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Acetylcholine (ACh) caused repetitive transient Cl- currents activated by intracellular Ca2+ in single rat submandibular grand acinar cells. As the concentration of ACh increased the amplitude and the frequency of the transient Cl- currents increased. These responses occurred also in the absence of extracellular Ca2+ but disappeared after several minutes. Repetitive transient Cl- currents were restored by readmission of Ca2+ to the extracellular solution. The higher the concentration of extracellular Ca2+ readmitted, the larger the amplitude of the transient Cl- currents. Ca2+ entry through a store-coupled pathway was detected by application of Ca2+ to the extracellular solution during a brief cessation of stimulation with ACh. In these experiments too, the higher the concentration of Ca2+, the larger the transient Cl- currents activated by Ca2+ released from the stores. The time course of decrease in total charge movements of repetitive transient responses to ACh with removal of extracellular Ca2+ depended on a decrease in charge movements of each transient event rather than a decrease in frequency of the repetitive events. The decrease of charge movements of each transient event was due to a decrease in its amplitude rather than its duration. The results suggest that in this cell type and amplitude-modulated mechanism is involved in repetitive Ca2+ release and that Ca2+ entry is essential to maintain the repetitive release of Ca2+. The results further suggest that the magnitude of Ca2+ entry determines the number of unitary stores filled with Ca2+ which can synchronously respond to ACh.

1 Effects of various drugs were examined on the mechanical responses of the circular smooth muscle of guinea-pig vas deferens evoked by field stimulation and ACh. 2 During field stimulation (0.1 msec, 3–80 Hz) a contraction consisting of two (initial and second) phases occurred. In addition, another contraction (after-response) occurred after the cessation of the stimulation particularly at higher frequencies (40–80 Hz). 3 Guanethidine (2–20 μM) suppressed both the initial and second phases of the field stimulation-evoked responses. Prazosin (0.1-1 μM) suppressed the second phase but minimally affected the initial phase. 4 Both phases were suppressed by atropine (0.1-1 μM.) and potentiated by physostigmine (5 μM). 5 dTC (0.5–20 μM) did not suppress either phase but potentiated the second phase. 6 ACh (0.28–280 μM) produced a contraction, which was reduced by atropine but was little affected by either dTC or TTX. 7 These results suggest that at the postsynaptic site of the neuromuscular junction in this tissue the sympathetic process activates the smooth muscle through two different types of receptor; one is non-noradrenergic and the other is an α-adrenoreceptor. In addition, a cholinergic process also activates the smooth muscle through muscarinic receptors.

1. The effects of various drugs on excitatory junction potentials (EJPs) elicited by field stimulation of the longitudinal smooth muscle of the guinea-pig vas deferens were examined using the sucrose-gap method. 2. Field stimulation with a single pulse (1.0 ms, 20 Vcm-1) produced a depolarization of 3-7 mV. When a single pulse was repetitively applied, the depolarization showed summation, resulting in action potential firing. 3. Tetrodotoxin (1 microM) and guanethidine (1 microM) completely suppressed EJPs whilst propranolol (1 microM) or atropine (0.1 microM) did not affect them. Phentolamine (1 microM) slightly potentiated EJPs. 4. d-Tubocurarine (dTC) at lower concentrations (0.5-2 microM) suppressed EJPs, but at a higher concentration (20 microM) the drug little affected them. 5. Hexamethonium (C6) suppressed EJPs at low concentrations (0.5-5 microM), but potentiated them at a higher concentration (50 microM). 6. Either dTC or C6, each at a concentration of 0.5 microM, did not affect the depolarization induced by exogenously applied ATP or noradrenaline. 7. These results suggest that in this tissue cholinergic nerve activity can contribute to the magnitude of EJPs, and that this effect is mediated through nicotinic receptors.

Effects of various drugs on the mechanical responses of the longitudinal smooth muscle of guinea-pig vas deferens evoked by ACh and field nerve stimulation were examined. ACh (28-280 microM) produced a contraction consisting of two phases. The first phase of the contraction was suppressed by guanethidine and by nicotinic antagonists. The second phase was suppressed only by atropine. Both phases were unaffected by TTX or prazosin. Field stimulation (0.1 msec, 40 Hz) evoked contractions which also consisted of two (early and late) phases. Guanethidine (0.1-1 microM) suppressed both phases whilst prazosin (1 microM) suppressed only the late phase. Atropine (0.1 microM) suppressed both phases whilst physostigmine (5 microM) potentiated both phases of field stimulation-evoked contractions. Pentolinium suppressed both phases of field stimulation-evoked contractions at low concentrations (2-10 microM), but potentiated them at a higher concentration (100 microM). dTC at a low concentration (0.5 microM) suppressed the early phase, but slightly enhanced the late phase of field stimulation responses. At a higher concentration (20 microM), dTC potentiated both phases of the response. Pentolinium and dTC did not affect the contractions induced by 90 mM-K ions, ATP or NA. These results suggest that cholinergic nerves possess an excitatory action not only directly at the smooth muscle but also at the noradrenergic nerve terminals. The role of each receptor is discussed further.

