Correlation of Rates of Calcium Entry and Release of Endogenous Norepinephrine in Rat Brain Region Synaptosomes

Voltage-dependent 45Ca2+ uptake and endogenous norepinephrine (NE) release were measured simultaneously in synaptosomes isolated from rat hypothalamus, brainstem, and cerebellum at 1, 3, 5, 15, and 30 s. In synaptosomes depolarized by 125 mM KCl, 45Ca2+ uptake and NE release exhibited fast and slow components. Rates of NE release and 45Ca2+ uptake were fastest from 0 to 1 s. NE release and 45Ca2+ uptake rates from 1 to 5 s were less than 15% of 0-1 s rates. Both resting (5 mM KCl) and depolarization-induced (125 mM KCl) NE release paralleled 45Ca2+ uptake from 1 to 30 s. Voltage-dependent NE release was approximately 1% and 2% of total synaptosomal NE content at 1- and 30-s measurement intervals, respectively, and did not differ between the three brain regions studied. Calcium and potassium dependence studies showed that NE release was stimulated by increased potassium and that depolarization-induced NE release was dependent on the presence of external calcium. These results show that calcium-dependent NE release from synaptosomes is correlated with calcium entry. Both processes exhibit fast and slow temporal components.     

PACAP enhances stimulation-induced norepinephrine release in canine pancreas in vivo

The present study was to investigate whether pituitary adenylate cyclase activating polypeptide (PACAP) can modify norepinephrine (NE) release in response to pancreatic nerve stimulation in anesthetized dogs. Plasma catecholamine concentrations in aortic and superior pancreaticoduodenal (SPD) venous blood were determined by a high performance liquid chromatography method. SPD venous blood flow was measured with an electromagnetic flowmeter. Pancreatic nerves were directly stimulated for 1 min (2 ms, 12 V) at various frequencies at the level of the SPD artery. Various doses of PACAP1-27 (PACAP27) were locally infused into the pancreas through the SPD artery. Nerve stimulation significantly increased both SPD venous NE concentration and its output from the pancreas in a frequency-dependent manner. With PACAP27 alone, neither SPD venous NE concentration nor its output changed significantly following the local administration of PACAP27 at any dose tested. In the presence of PACAP27, however, the net increases in NE concentration and its output in response to nerve stimulation at 2 Hz were significantly enhanced in a dose-dependent manner. The enhanced NE responses to nerve stimulation by PACAP27 were thus significantly greater than those obtained from the group receiving either PACAP27 or stimulation alone. Increases in NE concentration and its output induced by local administration of tyramine were virtually abolished by desipramine, a neural amine uptake inhibitor. However, the NE response to tyramine was not diminished by PACAP27. The results indicate that PACAP27 enhances the stimulation-induced NE release in the pancreas, and that this facilitatory effect of PACAP27 does not result from an inhibition of the neural amine uptake mechanism. The study suggests that PACAP receptor-mediated mechanisms may be involved either directly or indirectly in the local modulation of neural NE release in the canine pancreas in vivo.     

Atrial natriuretic factor inhibits norepinephrine biosynthesis and turnover in the rat hypothalamus

We have previously reported that atrial natriuretic factor (ANF) increased neuronal norepinephrine (NE) uptake and reduced basal and evoked neuronal NE release. Changes in NE uptake and release are generally associated to modifications in the synthesis and/or turnover of the amine. On this basis, the aim of the present work was to study ANF effects in the rat hypothalamus on the following processes: endogenous content, utilization and turn-over of NE; tyrosine hydroxylase (TH) activity; cAMP and cGMP accumulation and phosphatidylinositol hydrolysis. Results showed that centrally applied ANF (100 ng/microl/min) increased the endogenous content of NE (45%) and diminished NE utilization. Ten nM ANF reduced the turnover of NE (53%). In addition, ANF (10 nM) inhibited basal and evoked (with 25 mM KCl) TH activity (30 and 64%, respectively). Cyclic GMP levels were increased by 10 nM ANF (100%). However, neither cAMP accumulation nor phosphatidylinositol breakdown were affected in the presence of 10 nM ANF. The results further support the role of ANF in the regulation of NE metabolism in the rat hypothalamus. ANF is likely to act as a negative putative neuromodulator inhibiting noradrenergic neurotransmission by signaling through the activation of guanylate cyclase. Thus, ANF may be involved in the regulation of several central as well as peripheral physiological processes such as cardiovascular function, electrolyte and fluid homeostasis, endocrine and neuroendocrine synthesis and secretion, behavior, thirst, appetite and anxiety that are mediated by central noradrenergic activity.     

