Fluorescent imaging techniques showed intracellular vesicular compartmentation of LY in skate hepatocyte clusters. Studies in perfused rat livers also indicated that LY is taken up against a concentration gradient and concentrated in bile. LY uptake in isolated rat hepatocytes was saturable, but only at high concentrations, and demonstrated a K(m) of 3.7 +/- 1.0 mM and a V(max) of 1.75 +/- 0.16 nmol. min(-1). mg wet wt(-1). These results indicate that LY is transported into skate and rat hepatocytes and bile largely by carrier-mediated mechanisms, rather than by fluid-phase endocytosis.

Aortic atheroma is related to outcome but not numbers of emboli during coronary bypass.

BACKGROUND: The relation between aortic atheroma severity and stroke after coronary artery bypass grafting is established. The relation between atheroma severity and other outcome measures or numbers of emboli has not been determined. METHODS: Using transesophageal echocardiography, we determined the severity of atheroma in the ascending, arch, and descending aortic segments in 84 patients undergoing operations. Seventy patients were monitored using transcranial Doppler ultrasonography. RESULTS: The incidence of stroke was 33.3% among 9 patients with mobile plaque of the arch and 2.7% among 74 patients with nonmobile plaque (p = 0.011). Cardiac complications were not significantly related to atheroma severity in any aortic segment. Length of stay was significantly related to atheroma severity in the aortic arch (p = 0.025) and descending segment (p = 0.024). The presence of severe atheroma in both the arch and descending segments was associated with significantly longer hospital stays as compared with patients with severe atheroma in neither segment (p = 0.05). Numbers of emboli were greater in patients with severe atheroma at clamp placement, although the differences did not achieve statistical significance. CONCLUSIONS: Aortic atheroma severity is related to stroke and to the duration of hospitalization after coronary artery bypass grafting. The lack of correlation between numbers of emboli and atheroma severity suggests that m any emboli may be nonatheromatous in nature.

Determination of size of aortic emboli and embolic load during coronary artery bypass grafting.

BACKGROUND: Embolic signals have been detected within both the aortic lumen and the intracranial vasculature during coronary artery bypass grafting. Total numbers of these emboli have been reported. The present study examined the size of individual emboli and the total volume of embolization. METHODS: Using transesophageal echocardiography, we continuously monitored the aortic lumen of 10 patients undergoing isolated coronary artery bypass grafting. We manually analyzed 720,000 individual echo frames over a 4-minute period after the release of aortic clamps to track and to calculate the volume of 657 individual particles. The embolic load for the entire procedure was calculated from mean volume based on analysis of 1,508 particles. We simultaneously monitored the middle cerebral artery using transcranial Doppler ultrasonography and compared numbers of emboli detected by the two techniques. RESULTS: Particle diameter ranged from 0.3 to 2.9 mm (mean, 0.8 mm), and particle volume from 0.01 to 12.5 mm3 (mean, 0.8 mm3). Twenty-eight percent of particles measured 1 mm or more, 44% measured 0.6 to 1.0 mm, and only 27% measured 0.6 mm or less in diameter. Aortic embolic load for the procedure ranged from 0.6 cm3 to 11.2 cm3 (mean, 3.7 cm3). Estimated cerebral embolic load for the procedure ranged from 60 to 510 mm3 (mean, 276 mm3). The fraction of aortic emboli entering the cerebral circulation was very variable (3.9% to 18.1%). Seventy-six percent of the embolic volume after the release of clamps occurred over a 20-second period. Only 1 patient was encephalopathic perioperatively. This patient had the largest estimated cerebral embolic load (510 mm3) and the second largest aortic embolic load (8.4 cm3). CONCLUSIONS: We determined the size of individual intraaortic embolic particles and the total volume of embolization during coronary artery bypass grafting, and found the proportion entering the cerebral circulation to be very variable. The constitution of these particles and the neurologic impairment resulting from such embolization remains to be determined.

Impact of embolization during coronary artery bypass grafting on outcome and length of stay.

