Prognostic significance of L-type amino-acid transporter 1 expression in surgically resected pancreatic cancer.

BACKGROUND: The expression of L-type amino-acid transporter 1 (LAT1) is tumour-specific and has been shown to have essential roles in cell growth and survival. However, little is known regarding the clinical significance of LAT1 expression in pancreatic cancer. This study was conducted to determine the prognostic significance of LAT1 expression. METHODS: A total of 97 consecutive patients with surgically resected pathological stage I-IV pancreatic ductal adenocarcinoma were retrospectively reviewed. Tumour sections were stained by immunohistochemistry for LAT1, CD98, Ki-67 and vascular endothelial growth factor (VEGF), and microvessel density was determined by CD34 and p53. RESULTS: L-type amino-acid transporter 1 and CD98 were highly expressed in 52.6% (51/97) and 56.7% (55/97) of cases, respectively (P=0.568). The expression of LAT1 within pancreatic cancer cells was significantly associated with disease stage, tumour size, Ki-67, VEGF, CD34, p53 and CD98. L-type amino-acid transporter 1 expression was confirmed to be a significant prognostic factor for predicting poor outcome by multivariate analysis. CONCLUSION: L-type amino-acid transporter 1 expression is a promising pathological marker for the prediction of outcome in patients with pancreatic cancer.

A case of rhabdomyosarcoma of the vagina in an elderly woman.

Most rhabdomyosarcomas of the vagina (RMSV) occur in infants and children up to six years old. RMSV in elderly patients is extremely rare. We report a case of a 70-year-old woman with RMSV. She had received surgery for uterine endometrial cancer one year before and a vaginal polypoid tumor was noted during routine follow-up vaginal examination. She was referred to our department for radiation therapy following partial tumorectomy of the lesion. She was given three sessions of intra-vaginal radiation therapy, once a week with 6 Gy at 7.5 mm below the vaginal surface and external irradiation of 50 Gy to the pelvis. However, paraaortal lymph node metastasis developed during initial radiation therapy. Furthermore, multiple bone metastases appeared at the completion of the radiation therapy. Six months after initial treatment the patient died from progression of the disease. Autopsy demonstrated small residual tumor at the primary site as well as multiple systemic metastases.

Constitutive secretion of immunoglobulin A and other proteins into lumina of unstimulated submandibular glands in anaesthetised rats.

Salivary fluid secretion is dependent upon reflex stimuli mediated by autonomic nerves. In order to determine if immunoglobulin A (IgA) and salivary proteins are secreted in the absence of nerve stimulation, small volumes (< 2 microl) of saliva were consecutively collected from the submandibular duct of anaesthetised rats following rest pauses in order to sample the protein contents of the ductal system. Within the first 5 microl of such saliva collected by parasympathetic nerve stimulation, IgA and other salivary proteins reached peak concentrations that were over 20-fold greater than levels in parasympathetically stimulated saliva subsequently collected during a 5 min period of stimulation. Confocal microscopy of TRITC-labelled IgA added to live, acutely isolated submandibular acini indicated that it did not enter the lumina by paracellular leakage. IgG is thought to enter saliva by paracellular leakage but did not accumulate in luminal saliva in the present study. Electrophoresis suggested that the major proteins secreted in the absence of stimulation were the same as those present in subsequently stimulated saliva. It can be concluded that IgA and other major submandibular proteins are secreted into glandular lumina in the absence of nerve stimulation. The functional significance of such unstimulated protein secretion is at present unclear.

Quantitative comparison of anti-fading mounting media for confocal laser scanning microscopy.

Fading is one of the major obstacles to reliable observation in fluorescence microscopy. Using a confocal laser scanning microscope (CLSM) coupled to a computer, we quantitatively measured fading of fluorescence to formulate an equation, evaluated the anti-fading ability of several anti-fading media, and restored the faded images to the original level according to this equation. NIH 3T3 cells were stained with fluorescein isothiocyanate (FITC)-phalloidin, mounted with several commercial and homemade anti-fade media, and observed with CLSM under repeated illumination. With any mounting medium, attenuation of fluorescence intensity at a certain pixel occurred stepwise and the decrease was proportional to the intensity of the previous scan. From these results, we formulated an equation that has three coefficients: anti-fading factor (A), indicating the ability to retard fading; fluorescent intensity at the first scan (EM(1)); and background fluorescence (B). The fluorescent intensity at a certain point following nth scan is given as EM(n) = EM(1) * A ((n-1)). This equation was available for restoring faded images to their original states, even after the image had faded to only 60% of its original intensity.