Effect of temperature on the uptake, excretion and degradation of abrin and ricin by HeLa cells.

The effect of temperature on the uptake of abrin and ricin and on the subsequent excretion and degradation of the toxins was measured. Uptake was assessed either by monitoring the amount of cell-bound ¹²⁵I-labelled toxin that could not be released with lactose or by measuring the time required for transport of the toxins into a state where they were protected against neutralizing antibodies. The presence of toxin in this state was monitored by measuring inhibition of protein synthesis after a subsequent prolonged incubation period. In the case of abrin, straight lines were found in both cases when the data were plotted according to Arrhenius. The activation energies estimated was 18–21 kcal/mol (75–88 kJ/mol) in the case of uptake of [¹²⁵I]abrin and 15–19 kcal/mol (63–79 kJ/mol) when the indirect method was used.After internalization of [¹²⁵I]abrin and ricin a fraction of the radioactive material is released to the medium. Most of this material can be precipitated by trichloroacetic acid (TCA). There is a rapid release during the first 30 min and then over the next few hours the release occurs at a constant, but lower rate. The release of ricin was not affected by addition of colchicine, cytochalasin B (CB), ammonium chloride, sodium azide or bovine serum albumin, whereas the degradation of ricin was reduced by the above mentioned compounds (except albumin). The release of ricin was strongly temperature-dependent with a sharp transition at about 20 °C. The activation energies for the release above and below 20 °C were found to be 2.5 and 31 kcal/mol (10.5 and 172 kJ/mol), respectively.     

Effect of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) on monoamine neurotransmitters in mouse brain & heart

The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was studied on dopamine (DA), norepinephrine (NE), serotonin (5HT) and γ-aminobutyric acid (GABA) neurons in mouse brain and on NE neurons of mouse heart. MPTP (45 mg/kg) was administered s.c. to mice twice daily for 2 consecutive days. This dosage regimen produced a decrease in the forebrain concentrations of DA and NE at 7 and 20 days after injection. In contrast, the forebrain concentrations of 5HT and GABA were not significantly decreased at either time. MPTP administration also produced a marked decrease in the uptake of ³H-DA into striatal slices and ³H-NE into cerebral cortical slices. In contrast, the uptake of ³H-NE into hypothalamic slices and the uptake of ³H-5HT into slices from several brain regions were not altered. MPTP initially reduced the concentration of NE in the heart, but unlike the persistent decreases in the forebrain concentrations of NE and DA, the NE concentration in the heart returned to control levels at approximately 20 days after MPTP administration. These results, showing that MPTP can produce a long lasting and selective decrease in the forebrain concentrations of NE and DA and in the uptake of radioactive DA and NE into brain slices, suggest that MPTP can cause the destruction of catecholamine neurons in mouse brain. In contrast, MPTP administration does not appear to produce long term changes in either 5HT or GABA neurons.     

Angiotensin II-norepinephrine relationship in the central nervous system

The effects of angiotensin II (AII) and bilateral nephrectomy on [3H] norepinephrine (NE) uptake in hypothalamus and medulla oblongata were studied in male rats. The endogenous NE content in hypothalamus increased 4, 24 and 48 h after nephrectomy with a simultaneous decreasing of plasma renin activity. Intraventricularly infused [3H] NE uptake increased in hypothalamus and medulla oblongata of nephrectomized animals in cytoplasmatic compartment as in granular stores, while it decreased in hypothalamus of AII-infused animals. [3H] NE metabolites radioactivity decreased in nephrectomized animals if they are compared with AII-infused ones. These changes were independent of systolic arterial pressure that was not modified in none of the groups. The study of the ratio granular/cytoplasmatic [3H] NE and metabolites radioactivity shows that AII probably acts on cellular membrane uptake of NE. The modification of metabolites/NE ratio in both stores would be due to AII action on MAO activity. The effects of AII and nephrectomy on [3H] NE uptake can explain the inverse relationship between circulating AII levels and NE content in the central nervous system (CNS).     

