Studies on the effects of microgravity on the ultrastructure and function of cultured mammalian cells.

Four cultures of monkey kidney cell line, JTC-12, were flown on the Spacelab-J (SL-J) mission during 8 days. The results of the present study showed that the space flight gave no essential effect on morphology, cell cycle, glucose consumption and urokinase production of the mammalian culture cell. However, the cell proliferation slightly decreased under microgravity. Moreover, the lack of gravity induced the trypsin-treated dissociated cells to keep floating in the culture medium. Therefore, the attachment of the cells onto the substratum was delayed, and that caused difficulties in subculturing the cells. The present research also offered some important information on techniques for establishment of cell cultures in space laboratories.

Increase in basal level of Hsp70, consisting chiefly of constitutively expressed Hsp70 (Hsc70) in aged rat brain.

Alteration of proteins in the function and higher structure has been observed in aged organisms. Stress proteins, which have a role to protect proteins from denaturation, may respond to the protein denaturation with aging. We found that the basal level of Hsp70, consisting chiefly of constitutively expressed Hsp70 (Hsc70), in 24-month-old Wistar rats was significantly higher in some parts of the brain than that in 6-month-old rats. On the other hand, the basal level of Hsp70 was significantly lower in 24-month-old rats after restricted feeding, which is known to suppress aging, than those fed the diet ad libitum. In the pons, medulla, striatum, and thalamus of 24-month-old rat brain, the level of Hsp70 increased and the denaturation of the cytosol proteins was suppressed. These results suggest that the expression of Hsp70, mostly Hsc70, increases with aging and may have a role to suppress protein denaturation.

Stress fibers in the mesenteric mesothelial cells of the large intestine of the bullfrog, Rana catesbeiana.

Actin-containing cytoplasmic fibers were visualized in the mesenteric mesothelial cells of the large intestine of bullfrog tadpoles by rhodamine-phalloidin staining of en face preparations of mesothelial cells. These fibers were concurrently stained by immunofluorescence using antibodies to myosin or alpha-actinin. Electron microscopy showed the presence of bundles of microfilaments in the basal cytoplasm of the cells. Such fibers in the mesothelial cells may be comparable to the stress fibers present in cultured cells. The mesothelial cells initially formed axially oriented stress fibers when they changed from a rhombic to a slender spindle-like shape. On the other hand, stress fibers disappeared as cells transformed from elongated to polygonal shapes during the period of metamorphic climax. Expression of stress fibers in these cells appears to be related to the degree of tension loaded on the mesentery, which may be generated by mesenteric winding. These stress fibers in the mesothelial cells may serve to regulate cellular transformation. They may also help to maintain cellular integrity by strengthening the cellular attachment to subepithelial tissue against tensile stress exerted on the mesentery.

Morphological study of acute myocardial lesions experimentally induced by methamphetamine.

A single intraperitoneal injection of methamphetamine hydrochloride (10 mg/kg) given to rats in a warm (30 degrees C) and humid environment caused an uncommon form of cardiac lesion during the first few hours. Disseminated loss of myoglobin was demonstrated immunohistochemically in the ventricular myocardium. Ultrastructurally, the myocardial cells with myoglobin loss were characterized by the presence of swollen mitochondrial and packed cellular constituents, but showed no cytoplasmic edema. Sarcolemmal damage was also noted by freeze-fracture electron microscopy. It was considered that the mitochondrial dysfunction was the first alteration to appear in the myocardial cells, and that this finally led to sarcolemmal rupture.

Immunohistochemical study on the excretion of a drug (methamphetamine) by salivary glands.

We have succeeded in the immunohistochemical demonstration of methamphetamine; and in the present study, we used this technique to determine the topographic distribution of exogenously administered methamphetamine in mouse major salivary glands. Positive reactions for methamphetamine were localized in the striated duct portion of the major salivary glands and granular duct in the submandibular glands. Time course of the decrease in immunohistochemical reactivity of the ducts was very similar to that for reported methamphetamine levels in plasma or saliva. These results show that through the striated ducts of the major salivary glands and the granular ducts of the submandibular glands, MA is excreted into saliva; however, the mechanism by which MA is excreted into saliva is unknown.

[Immunohistochemical study on the mechanism of excretion of methamphetamine].