1. Two noradrenergic components of contraction were induced by field nerve stimulation of the circular smooth muscle of guinea-pig vas deferens. One component occurred during the stimulation (former-response) and the other component occurred after stimulation had ceased (after-response), with a distinct tension fall between these two responses. 2. The effects of stimulation frequency and duration upon these components were examined. 3. Prazosin (0.05-0.5 microM) suppressed both responses, whereas nifedipine (0.02 microM) suppressed only the after-response. 4. It is suggested that in this tissue there are two neurogenic alpha-adrenergic contractions which possess different Ca2+ activation mechanisms. The former-response is due to an increase in cytoplasmic Ca2+ concentration through a pathway independent of action potentials, while the after-response is attributed to action potentials arising as a secondary response to the released noradrenaline.

The distribution of a novel difucoganglioside (6B ganglioside, NeuAc alpha 2----3Gal beta 1----4[Fuc alpha 1----3]GlcNAc beta 1----3Gal beta 1----4[Fuc alpha 1----3]GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1Cer) in various normal adult and fetal tissues, as well as in cancer tissues, has been studied by immunoperoxidase staining with a specific monoclonal antibody, FH6, directed to this antigen. A large variety of embryonic and fetal tissues (stomach, colon, small intestine, pancreas, esophagus, lung, and heart) showed a diffuse, weakly positive staining, particularly in the epithelial layer, up to the 70th to 80th day of gestation. However, no staining was observed in various normal adult tissues, including gastrointestinal and glandular epithelial tissues which were stained positively by antibody N-19-9 (directed to sialyl-Lea) or CSLEXI (directed to sialyl-Lex). FH6-positive loci were limited to the proximal convoluted tubuli in kidney and granulocytes. In contrast, 44 of 76 cases of cancer tissue tested, including gastric, colonic, lung, breast, and renal cancers, showed clearly positive staining. The intensity of staining in gastric and colonic cancer tissues by FH6 antibody was weaker and less frequent, although the incidence of positive staining for lung (50%) and breast cancer (86%) was significantly higher than that of the antigen stained by monoclonal antibody FH4 (Y. Fukushi, S. Hakomori, and T. Shepard, J. Exp. Med., 159: 506-520, 1984), which is directed to the asialo core of the FH6 antigen. The antigen levels in the serum of patients with various cancers, inflammatory diseases, and normal subjects were determined by radioimmunoassay. The antigen level was found to be significantly higher in the serum of some patients with cancer, particularly lung, liver, and pancreatic cancers, as compared with the serum levels in other types of cancer, noncancerous diseases, and normal subjects.

SSEA-1 antigen (stage-specific embryonic antigen-1) has been shown to be a series of carbohydrate antigens having type-2 chain and X-hapten structures. These carbohydrate antigens are remarkably accumulated in various human cancer tissues, and activity secreted into the blood stream. Frequently SSEA-1 antigens are further modified with fucoses or sialic acid in human cancer tissues, thus forming various subgroups of antigens such as fucosyl SSEA-1, sialyl SSEA-1 or polyfucosylated antigens. Many monoclonal antibodies are established which can discriminate each subgroup of antigens. Assay systems for these antigens in the sera of cancer patients have been developed using these monoclonal antibodies. Sialyl SSEA-1 is especially elevated in the sera of patients with adenocarcinoma of the lung. The antigens detected with these monoclonal antibodies are mucin-like glycoproteins(cancer-associated mucin, CAM). Various types of cancer-associated mucins can be characterized by respective monoclonal antibodies. It is therefore possible to classify cancer-associated mucins according to the structure of their carbohydrate side chains using these monoclonal antibodies.

Interaction between rat bladder epithelium and Escherichia coli was observed using the transmission electron microscope. As a first step, the bacterial cell coat attaches to the cell coat of the luminal membrane. Then, the luminal membrane enfolds to envelope the bacterium and take it within the cytoplasm. The unit membranes covering the bacteria are destroyed and some of these bacteria are found to be deformed and probably dead in the cytoplasm. Finally, the superficial epithelial cells invaded by many bacteria are collapsed and desquamated into urine. However, these epithelia show a rather prompt recovery and form the normal three layers within 24 hr after inoculation. This experiment suggests one of the defense mechanisms of the vesical epithelium againts E. coli. It is likely that the onset of clinical cystitis may be prevented by the same mechanism during the occasional bacterial invasion of the bladder.