Effect of anti-inflammatory agents on ricin-induced macrophage toxicity

The toxicity of ricin in susceptible cells is well characterized biochemically, but the pathophysiological implications of its toxicity and the immune response to ricin challenge in the lung are unknown. Incubating macrophage cell line with ricin (1 pM-10 nM) for 4 hours markedly inhibited ³H-leucine incorporation (acid insoluble) into protein (>95%, at 1 nM) without affecting the acid-soluble radioactivity. In spite of increased uptake of total thymidine (141×13.5%) and total uridine (135×17.2%), DNA synthesis in ricin-treated cells was progressively inhibited although RNA synthesis was not affected. Fluocinolone (an anti-inflammatory glucocorticoid) pretreatment increased the ricin-induced inhibition of protein synthesis. The synergistic effect of fluocinolone on ricin-induced protein synthesis inhibition was due to an increased binding (167%, p < 0.01) and internalization (134×12%, p < 0.025) of ricin. Partial protection from ricin-induced inhibition of protein synthesis by indomethacin (nonsteroidal, anti-inflammatory agent) was due to decreased binding and internalization of ricin. These results show that macrophages are sensitive to ricin and that pharmacologically active drugs may regulate ricin's toxicity, perhaps by controlling synthesis and release of certain mediators of fast death.     

Norepinephrine release in the heart atria of diabetic rats

The content, release and uptake of norepinephrine (NE) in the sympathetic nerves of the rat heart atria were studied in the course of diabetes and in age-matched controls. Diabetes was induced by streptozotocin (STZ) and rats were subjected to further experiments 1, 4 or 7 months later (STZ1, STZ4, STZ7). Isolated atria were superfused with oxygenated Krebs-Henseleit (KH) solution. After equilibration, four 10-min fractions were collected: B1, basal release of NE; S1, potassium-evoked release (KER), where NE outflow was stimulated by depolarisation with 50 mmol/l KCl; B2, basal release of NE under the influence of the neuronal uptake blocker desipramine (DES); S2, KER under the influence of DES. The content of NE was measured by radioimmunoassay. In STZ4 and STZ7 rats, NE concentrations were significantly lower in both atria compared to controls. B1 and S1 were significantly higher in STZ4 than in control atria. DES increased KER of NE in controls only. In contrast, DES caused a significant decrease in B2 and S2 in STZ4 atria, suggesting that a substantial portion of NE release was due to a calcium-independent carrier-mediated process. In experiments with calcium-free KH solution in fractions B2 and S2, KER ill controls was nearly abolished. However, in STZ4 and STZ7 atria, S2 was still significantly higher than B2. In conclusion, the NE-releasing mechanism may be different in the chronically diabetic animals than in healthy subjects and may contribute to the decreased NE concentration in the STZ atria.     

Vascular-dependent effects of elevated glucose on postganglionic sympathetic neurons

Perivascular sympathetic nerves are important determinants of vascular function that are likely to contribute to vascular complications associated with hyperglycemia and diabetes. The present study tested the hypothesis that glucose modulates perivascular sympathetic nerves by studying the effects of 7 days of hyperglycemia on norepinephrine (NE) synthesis [tyrosine hydroxylase (TH)], release, and uptake. Direct and vascular-dependent effects were studied in vitro in neuronal and neurovascular cultures. Effects were also studied in vivo in rats made hyperglycemic (blood glucose >296 mg/dl) with streptozotocin (50 mg/kg). In neuronal cultures, TH and NE uptake measured in neurons grown in high glucose (HG; 25 mM) were less than that in neurons grown in low glucose (LG; 5 mM) (P < 0.05; n = 4 and 6, respectively). In neurovascular cultures, elevated glucose did not affect TH or NE uptake, but it increased NE release. Release from neurovascular cultures grown in HG (1.8 ± 0.2%; n = 5) was greater than that from cultures grown in LG (0.37 ± 0.28%; n = 5; P < 0.05; unpaired t-test). In vivo, elevated glucose did not affect TH or NE uptake, but it increased NE release. Release in hyperglycemic animals (9.4 + 1.1%; n = 6) was greater than that in control animals (5.39 + 1.1%; n = 6; P < 0.05; unpaired t-test). These data identify a novel vascular-dependent effect of elevated glucose on postganglionic sympathetic neurons that is likely to affect the function of perivascular sympathetic nerves and thereby affect vascular function.     