Fluid and amylase secretion by perfused parotid gland: physio-morphological approach.

Whole gland perfusion technique was applied to rat parotid glands to assess whether amylase affects fluid secretion. Control perfusion without any secretagogue evoked no spontaneous secretion. Carbachol (CCh 1 microM) induced both amylase and fluid secretion with distinctive kinetics. Fluid secretion occurred constantly around 60 microL/g-min, whereas amylase secretion exhibited an initial peak, followed by a rapid decrease to reach a plateau. Isoproterenol (Isop 1 microM) alone did not induce fluid secretion although it evoked amylase secretion as measured in isolated perfused acini. Addition of Isop during CCh stimulation evoked a rapid and large rise in amylase secretion accompanied by small increase in oxygen consumption. Morphological observations carried out by HR SEM and TEM revealed exocytotic profiles following Isop stimulation. CCh stimulation alone seldom showed exocytotic profiles, suggesting a low incidence of amylase secretion during copious fluid secretion. Combined stimulation of CCh and Isop induced both vacuolation and exocytosis along intercellular canaliculi. These findings suggest that control of salivary fluid secretion is independent of the amylase secretion system induced by CCh and/or Isop.

Cell biology of human salivary secretion.

We treated surgical specimens of human parotid and submandibular glands in vitro to manipulate the receptor-signaling cascade pharmacologically and analyzed cellular responses by light microscopy on epoxy embedded sections. Treatment of specimens with the b-agonist, isoproterenol, and with the second messenger analog, dibutyryl cyclic AMP, stimulated serous acinar cells to engage in exocytosis and degranulation. The muscarinic agonist, carbachol, and the calcium ionophore, A23187, on the other hand, elicited formation of "vacuoles" in the cytoplasm of serous acinar cells. Taking previous in vivo human and animal studies into account, these changes are suggested as the morphological expression of enzyme release and fluid secretion, respectively. Specimens obtained from patients over 70 years old exhibited poor response even though their morphological appearance remained intact. Aged salivary glands are thus suggested to experience a decline in their secretory activity at the cellular level, probably by impairment of the signaling processes downstream to the receptor activation and second messenger production.

Relationship between amylase and fluid secretion in the isolated perfused whole parotid gland of the rat.

Whole gland perfusion technique was applied to rat parotid glands to assess whether amylase affects fluid secretion. Control perfusion without any secretagogue evoked no spontaneous secretion. Carbachol (CCh 1 microM) induced both amylase and fluid secretion with distinctive kinetics. Fluid secretion occurred constantly at 40-120 microliter/g-min (average plateau was 60 microliter/g-min), whereas amylase secretion exhibited an initial peak (10 mg maltose/30 s per g wet w. of the gland), followed by a rapid decrease to reach a plateau level of 1 mg maltose/30 s later than 1.5-2 min. Isoproterenol (Isop 1 microM) alone did not induce fluid secretion although it evoked amylase secretion as measured in isolated perfused acini. Addition of Isop during CCh stimulation evoked a rapid and large rise in amylase secretion to 15 mg maltose/30 s accompanied by the increase in oxygen consumption. However, the fluid secretion exhibited a rather gradual decrease. These findings suggest that control of salivary fluid secretion is independent of the amylase secretion system induced by CCh and/or Isop. Morphological observations carried out by HR SEM and TEM revealed exocytotic profiles following Isop stimulation. CCh stimulation alone seldom showed -exocytotic profiles, suggesting a low incidence of amylase secretion during copious fluid secretion. Combined stimulation of CCh and Isop induced both vacuolation and exocytosis along intercellular canaliculi. During washout of secretagogues, lysosomal digestion of excess membrane took place.

Carbachol-induced [Ca2+]i increase, but not activation of protein kinase C, stimulates exocytosis in rat parotid acini.