BACKGROUND: Transcranial Doppler ultrasonography detects emboli in most patients during coronary artery bypass grafting. However, the significance of these emboli has not yet been established. METHODS: We monitored 82 patients during coronary artery bypass grafting with this technique and related the numbers of emboli to the outcomes and length of hospital stay. RESULTS: We detected cerebral emboli in all patients. Patients with stroke (n = 4; 4.9%) had a mean of 449 emboli, as compared with 169 emboli in patients without stroke (n = 78) (p = 0.005). Patients with major cardiac complications (n = 7) had a mean of 392 emboli, as compared with 163 in patients without such complications (n = 75) (p = 0.003). The mean hospital stay of survivors was 8.6 days in patients with fewer than 100 emboli (n = 40), 13.5 days in patients with 101 to 300 emboli (n = 23), 16.3 days in those with 301 to 500 emboli (n = 16), and 55.8 days in patients with more than 500 emboli (n = 6) (p = 0.0007). This relation was unchanged when patients with complications were excluded. The correlation between embolization and outcome was independent of the extent of aortic atheroma or age. CONCLUSIONS: Emboli detected during coronary artery bypass grafting are significantly related to major cardiac and neurologic complications and affect length of stay in all patients, even in the absence of such specific complications.

Active microtubule-dependent secretion of a fluorescent bile salt derivative in skate hepatocyte clusters.

Fluorescence microscopy and video image analysis were used to study the transport of a fluorescent bile acid derivative [N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-7 beta-amino-3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oyl-2-aminoethanesulfonate (NBD-TC)] in isolated clusters of hepatocytes from the little skate Raja erinacea. Analysis of images of hepatocyte clusters that were incubated in medium with 0.5-1 microM NBD-TC showed that the fluorescent derivative accumulated in the cells and that the clusters retained a patent canalicular lumen as well as the ability to actively transport the bile acid derivative from the cells into the lumen; i.e., the lumen-to-cell fluorescence ratio greatly exceeded unity. NBD-TC uptake by hepatocytes was inhibited by several organic anions, of which taurocholate was the most effective. Uptake was also blocked by metabolic inhibitors and by incubation in the cold. Neither Na replacement nor increased medium K, which depolarizes the membrane electrical potential [potential difference (PD)], affected NBD-TC accumulation by hepatocytes. Transport of NBD-TC into the canalicular lumen was inhibited by incubation in the cold and was substantially reduced by high-K medium; these blocks were removed by warming and transfer to normal-K medium, respectively. Treatment of hepatocytes with 20-40 microM nocodazole, a drug that reversibly depolymerizes microtubules, reduced cellular NBD-TC accumulation and blocked its secretion into the canalicular lumen; nocodazole effects were reversed by washing the hepatocyte clusters in drug-free medium. Thus uptake of NBD-TC by skate hepatocytes is active and carrier mediated but not dependent on the PD or Na gradient. NBD-TC secretion from cell to canalicular lumen also appears to be active and carrier mediated. Canalicular secretion appears to be driven at least in part by the PD and is highly dependent on an intact microtubular system in this marine species.

Schizosaccharomyces pombe rad23 is allelic with swi10, a mating-type switching/radioresistance gene that shares sequence homology with human and mouse ERCC1.

Schizosaccharomyces pombe (Sp) rad23-1 mutant cells are extremely sensitive to UV light and ionizing radiation. A genomic DNA fragment that contains wild-type (wt) rad23 has been cloned. The DNA sequence of this cloned gene has been determined and was found to be identical to the previously characterized mating-type switching/radioresistance gene, swi10. Complementation tests between rad23-1 and swi10-154 mutant cells exclusively produce UV-sensitive progeny and confirm that these two genes are allelic. The DNA sequences of rad23-1 and swi10-154 reveal that each contains a single, unique point mutation. In rad23-1, Glu231 changes to a stop codon, resulting in the production of a truncated protein. In swi10-154, a G to A transition mutation is within a splice consensus sequence for intron 1. Therefore, the corresponding mRNA is incapable of being processed appropriately.

Detection of aortic emboli by transesophageal echocardiography during coronary artery bypass surgery.