Regulation of hepatic malic enzyme by perfluorodecanoic acid

Perfluorodecanoic acid (PFDA) administration to adult male rats increased both the activity of hepatic malic enzyme and liver weight in a dose-dependent manner. Hepatomegaly and augmented activity of malic enzyme in liver were apparent within one day following PFDA administration and reached a plateau by three days posttreatment. Malic enzyme quantity per liver in PFDA-treated rats was elevated within one day following dosing and increased continually throughout five days posttreatment. Administration of PFDA to rats in the fed state also led to an increase in the specific activity of hepatic malic enzyme that peaked at three days following dosing. When compared to the fed condition, rats fasted for 48 hours had a decrease in both relative liver weight and the quantity of supernatant protein per liver. The total activity (U/liver) and specific activity of malic enzyme in the liver were also reduced in the fasted state. During the 24 hours after treatment in rats fasted for 48 hours, the body weight as well as the absolute and relative liver weight of animals receiving vehicle declined continuously in the absence of feed. Following the administration of PFDA to fasted rats, body weight was maintained until eight hours posttreatment but then declined at a rate similar to that found with the vehicle-treated group. Absolute and relative liver weight in PFDA-treated rats were increased significantly at eight hours posttreatment when compared to those receiving vehicle, and this increment was maintained throughout the rest of the 24 hours following dosing. While the activity and enzyme content of hepatic malic enzyme decreased in the vehicle-treated group, administration of PFDA to rats fasted for 48 hours prevented their decline. The specific activity of hepatic malic enzyme in 48 hours fasted rats receiving PFDA was also elevated significantly at 16 hours posttreatment. Thus, the administration of PFDA to the adult male rat in both the fed and fasted nutritional states was found to regulate hepatic malic enzyme by not only increasing enzyme quantity but also by augmenting the specific activity, (ie, catalytic state) of the enzyme.     

Somatostatin Content Increases Following Norepinephrine Depletion in Frontal Cortex of l-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Treated Mice

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on somatostatin (SS)-containing neurons were examined by measuring dopamine, norepinephrine (NE), SS, and SS mRNA in striatum and frontal cortex of C57/B16 mice at various times following treatment with MPTP-HCl (96 mg/kg i.p.). MPTP caused a 70% depletion of dopamine in striatum by 1 day and a 40% depletion of NE in frontal cortex within 3 days. SS content was increased in frontal cortex 4 days later, but not in striatum; there were no changes in SS mRNA. Maprotiline, a specific NE-uptake blocker, prevented both the depletion of NE and the increase of SS in frontal cortex due to MPTP administration. These results support the possibility that NE can regulate SS in frontal cortex and are discussed in terms of the decrease of SS seen in parkinsonian patients with dementia.     

Augmented behavioral response and enhanced synaptosomal calcium transport induced by repeated cocaine administration are decreased by calcium channel blockers

Recent studies suggest that calcium influx via L-type calcium channels is necessary for psychostimulant-induced behavioral sensitization. In addition, chronic amphetamine upregulates subtype Cav1.2-containing L-type calcium channels. In the present studies, we assessed the effect of calcium channel blockers (CCBs) on cocaine-induced behavioral sensitization and determined whether the functional activity of L-type calcium channels is altered after repeated cocaine administration. Rats were administered daily intraperitoneal injections of either flunarizine (40 mg/kg), diltiazem (40 mg/kg) or cocaine (20 mg/kg) and the combination of the CCBs and cocaine for 30 days. Motor activities were monitored on Day 1, and every 6th day during the 30-day treatment period. Daily cocaine administration produced increased locomotor activity. Maximal augmentation of behavioral response to repeated cocaine administration was observed on Day 18. Flunarizine pretreatment abolished the augmented behavioral response to repeated cocaine administration while diltiazem was less effective. Measurement of tissue monoamine levels on Day 18 revealed cocaine-induced increases in DA and 5-HT in the nucleus accumbens. By contrast to behavioral response, diltiazem was more effective in attenuating increases in monoamine levels than flunarizine. Cocaine administration for 18 days produced increases in calcium uptake in synaptosomes prepared from the nucleus accumbens and frontal cortex. Increases in calcium uptake were abolished by flunarizine and diltiazem pretreatment. Taken together, the augmented cocaine-induced behavioral response on Day 18 may be due to increased calcium uptake in the nucleus accumbens leading to increased dopamine (DA) and serotonin (5-HT) release. Flunarizine and diltiazem attenuated the behavioral response by decreasing calcium uptake and decreasing neurochemical release.     