Many methods of analysis are available to the forensic toxicologist for determining the amount of methamphetamine within human tissues, but few have the potential of histochemistry for enabling the precise site of excretion of methamphetamine to be defined. We have established a method for the demonstration of methamphetamine by immunohistochemistry, and applied this method for showing morphologically the disposition of methamphetamine. The following cells in the tissues of methamphetamine-intoxicated mice gave a strong positive reaction of the localization, which was thought to be the histological evidence of excretion of this drug: epithelial cells of the distal part of the renal tubule and of the collecting tubule, transitional epithelial cells of the bladder, liver parenchymal cells, epithelial cells of the striated duct of the salivary gland, parietal cells of the gastric gland, part of epithelial cells of the distal portion of the large intestine, secretory cells and part of epithelial cells of the ductal portion of the sweat gland, alveolar cells of the mammary gland, secretory cells of the sebaceous gland and hair medulla and cortex. These results indicated passive diffusion of methamphetamine across membranes of the cells of the distal tubule and collecting tubule of the kidney, of the bladder and of the striated duct of the salivary gland. In the parietal cells of the gastric gland, part of epithelial cells of the distal portion of the large intestine and secretory cells of the sweat gland, methamphetamine was thought to be stored and subsequently released. In the mammary gland, methamphetamine was found to be combined with casein and excreted by exocytosis. Accumulation of methamphetamine in the hair was supposed to be chiefly due to the penetration of this drug derived from tissue fluid and sebum.

Cell death in the anuran tadpole tail: thyroid hormone induces keratinization and tail-specific growth inhibition of epidermal cells.

The mechanism of thyroid hormone-induced and glucocorticoid-modulated death of tail epidermal cells from tadpoles of bullfrog, Rana catesbeiana, was investigated by comparing tail epidermal cells with dorsal body epidermal cells. From morphological and biochemical criteria, there were two types of epidermal cells: basal cells and skein cells. The abundance of these cells was different between the tail and the body skin. Fifty percent of body cells and more than 95% of tail cells were skein cells. Effects of 3,3',5-triiodo-L-thyronine (T3, 10(-8) M) and cortisol (5 X 10(-7) M) were investigated with cultured epidermal cells. T3 differently regulated the keratinization of the tail and body cells. The keratinization of the tail epidermal cells was not observed without T3. T3 induced the keratinization dramatically. On the other hand, body epidermal cells were constantly undergoing keratinization without the hormone: T3 merely accelerated the rate of keratinization. Cortisol generally did not show any significant effect on keratinization. T3 showed opposite effects on DNA synthesis of the tail and body cells: suppression of tail cells and stimulation of body cells. Cortisol weakened the inhibitory effect of T3 on DNA synthesis in tail cells. Immunofluorescent micrographs with anti-BrdU showed that T3 decreased the number of cells in the S phase of the cell cycle in the case of tail cells but not of body cells. Thus, thyroid hormone plays dual roles for the tadpole epidermal cells: one is an induction and a promotion of keratinization in tail and body cells, respectively, and the other is an opposite regulation for the proliferation of both epidermal cells. These roles seem to have crucial connections to a tail-specific cell death induced by thyroid hormone.

Immunohistochemical investigation of topographic localization of phenobarbital and methamphetamine in the rat retina.

The topographic localization of methamphetamine and phenobarbital in the rat retina was studied. It is interesting that the secondary and primary ganglionic cells were stained predominantly only in the case of methamphetamine while in phenobarbital these were not stained. The staining patterns of the inner plexiform layer (strong reaction in phenobarbital and weak reaction in methamphetamine), outer nuclear layer (weak reaction in phenobarbital and strong reaction in methamphetamine) and choroid (very strong reaction in phenobarbital and no reaction in methamphetamine) also showed some differences.

Histochemical demonstration of phenobarbital by immunocytochemistry.