1. A column perfusion system was applied to rat parotid acinar cells to clarify the roles of Ca2+ and protein kinase C (PKC) in the mechanisms of carbachol (CCh)-induced amylase secretion. 2. CCh evoked a biphasic response of amylase secretion with an initial rapid and large peak that reached maximum at about 10 s followed by a sustained plateau. The time profile and the dose-response relationship paralleled with those of cytosolic free Ca2+ concentration ([Ca2+]i). 3. The CCh-induced sustained response of amylase secretion maintained by Ca2+ influx into cells was ATP dependent, while the initial peak response regulated by Ca2+ released from InsP3-sensitive stores was relatively ATP independent. 4. Restoration of extracellular Ca2+ during continuous stimulation with CCh in Ca(2+)-free medium evoked a second rapid and large peak of amylase secretion. 5. Phorbol 12,13-dibutyrate (PDBu) potentiated the CCh-induced amylase secretion in both the initial peak and the sustained plateau without enhancing CCh-induced [Ca2+]i changes. 6. PKC inhibitors such as Ro 31-8220 inhibited the potentiating effect of PDBu but only slightly reduced amylase secretion induced by CCh alone. 7. These results suggest that a CCh-induced rise in [Ca2+]i triggers the final fusion and/or exocytosis of amylase secretion. CCh also has some ability to promote ATP-dependent priming of secretory granules that, together with Ca2+ influxed into cells, contributes to the CCh-induced sustained plateau of amylase secretion. PDBu-induced activation of PKC promotes the priming of secretory granules, thereby enhancing the efficacy for Ca2+ to trigger fusion/exocytosis.

High levels of GM(1)-ganglioside and GM(1)-ganglioside beta-galactosidase in the parotid gland: a new model for secretory mechanisms of the parotid gland.

A new model for the subcellular basis of parotid secretion is presented in this article. GM(1)-ganglioside, typically found in neural tissues, is shown to be abundant in the parotid gland. This ganglioside may play a central role in membrane turnover mechanisms underlying exocytosis/endocytosis in its role as a promoter of membrane fusion or a fusogen. The lysosome and lysosomal hydrolases also play a central role in this model in catabolism of GM(1)-ganglioside. Consequently, high levels of the lysosomal hydrolase acidic beta-galactosidase are demonstrated in the salivary gland. GM(1)-gangliosidosis of the parotid glands, as described in mice, appears to be the first single-gene heritable disease found so far in the salivary glands.

Millisecond analyses of Ca2+ initiation sites evoked by muscarinic receptor stimulation in exocrine acinar cells.

High speed laser confocal microscopy (8 ms/image) was applied to the dissociated parotid acini as a model to study Ca2+ signaling mechanisms in non-excitable exocrine secretory cells. Immunofluorescence microscopy showed the localization of IP3 receptor type 2 along the apical membrane region. Muscarinic stimulation with carbachol evoked a rise in [Ca2+]i that was initiated from apical region and propagated into basal region as Ca2+ waves. This was most clearly observed when extracellular Ca2+ was omitted. Carbachol also triggered the abrupt increase of [Ca2+]i simultaneously at both basal and apical regions in many acini. Within an acinus, each cell responded synchronously. The present results suggest that one Ca2+ initiation site in the rat parotid acinar cell is apical region, corresponding to the localization of IP3 receptors. Another Ca2+ initiation site is basal region, which seems to be related to Ca2+ entry from extracellular medium and/or Ca2+ release from basally located organelles such as nuclei and endoplasmic reticulum.

Human salivary gland parenchymal cells seen by SEM from the cytoplasmic side using a new osmium maceration method.

By removing all or most organelles, we have exposed the cytoplasmic side of the plasmalemma and its specializations in serous cells and in cells of striated and excretory ducts of human major salivary glands. The areas of plasmalemma located beneath the lumen and those bordering the intercellular canaliculi are covered by evenly distributed particles arranged in a continuous band and, below it, in regularly spaced clusters. A similar pattern of particles is seen on the internal aspects of the juxtaluminal plasmalemma of cells of both striated and excretory ducts. Small isolated clusters of particles are seen in other regions of serous and ductal cells as well, being particularly numerous along the basal processes of cells of striated ducts. A distribution of particles resembling that present along intercellular canaliculi of serous cells also is seen on the plasmalemma bordering the biliary canaliculi where, however, the clusters look smaller and farther apart. Large clusters of particles, matching those seen on salivary glands and on liver, are present at the base of the short processes of cells of the stratum spinosum of squamous stratified epithelia. Since the sites of location of the clusters closely correspond to the areas where transmission electron microscopy (TEM) reveals the presence of desmosomes, we believe that the clusters may be related to these cellular junctions. Of more difficult interpretation are the particles present on the juxtaluminal band corresponding both to the zonula occludens and to the zonula adhaerens.

Cytoskeletal regulation of human salivary secretion studied by high resolution electron microscopy and confocal laser microscopy.