The present study was undertaken in order to examine whether dilazep (1,4-bis-[3-(3,4,5-trimethoxybenzoyloxy)propyl]perhydro-1, 4-diazepine dihydrochloride monohydrate) attenuates myocardial acidosis induced by coronary artery occlusion in dogs. In dogs with nonischemic normal heart, dilazep (300 or 500 micrograms/kg i.v.) increased blood flow in the left anterior descending coronary artery (LAD) with a decrease in heart rate and diastolic blood pressure. In other dogs, LAD flow was reduced by an occluder by 57 to 68% (partial occlusion) for 90 min. Partial occlusion for 30 min decreased myocardial pH by 0.67 to 0.87 pH units, increased ST segment of the surface electrocardiogram, and decreased regional myocardial contractile force. Dilazep was injected i.v. 30 min after partial occlusion. The decrease in myocardial pH induced by partial occlusion was attenuated by the injection of 300 micrograms/kg of dilazep insignificantly and by that of 500 micrograms/kg of dilazep significantly. Restoration of myocardial [H+] induced by dilazep was calculated from the myocardial pH data. Dilazep (500 micrograms/kg) restored myocardial [H+] induced by partial occlusion by 56.7%, and saline solution restored it by 27.4% 60 min after the drug injection, the actual restoration induced by dilazep being 29.3%. Dilazep, however, did not restore the ST segment elevation and contractile force decrease. It is concluded that dilazep attenuates myocardial acidosis during ischemia.

Bacterial adherence to mucosa is thought to be an initial and important stage to cause urinary tract infection. Among some mechanisms of bacterial adherence, the role of fimbriae and its receptor is worthy of notice. In particular, type 1 fimbriae, for which mannose is assumed as a receptor, is reported as the most common type and called "common fimbriae". Therefore if a certain amount of mannose is present in urine, it will cover the fimbriae of bacteria and competitively block the bacterial adherence to bladder mucosa. As the first step, we tried to detect mannose in urine by high performance liquid chromatography (HPLC). Sugar can be measured by detecting the fluorescence which is produced by a sugar separated by ion exchange, reacting with arginine at high temperature. The results using standard sugar samples should have highly stable retention time and concentration curve with the minimum detectable mannose concentration of 0.02 microgram. We investigated mannose in urine from 186 cases. Since the mannose peak was often masked by near unidentified peaks, the peak of mannose could be detected only in 80 cases and its concentration could be measured only in 24 cases. Mannose concentration in the urine of the 24 cases was between 2.6 and 108.7 micrograms/ml and in most of cases it was lower than 20 micrograms/ml. Secondary, we examined the possibility of a mannose in urine to prevent bacterial adherence to mucosa by the hemagglutination test using guinea pig erythrocytes and type 1 fimbriated E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron and light microscopic observations were made on experimentally induced cystitis in rats with particular emphasis on the role of polymorphs in the wall of the urinary bladder. Polymorphs appear from the time of invasion of bacteria to the epithelial cells of the bladder, aggregate primarily around infected epithelial cells, which become surrounded and isolated from healthy epithelial cells, and accelerate the desquamation of the infected epithelial cells. In the submucosal layer, all invading bacteria are phagocytized by polymorphs. Although polymorphs found in the intercellular space of the epithelial cells do not show phagocytosis, those that are released into the lumen of the urinary bladder do.

Induction of rat heme oxygenase is mediated by at least two factors: its substrate heme and heat shock (Shibahara, S., Müller, R.M., and Taguchi, H. (1987) J. Biol. Chem. 262, 12889-12892). We have identified the cis-acting element of the rat heme oxygenase gene (HO gene), that was specifically bound by a nuclear protein prepared from rat glioma cells. We have termed this protein as heme oxygenase transcription factor (HOTF), whose estimated molecular weight is about 40,000. The element identified is CCACCACGTGACTCGAG (-51/-35) located just upstream of TATA-like sequence. The functional studies indicate that this sequence is required for the accurate and efficient initiation of transcription from the HO gene promoter. Since the HOTF-binding element is similar to the upstream promoter sequence (UPS) of the adenovirus 2 major late promoter (Ad2MLP), we examined whether rat HOTF is homologous to the upstream stimulatory factor (USF) or major late transcription factor previously identified in HeLa cells, which interacts with the UPS and activates the transcription from the Ad2MLP. HOTF specifically binds to the UPS of Ad2MLP, whereas HOTF failed to form a stable complex with the mutated HOTF-binding element lacking a GTGA sequence (-44/-42 of the HO gene), the sequence of which is identical to a core sequence of the USF-binding sites. Moreover, we show that like USF, HOTF is heat stable. These results suggest that HOTF may be homologous to USF or the major late transcription factor. Since USF is present in uninfected cells, it is conceivable that the expression of the HO gene is regulated at least in part by USF.