A method for the demonstration of the topographical distribution of phenobarbital at the cellular level in various tissues was established. Mice that had been exposed to various doses of phenobarbital by intraperitoneal injection were killed, and their tissues were fixed with 0.1 M phosphate buffer solution (pH 7.4) containing paraformaldehyde and glutaraldehyde. Thereafter, paraffin and frozen sections were made and stained by the indirect immunoperoxidase method using antisera obtained from commercial sources and used for the immunochemical assay of the blood level of phenobarbital in clinical medicine. A specific positive reaction was observed solely in testing the intoxicated tissues, and this reaction was inhibited when phenobarbital was added to the antisera. The minimal sensitivity of the positive reaction, which can be discerned by observing the stained slides macroscopically, was in the range of 10 mg/kg. Thus, the diagnosis of phenobarbital intoxication in the forensic autopsy can be made by immunohistochemistry. A positive reaction was found in various tissue cells, including nerve cells, myelin sheaths, glia cells, hepatocytes, cells of the alveolar and bronchial wall, epithelial cells of the distal part of the renal tubules, and so forth. Endothelial cells of the capillaries in all tissues gave a strong positive reaction. The immunocytochemical electron microscopy of the hepatocytes revealed that the positive reaction in the cytoplasm was located solely in the intraluminal space of the smooth endoplasmic reticulum. These results indicate some interesting aspects of the pharmacokinetics of phenobarbital in vivo. It is expected that the antisera, which are used widely for the assay of the blood concentration of various drugs (phenobarbital, amphetamines, morphine, and so forth), may be regarded as excellent reagents for immunocytochemistry. This clearly indicates that morphological evidence in toxicology, which had so far remained obscure, can be easily obtained by applying these antisera against various drugs.

Heterogeneity in the myoglobin content of chicken heart Purkinje fibers.

The myoglobin content of chicken myocardial cells was studied using indirect-immunoperoxidase histochemistry. While ordinary myocardial cells exhibited a homogeneous reaction pattern, the reactions for ventricular Purkinje fibers were remarkably heterogeneous. On the basis of the degree of staining, three types of cells, i.e., dark, intermediate, and clear, were distinguishable. In addition, the cytological heterogeneity of Purkinje cells was confirmed using conventional and immunological electron microscopy. The dark cells contained more myofibrils, mitochondria, and other organelles (e.g., ribosomes) than the clear cells.

Pharmacological properties of presynaptic beta-adrenoceptors in guinea-pig pulmonary arteries.

Pharmacological properties of the facilitatory presynaptic beta-adrenoceptor mechanism were studied in superfused spiral preparations of guinea-pig pulmonary arteries preloaded with 3H-norepinephrine. (-)-Isoproterenol (0.3 microM)-induced increases in total 3H efflux per pulse evoked by transmural field stimulation (1, 5, 10 and 20 Hz, 10 V, 2 msec pulse width, 100 pulses and 30 min intervals) were neither dependent on impulse-frequencies nor selective at lower frequencies. Isoproterenol increased 3H efflux at 5 Hz in a concentration-dependent manner (1 nM to 1 microM): pD2 was 7.7. Salbutamol increased 3H efflux in a similar manner to isoproterenol: pD2 was 7.4. Prenalterol at 3 microM only slightly increased 3H efflux. Tazolol (10 nM to 3 microM) produced no increases. Atenolol (3 microM) and practolol (3 microM) did not antagonize isoproterenol (0.3 microM)-induced increases in 3H efflux. Butoxamine (3 microM) and H 35/25 (3 microM) did antagonize this parameter. (-)-Epinephrine (1 nM to 0.1 microM) decreased 3H efflux at 5 Hz and concentration-dependently increased this parameter in the presence of 10 microM phentolamine. (-)-Norepinephrine (10 nM to 1 microM) concentration-dependently inhibited evoked 3H efflux and did not increase the parameter in the presence of 10 microM phentolamine, 10 microM cocaine and 10 microM normetanephrine. Thus, there exist presynaptic beta 2-subtype receptors on noradrenergic nerve endings innervating guinea-pig pulmonary arteries.

Effects of disruption of microtubules on translocation of particles and morphology in tissue cultured neurites.

The role of microtubules in axoplasmic transport was studied by using colchicine and vinblastine in tissue cultured dorsal root ganglion cells dissected from chick embryos. After 3-4 days in culture, the effects of the agents on particle movement were examined under Nomarski optics. The movements of the particles were analyzed with high-contrast video systems. The number of transported particles passing through one region decreased after exposure to colchicine or vinblastine in concentrations greater than 1 X 10(-7) M, but these agents did not affect the velocity of the transported particles. After treatment with the agents swelling occurred in regions where particles sometimes stopped or slowed down in normal conditions. The intervals between swollen regions were 5-25 micron and transported particles finally stopped in the swollen regions. After 1 h of treatment with 1 X 10(-5) M colchicine, electron micrographs showed many organelles but no microtubules in the swollen regions. It is thought from these results that the disruption of microtubules in neurites might occur at the ends of the microtubules, followed by swellings in the neurites and particle transport blockade, but the morphological change itself might not have an effect on the transport of particles.