The influence of exogenous PMS and HCG on the arachidonic acid content of the immature rat ovary.

The influence of exogenous PMS and/or HCG, on the arachidonic acid (C 20:4omega6) content of the immature rat ovary was examined. Changes in ovarian arachidonate content associated with hormone administration were assessed in total lipid extracts, and in several neutral and phospholipid fractions. Both relative percentage and absolute amounts of arachidonic acid in several lipids were measured as well as uptake of radioactivity into total lipid resulting from the administration of 3H-labeled arachidonic acid in vivo. On the basis of these studies, we conclude (1) PMS, with or without HCG promotes increased uptake of exogenous arachidonic acid into ovarian total lipids; (2) Arachidonic acid is a mojor fatty acid constituent from noncholine containing phosphatides at the onset of normal estrous (ca. 38 days) even in the animals which received no PMS or HCG; (3) Changes in ovarian arachidonic acid levels following gonadotropin administration are more striking in the two phospholipid fractions than in the two neutral lips examined; (4) PMS is associated with a rapid outpouring of ovarian lipid, accompanied by a high turnover of arachidonic acid which is enhanced or modified temporally by added HCG in vivo. These results provide the first quantitative evidence that gonadotropins may regulate prostaglandin biosynthesis in the ovary by their effects on the uptake, storage, or release of arachidonic acid, a major PG precursor, from specific ovarian lipids. While the data strongly suggest that the regulation of one or more ovarian esterases (cholesterol esterase, lipase, phospholipase) is the mechanism by which gonadotropins regulate PG biosynthesis, a direct action on PG synthetase is not ruled out.     

Ricin disturbs calcium homeostasis in the rabbit heart

Ricin, a toxic lectin from the castor bean, affects the cardiovascular system. Because calcium is very important in cardiotoxicity and cell intoxication, we studied the effects of ricin pretreatment to rabbits on basal intracellular calcium levels and calcium uptake and release from isolated papillary muscle, microsomes, and mitochondria. An increase in basal intracellular calcium levels was observed. Ricin pretreatment nearly doubled the intracellular-free Ca²⁺ concentration as measured by fura-2 fluorescence microscopy in isolated myocytes (p = 0.002). Ricin did not alter basal calcium efflux in isolated papillary muscles. However, ricin inhibited the NE-induced calcium efflux (expressed as fractional efflux ratios) in papillary muscles from rabbits receiving the minimum lethal dose of ricin at 25–35 minutes (p = 0.002 and 0.003, respectively). Ricin depressed basal calcium uptake into isolated papillary muscles at 5 minutes (mean ± SEM, μmol/g wet weight) (control: 3.68 ± 0.57; ricin: 2.31 ± 0.28, p = 0.045, n = 6). Ricin pretreatment significantly depressed calcium uptake into microsomes (mean ± SEM, μmol/g protein) (control: 9.9 ± 1.9; ricin: 3.1 ± 1.9, p = 0.025, n = 6). Calcium uptake into mitochondria was increased at the beginning (2 minutes, p = 0.048), but not thereafter. Thus, administration of ricin disturbed calcium homeostasis in the rabbit heart, which may be at least partially responsible for altering cardiac function and myocardial cell death. © 1996 John Wiley & Sons, Inc.     