OBJECTIVE: The purpose of this study was to determine whether emboli can be detected within the aortic lumen in patients undergoing coronary artery bypass surgery (CABG) and to relate the appearance of emboli to specific operative events. DESIGN: Twenty patients were prospectively studied intra-operatively. SETTING: Subjects were inpatients in an academic medical center. PARTICIPANTS: All participants were scheduled for elective, isolated CABG. INTERVENTIONS: Patients were continuously monitored using transesophageal echocardiography (TEE) from aortic cannulation to bypass discontinuation. After completion of the aortic examination, the probe was focused at the level of the aortic arch, just before the takeoff of the left subclavian artery. Emboli were defined as echogenic intraluminal signals not present in the same position on consecutive cross-sectional frames. RESULTS: Intraluminal emboli were detected in all subjects, with a mean number of 535 and range of 8 to 1,885. Embolization was unevenly distributed through the procedure. A mean of 224 (42%) of 535 were detected within 4 minutes of aortic cross-clamp release and another 140 (24%) appeared after partial occlusion clamp release. Together, clamp placement and release represented 84% of all emboli. Emboli detected after clamp release were large, echodense particles easily distinguishable from the small, indistinct, poorly echogenic signals observed at bypass initiation. CONCLUSIONS: Emboli can be visualized within the aortic lumen during CABG. Confirming previous reports, the majority of emboli detected are related to manipulation of aortic clamps. The composition and clinical significance of embolic material are unclear. The value of intraoperative TEE monitoring in predicting neurologic outcome remains to be determined.

Comparison of transcranial Doppler ultrasonography and transesophageal echocardiography to monitor emboli during coronary artery bypass surgery.

BACKGROUND AND PURPOSE: Transcranial Doppler ultrasonography (TCD) is the standard technique for monitoring emboli in the cerebral circulation. Embolic signals have been detected with the use of this technique in most patients undergoing coronary artery bypass surgery. We previously reported that the majority of emboli are detected after release of aortic cross-clamps and partial occlusion clamps. In this study we compare the intraoperative use of TCD with transesophageal echocardiography (TEE) to monitor cerebral emboli. METHODS: We simultaneously monitored 20 patients undergoing coronary bypass surgery with TCD and TEE. All patients also underwent routine TEE examination of the aorta. RESULTS: Embolic signals were detected in all patients by both techniques. Mean total number of emboli was 535 +/- 109 by TEE compared with 133 +/- 28 by TCD. We found correlation between numbers of emboli detected by the two techniques at clamp placement and release (r = .65, P = .002). Clamp placement and release accounted for 84% of all emboli by TEE and 83% by TCD. By TEE, large, highly echogenic particles were detected after clamp release compared with small, barely echodense particles at the onset of bypass. No such distinction was apparent by TCD. We found correlation between severity of aortic atheroma and both TEE- (P = .003) and TCD-detected (P = .009) emboli. CONCLUSIONS: TEE and TCD can both be used to continuously monitor emboli during coronary artery bypass surgery. However, TEE is invasive and justified only if it is being performed for intraoperative assessment of aortic atheromatosis or cardiac function.

Cytoskeletal organization in clusters of isolated polarized skate hepatocytes: structural and functional evidence for microtubule-dependent transcytosis.

Isolated hepatocytes from the marine vertebrate Raja erinacea (the little skate) retain their structural and functional integrity as clusters of cells formed around a single tubular bile canaliculus, and therefore can be used as a model of polarized hepatocytes in situ. In this study we used confocal and conventional epifluorescence microscopy in conjunction with fluorescent markers and immunocytochemistry to examine the structure and function of the cytoskeleton in these cells. Actin filaments in the hepatocyte clusters were found cortically and also concentrated in a pericanalicular array, while microtubules appeared to radiate away from a concentration near the apical membrane of the biliary pole towards the basolateral sinusoidal surfaces. Treatment of clusters with the microtubule disrupting agent, nocodazole, resulted in the microtubules depolymerizing from the basolateral surfaces towards the apical surface, indicating that the microtubules were oriented with their plus ends at the basolateral surface and their minus ends at the apical surface. Nocodazole was also found to disrupt the ability of clusters to transcytose a fluorescent bile salt derivative into their canalicular lumens. We detected cytoplasmic dynein in skate hepatocyte homogenates by Western blotting using an anti-dynein intermediate chain antibody, and immunofluorescent staining of intact hepatocytes revealed a punctate vesicular pattern. The polarized arrangement of microtubules, the presence of cytoplasmic dynein, and the inhibition of bile salt secretion by nocodozole are consistent with the microtubule cytoskeleton playing a fundamental role in the mediation of transcytosis, endocytosis, and bile excretory function in these hepatocytes. These polarized isolated skate hepatocytes represent an excellent experimental model for the in vitro study of hepatic transport, and allow for important comparative studies aimed at elucidating the evolutionarily conserved nature of various hepatocyte structures amongst the vertebrates.