Further characterization of presynaptic beta-adrenoceptors in guinea-pig pulmonary arteries.

Presynaptic beta-adrenoceptors were further characterized in spiral strips of guinea-pig pulmonary arteries preloaded with [3H]norepinephrine. l-Metoprolol (3 X 10(-6) M) inhibited isoproterenol (3 X 10(-7) M)-induced increases in 3H efflux by transmural field stimulation, whereas the d-isomer produced no inhibition. However, IPS 339, H 35/25, butoxamine and metoprolol (3 X 10(-6) M) antagonized salbutamol (3 X 10(-7) M)-induced increases in the parameter, whereas acebutolol, bevantolol and practolol (3 X 10(-6) M) produced no antagonism. Presynaptic beta-adrenoceptors in guinea-pig pulmonary arteries appear to have characteristics similar to those postsynaptic classical beta-adrenoceptors.

Comparative studies of (--)-, (+/-)-propranolol, atenolol, guanethidine, bretylium and tetracaine on adrenergic transmission.

1 Effects of (--)-, (+/-)-, and (+)-propranolol, atenolol, guanethidine, bretylium and tetracaine were studied on relaxation responses of rabbit ileum and contractile responses of rabbit pulmonary artery and guinea-pig vas deferens to electrical nerve stimulation (2 to 50 Hz). 2 In the ileum, inhibition by tetracaine 3.3 x 10(-6) M occurred at high frequencies of stimulation, while bretylium 1.2 x 10(-4) M and guanethidine 2 x 10(-5) M inhibited responses at all frequencies, the latter producing greater inhibition at low frequencies. 3 (+/-)-Propranolol 10(-5) M produced a tetracaine-type inhibition after 1 h and a bretylium-pattern after 2 h in the ilea and pulmonary arteries and a transition from bretylium- to guanethidine-pattern in the vas deferens, while atenolol 2 x 10(-5) to 10(-4) M produced guanethidine-type inhibition in all preparations. 4 (--)-, (+/-)-, and (+)-Propranolol 3 x 10(-6) to 3.3 x 10(-5) M were equipotent in the vas deferens and ileum. However, inhibition by (--)-propranolol 3.3 x 10(-5) M persisted in the ileum, while that by the (+)-isomer was partially restored by washing. 5 (--)- or (+)-Propranolol 3.3 x 10(-5) M or atenolol 2 x 10(-5) M did not inhibit relaxation of the ileum after the bath temperature was maintained at 4 degrees C for 2 h during drug application. 6 In conclusion, propranolol and atenolol both have gradually developing guanethidine-like adrenergic neurone blocking actions.

Adrenergic transmission failure via the blockade of presynaptic beta receptors in guinea-pig pulmonary arteries.

Mechanisms related to the inhibitory actions of low concentrations of l- and d-propranolol on adrenergic transmission were investigated in isolated preparations of guinea-pig pulmonary arteries. In sympathetic nerve-radial muscle preparations, l-propranolol (3.3 X 10(-8) and 10(-7) M) dose-dependently inhibited by 15 to 20% contractile responses to nerve stimulation (1 Hz, 2-msec pulse width, 100-sec period and 30-min intervals) 30 and 60 min after the addition, whereas 3.3 X 10(-7) M produced a maximal inhibition. Contractile responses to cumulatively applied norepinephrine were not modified by pretreatment with l-propranolol (10(-7) - 10(-6) M). d-Propranolol (10(-7) M) produced no inhibition of adrenergic transmission. In superfused spiral preparations preloaded with [3H]norepinephrine, l-isoproterenol (3 X 10(-8) - 10(-6) M) dose-dependently facilitated total 3H efflux by transmural field stimulation by 10 to 35% under the same conditions; this facilitation was antagonized by l-propranolol (10(-7) M) but not by d-propranolol (10(-7) M). Phentolamine (3 X 10(-6) M) increased 3H efflux by approximately 3-fold. In the presence of phentolamine, l-propranolol (10(-7) M) significantly inhibited 3H efflux, whereas d-propranolol (10(-7) M) produced no effect. Presynaptic beta adrenoceptors are present on sympathetic nerve endings which innervate guinea-pig pulmonary arteries and low concentrations of propranolol inhibit adrenergic transmission via blockade of these receptors.