Ricin-induced toxicity in the macrophage J744A.1 cells: the role of TNF-alpha and the modulation effects of TNF-alpha polyclonal antibody

Ricin is a natural toxin of the castor beans (Ricinus communus). We studied the time- and concentration-dependent effects of ricin on the release of TNF-alpha and lactate dehydrogenase (LDH), as well as the modulation of the ricin-induced effects by TNF-alpha antibody in the J774A.1 cells. When added at concentrations ranging from 0 to 1000 ng/mL, ricin caused concentration-dependent increases in the release of TNF-alpha after incubation for 12 to 24 hours. Concentration-dependent increases in the leakage of LDH were also observed after incubation of the cells with those concentrations of ricin for 24 to 48 hours. Addition of 5 units/mL of rabbit anti-mouse TNF-alpha polyclonal antibody (TNF-alpha antibody) 2 hours prior to the addition of ricin resulted in a decrease in the ricin-induced toxicity, indicated by the release of LDH by the cells. However, when added at concentrations higher than 5 units/mL, the antibody resulted in either no effect or an increase in the ricin-induced LDH leakage. These results suggest that secretion of TNF-alpha by the macrophages in response to ricin plays a significant role in the toxicity of ricin and that TNF-alpha antibody can antagonize the effects of ricin in this cell line when added at relatively low concentrations.     

Levonorgestrel and norethindrone alter insulin action on skeletal muscle of the female rat

Prospective studies of women receiving oral contraceptives suggest that the progestin component may induce insulin resistance and variable deterioration of glucose tolerance. Because the tissue sites and nature of this insulin antagonism are not well-defined, we studied the effects of two parenterally administered progestins, levonorgestrel (NG) and norethindrone (NE), on insulin-regulated glucose uptake and phenylalanine release by the perfused rat hindquarter. Female rats were injected sc for 14 days with NG or NE (10 or 30 micrograms/kg/day). Low-dose NG and high-dose NE approximate the per kg dose received by women taking a high-dose progestin oral contraceptive. Phenylalanine release and glucose uptake (nmole/min/g) by the perfused hindquarters were calculated from the A-V difference for each. Progestin treatment (30 micrograms/kg/d) significantly reduced phenylalanine release from hindquarters perfused without exogenous insulin. Hindquarters from the high dose NG and low and high dose NE rats perfused with insulin (100 microU/ml) released 22% less phenylalanine than control rats perfused with the same insulin concentration (P less than 0.01) but the net suppression below baseline was similar in the control and steroid-treated groups. High-dose progestin treatment did not alter glucose uptake by hindquarters perfused without exogenous insulin. Insulin (100 microU/ml) increased glucose uptake by hindquarters of control and progestin-treated rats as compared to animals in the same treatment group perfused without exogenous insulin (P less than 0.01). High dose NE impaired insulin-stimulated glucose uptake 24% below values of the control group (P less than 0.01). The other NE and NG doses had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphologic and biochemical variability of tissue and cultured cells from human pheochromocytoma

Primary cell cultures from 18 human pheochromocytomas were maintained in culture for 10 to 12 days and characterized. The cell yields ranged from 1.0 to 60.1 X 10(6) cells/g wet weight of tissue. Cell size, as determined by histofluorescent microscopy, varied as much as seven-fold among cells derived from a given tumor and ten-fold between cells from all tumors. Cell catecholamine content, norepinephrine (NE) plus epinephrine, ranged from 0.4 to 89.5 nmol/10(6) cells at day 5 in culture and did not correlate with catecholamine content of the tissue from which the cells were obtained. Cell catecholamine content decreased with time in culture, but this decrease could not be related to a change in cell viability, the type of media used, an inability to convert dopamine to NE, or an alteration in the uptake of 3H-NE. Cellular uptake of 1.0 microM 3H-NE varied as much as 230-fold between all cell dispersions. The basal and acetylcholine stimulated release of both preloaded 3H-NE and the endogenous catecholamines was quite variable. There was no correlation between the release rate, either basal or stimulated, of preloaded 3H-NE and the endogenous catecholamines. This study represents the largest existing data base on culturing cells from these tumors and describes many of the morphologic and biochemical characteristics of this cell system.     