To study the cell regulation mechanisms of human salivary secretion, surgical specimens of human parotid and submandibular glands were treated in vitro with isoproterenol (beta-agonist), carbachol (muscarinic agonist), and cytochalasin D (microfilament disruptive agent), and morphological changes occurring in serous acinar cells were observed. Control acinar cells treated without secretagogues exhibited only occasional examples of exocytosis. Microfilaments, revealed by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) of F-actin fluorescence stained by rhodamine-phalloidin, were localized underneath the luminal membrane to separate the secretory granules from the luminal membrane. Following isoproterenol treatment, secretory granules made direct contact with the luminal membrane and many omega-shaped exocytotic profiles appeared. TEM and scanning electron microscopy (SEM) showed these profiles to be of granule size or somewhat smaller and to be provided on their cytoplasmic surface with coated pits. Furthermore, CLSM detected the appearance of F-actin fluorescence around the exocytosed granule membranes. Carbachol treatment also evoked the formation in acinar cells of omega-shaped exocytotic profiles some of which were larger than the granules and which exhibited neither coated pits nor associated F-actin fluorescence. To determine if microfilaments regulate the post-exocytotic process of membrane retrieval, we combined isoproterenol treatments with cytochalasin D or carbachol. Following these treatments, F-actin fluorescence surrounding the exocytosed membrane was dispersed or diffused and the exocytotic profiles enlarged remarkably. These results led to the hypothesis that exo/endocytotic processes in human salivary serous acinar cells are regulated differently under autonomic receptor control mediated by microfilaments.

Apical vesicles bearing inositol 1,4,5-trisphosphate receptors in the Ca2+ initiation site of ductal epithelium of submandibular gland.

In polarized epithelial cells, agonists trigger Ca2+ waves and oscillations. These patterns may be caused by the compartmentalization of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools into specific regions. We have investigated the relationship between the distribution of IP3 receptors (IP3Rs) and the spatiotemporal pattern of Ca2+ signaling in the duct cells of the rat submandibular gland (SMG). Using immunofluorescence, although labeling was somewhat heterogeneous, the IP3Rs were colocalized to the apical pole of the duct cells. Immunoelectron microscopy identified small apical vesicles bearing IP3R2 in some types of duct cells. Real-time confocal imaging of intact ducts demonstrated that, after carbachol stimulation, an initial Ca2+ spike occurred in the apical region. Subsequently, repetitive Ca2+ spikes spread from the apical to the middle cytoplasm. These apical Ca2+ initiation sites were found only in some "pioneer cells," rather than in all duct cells. We performed both Ca2+ imaging and immunofluorescence on the same ducts and detected the strongest immunosignals of IP3R2 in the Ca2+ initiation sites of the pioneer cells. The subcellular localization and expression level of IP3Rs correlated strongly with the spatiotemporal nature of the intracellular Ca2+ signal and distinct Ca2+ responses among the rat SMG duct cells.

Exocytosis in human salivary glands visualized by high-resolution scanning electron microscopy.

The luminal membrane of salivary acinar cells creates a specialized cell surface area that accepts exocytosis and undergoes dynamic changes during secretion. These changes were visualized three-dimensionally from both the inside and outside of the cell in human parotid and submandibular glands, by application of in vitro secretory stimulation and then of OsO4 maceration to remove cytoplasmic organelles by varying degrees. In control glands treated without secretagogues, the luminal surface of serous acinar cells bore well-developed microvilli with only an occasional incidence of exocytotic profiles. Following treatment with the beta-adrenergic agonist, isoproterenol, considerable shortening and loss of microvilli occurred along the luminal membrane where, on its cytoplasmic side, many protuberances of sizes similar to or smaller than those of single secretory granules (approximately 1 micron in diameter) appeared. The cytoplasmic surface of these protuberances exhibited small vesicles (approximately 100-150 nm in diameter) that, by transmission electron microscopy, were shown to be coated pits or vesicles present on or around the exocytosed granule membranes. Treatment of tissues with the muscarinic agonist carbachol also caused a decrease of microvilli and the appearance of protrusions at the luminal membrane. However, unlike isoproterenol treatment, many of these protrusions were devoid of small pits or vesicles and were much larger than a single secretory granule. These results indicate that (1) secretory stimulation causes the dynamic transformation of microvilli at the luminal membrane, where granule docking and membrane fusion take place, and (2) after fusion, the exocytosed membranes are processed differently, by coated pit/vesicle mediated or non-mediated mechanisms, according to the autonomic receptor control.

Dynamics of salivary secretion studied by confocal laser and scanning electron microscopy.