Endocytosis, intracellular transport and transcytosis of the toxic protein ricin by a polarized epithelium

The toxic plant protein ricin binds to both the apical and basolateral surface domains of MDCK (strain I) cells grown on polycarbonate filters. Endocytosis of 125I-labeled ricin was not only higher from the basolateral than from the apical surface--an observation which can be explained by the higher surface area of the basolateral surface--but it also appeared to be more efficient when measured as a percentage of total cell-associated ricin. Monovalent ricin-horseradish peroxidase (Ri-HRP), which is known to behave like native ricin with respect to intracellular transport, also binds to, and is taken up from, both the apical and the basolateral surfaces. Initially, after 10 to 15 min, molecules taken up from the two surface domains at 37 degrees C are present in two separate (basolateral and apical) early endosomal populations. This can also be obtained by incubating for 60 min at 18 degrees C. However, after 30 to 60 min at 37 degrees C, most internalized ligand is found in apical lysosomes, regardless from which surface endocytosis took place. Experiments with endocytosis of cationized ferritin from the apical pole and HRP or Ri-HRP from the basolateral pole showed that intermixing in apical lysosomes (or prelysosomes) of molecules taken up from the two poles occurs. Bidirectional transcytosis involving coated pits of both 125I-labeled ricin and Ri-HRP was demonstrated and was found to be most efficient (as measured in per cent of endocytosed toxin) from the apical pole. Transcytosis was strongly reduced at 18 degrees C, and no transepithelial transport of ricin could be measured at 4 degrees C. Transcytosed ricin was intact and could intoxicate new cells. Finally, delivery of ricin internalized from both the apical and the basolateral surface to the apically localized trans-Golgi network occurred at 37 degrees C but not at 18 degrees C, and ricin inhibited protein synthesis largely with the same kinetics following uptake from the two poles. Incubation at 18 degrees C strongly inhibited the toxic effect of ricin. These data show that ricin can intoxicate epithelia from both sides and also penetrate tight epithelial barriers in intact form.     

Effects of estrogens and progesterone on the catecholaminergic activity of the adrenal medulla in female rats

Some reports in the literature allow to suspect the existence of an effect of sexual steroids on the adrenal catecholamines. To test this possibility, we have examined the catecholaminergic activity in the adrenal medulla of normal cycling rats in three phases of estrous cycle and of ovariectomized (OVX) rats injected with pharmacological doses of estradiol (ES), 2-hydroxyestradiol (HE) and/or progesterone (P). Adrenomedullary content of norepinephrine (NE) was similar during the estrous cycle, while epinephrine (E) content was increased during diestrous. This increase was concomitant with an increased phenylethanolamine-N-methyltransferase (PNMT) activity. Moreover, the monoamine oxidase (MAO) activity was significantly increased during proestrous, while the catechol-O-methyltransferase (COMT) activity was significantly decreased during estrous. In addition to these observations, ovariectomy caused a significant reduction of the E/NE ratio and of COMT and MAO activities. Administration of ES to OVX rats increased the E content, the E/NE ratio and the COMT activity as compared to vehicle-treated OVX rats. Administration of P to OVX animals led also to a significant increase of the E/NE ratio and of the COMT activity but not of the E content, while the administration of this steroid to OVX rats previously treated with ES only increased the COMT activity. Finally, administration of HE caused non-significant changes in NE and E contents and in MAO, COMT and PNMT activities. We can conclude that sexual steroids seem to be able to modify the catecholamine metabolism in the adrenal medulla and, hence, they could alter the ability of this gland to store and release these amines.     

Contractile function, glucose consumption and lactate release by the isolated rat heart during different regimens of alcohol administration and after discontinuation of ethanol

Acute or chronic intoxication of rats with ethanol (intragastric administration at a dose of 8 g/kg or free-choice drinking of 10% ethanol for 3 months) produced no significant changes in contractile function, glycogen content, glucose uptake and lactate release in isolated hearts. Withdrawal syndrome simulated in rats following a short period of severe intoxication with ethanol at a dose of 4-5 g/kg twice daily has demonstrated a 15 and 28% decrease in peak systolic pressure and tension time index, respectively. In this case glucose uptake and lactate release were 2 times higher. Changes in glycogen level were observed three days after the last ethanol administration. The rats, survived after the abstinence period, revealed areas of perivascular myocardial necrosis. It is concluded that withdrawal syndrome plays an important role in pathogenesis of alcoholic cardiomyopathy.