Plasma membrane events during secretion of the parotid and submandibular glands of humans and rats were observed in living cells by confocal microscopy and from the cytoplasmic side by scanning electron microscopy after removal of the cell organelles by the OsO4 maceration method. These new microscopic techniques revealed in acinar cells two distinct exocytosis-endocytosis coupling mechanisms elicited in response to different secretory stimuli. Beta-adrenergic stimulation with isoproterenol and its second messenger analog dibutyryl cyclic AMP evoked exocytosis after which fused granule membranes were individually removed from the luminal plasma membrane within several minutes. On the fused membrane area, small endocytotic vesicles about 100-150 nm in diameter were abundant. Muscarinic stimulation with carbachol also induced exocytosis, but in this case the fused granule membranes coalesced to form enlarged invaginations which stayed on the luminal membrane for more than 30 min. These invaginations, almost devoid of small endocytotic vesicles, were then pinched off from the luminal membrane and dispersed into the cytoplasm in the form of light microscopically detectable vesicles. After treatment with isoproterenol and carbachol, acinar cells exhibited different distributional changes of F-actin around the fused membrane area, suggesting the involvement of microfilaments in regulating the membrane events following exocytosis.

Scintigraphic detection of xenografted renal tumor by anti-renal cancer monoclonal antibody radiolabeled with technetium-99m.

Accumulation in the tumor of the RCS-1 monoclonal antibody, which recognizes the cell surface antigen of renal cancer cells was examined. The antibody purified by affinity chromatography (protein-A column) and gel fractionation was labeled with technetium-99m (99mTc) by a direct method. High labeling efficiency (> 98%) could be routinely obtained. However, the 99mTc labeling of the antibody did not reduce the reactivity of the RCS-1 antibody. The labeled antibody was injected into nude mice transplanted with human renal and gastric tumors, and the accumulation of the antibody in each tumor and various tissues was compared at 48 hours after injection. The highest accumulation of radiolabeled RCS-1 antibody was observed in the AM-RC-3 renal tumors; at 8.0% of the injected dose per gram and a tumor-to-blood ratio of 1.05, respectively. However, the radiolabeled RCS-1, did not show specific accumulation in the gastric tumor nor in any tissues tested. The xenografted tumor, AM-RC-3 was successfully visualized with the radiolabeled RCS-1 antibody by scintigraphy.

[Function of myoepithelial cells in salivary secretion: reevaluation of the expulsion theory].

Although myoepithelial cells have been suggested to provide the expulsive force to secrete saliva into the mouth, direct evidence to support this idea has been lacking. We examined the structure and distribution of myoepithelial cells in rat parotid and sublingual glands, and observed whether contraction occurs in living acini during secretion in vitro. Scanning electron microscopy and the whole mount alkaline phosphatase histochemistry revealed well-developed myoepithelial cells around the sublingual acini. In the parotid gland, myoepithelial cells were abundant along the intercalated ducts but were sparse around the acini. A stress fiber-like distribution of actin fibers was demonstrated by rhodamine phalloidin staining in the myoepithelial cells of sublingual glands. Under a video-enhanced microscope, a rapid shrinkage of sublingual acini was observed within 2 min of secretory stimulation. However, the shrinkage was slight and transient, and thereafter the acinar shape was almost maintained over 30 min, during which time considerable amounts of secretory granules were released. It was suggested that the major function of myoepithelial cells in salivary secretion is the support for the glandular structure through isometric contraction. This conclusion is compatible with our previous morphometric analyses on fixed cells (Noriko Shoi: Kitasato Med 24: 432-434, 1994).

Tight junctional permeability in living cells: dynamic changes directly visualized by confocal laser microscopy.

Confocal microscopy was used to study the tight junctional permeability in living rat parotid and submandibular glands. The interstitial space of the tissue was perfused with medium containing fluorescent tracers Lucifer Yellow (anionic: MW 457), Propidium Iodide (cationic: MW 668) and dextrans labeled with FITC or RITC (anionic and neutral: MW 3K, 10K, 40K, 70 K and 500 K) to monitor whether or not these tracers permeate into the lumen across the junction. In the acini of normal glands, fluorescence was detected in the basolateral space but not in the luminal space up to 30 min. However, when secretion was induced by isoproterenol or carbachol, fluorescence appeared in the luminal space within 2 to 5 min. This did not involve the disruptive changes in tight junction ultrastructure, nor was it irreversible; the luminal fluorescence disappeared again when the secretagogues were removed. Tracers up to MW 40 K for isoproterenol and MW 10 K for carbachol revealed the luminal fluorescence in parotid acini, with little indications of the charge preference characteristics. The luminal fluorescence also appeared by anoxia, enzymatic cell dissociation and the cytochalasin D treatment. It was suggested that the tight junctions in salivary acini dynamically alter their permeability and modulate the passage of large molecules through the paracellular pathway. Oxygen supply, extracellular matrices and cytoskeletons were suggested to influence these regulations.