The effect of estradiol on collagen structure and organization in the immature rat uterus

An examination of collagen ultrastructure in the lamina propria of the immature ovariectomized rat uterus revealed that a single injection of estradiol (40 micrograms/kg) produced a biphasic effect on collagen structure and organization. In saline-treated animals (controls) dense populations of collagen bundles were seen throughout the extracellular matrix (EX). Cross-sections of these bundles suggested that the bundles run parallel to the long axis of the uterus and thin filaments seemed to form cross-links between collagen fibers. In contrast, large clear spaces, collagen fragments, and loosely packed bundles of collagen were observed in the EX of animals injected with estradiol 24 hr earlier. In a time course study (0, 1, 2, 4, 24, and 48 hr), estradiol treatment altered collagen structure and organization 1 hr following administration. Collagen bundles did not appear to be as densely packed as in control tissues and large clear spaces were evident in the EX. Two hours following estrogen administration, collagen fibers appeared to be fragmented and seemed to be separating from the plasma membrane of stromal cells. Four hours following estrogen administration, large clear spaces occupied most of the EX in the lamina propria. Collagen fragments were diffusely distributed throughout the EX and small cross-sectional patches of collagen bundles were present. In 48-hr-treated animals, collagen bundles reappeared and were often closely associated with the plasma membrane of stromal cells. The collagen was not as abundant as in control animals. An overview of the cellular organization of the lamina propria revealed that stromal cells in control tissues were more densely packed than in estradiol-treated tissues (4, 24, and 48 hr) and the stromal cell size appeared to increase in hormone-treated tissues. These responses provide a good model system to study the role of estradiol in the control of collagen structure and organization in the uterus.     

Influence of VIP on the number of enterochromaffin and mucosal mast cells in the colon of the rat.

The possibility that VIP (Vasoactive intestinal peptide) could influence the enterochromaffin (EC) cell secretion of serotonin (5HT) and the action of VIP on the mast cell population of lamina propria were investigated in Wistar rat colon infused with a short chain fatty acid solution (sodium acetate), during a 1 h period. Under the action of an intravenous injection of synthetic porcine VIP, 14 micrograms/kg/h), the number of EC cells diminished significantly in the cecum and left colon, when compared to non-injected animals, both infused with a sodium acetate solution. At the same time, the number of mucosal mast cells in the crypts and lamina propria decreased significantly in the cecum. The postulate we put forward is that these VIP-induced changes are exerted through the stimulation of 5HT released from EC cells not only under normal physiological conditions but probably also under pathological conditions.     

Changes in the activities of various lipid and carbohydrate biosynthetic enzymes in the diatom Cyclotella cryptica in response to silicon deficiency

The effects of silicon deficiency on the activities of several enzymes involved in lipid and storage carbohydrate synthesis in the diatom Cyclotella cryptica were determined. The activity of UDPglucose pyrophosphorylase was not affected after 4 h of silicon-deficient growth, but the activity of UDPglucose: beta-(1----3)-glucan-beta-3-glucosyltransferase (chrysolaminarin synthase) was reduced by 31% during this period. Acetyl-CoA synthetase, acetyl-CoA hydrolase, and citrate synthase activities were present in cell-free extracts of C. cryptica, but did not change in response to 4 h of silicon deficiency. However, the activity of acetyl-CoA carboxylase increased approximately two- and fourfold after 4 and 15 h of silicon-deficient growth, respectively. This induction could be blocked by cycloheximide (20 micrograms/ml) and actinomycin D (10 micrograms/ml), suggesting that silicon deficiency may induce an increase in the rate of acetyl-CoA carboxylase synthesis. These changes in enzymatic activity may be partially responsible for the accumulation of lipids that has been observed in C. cryptica and other diatoms in response to silicon deficiency.     

Replenishment of uterine estrogen receptor in chronically estrogenized rats

Replenishment of uterine estrogen receptor (ER) following a single injection of estradiol-17 beta (E2) was examined in chronically estrogenized rats. Subcutaneous implantation of E2-pellet for 7 days in ovariectomized rats resulted in a significant stimulation of uteri with regard to wet tissue weight, DNA content and progesterone receptor content, with a shift of ER distribution. An intraperitoneal injection of 5 micrograms E2 in the E2-implanted rats induced a significant decrease in soluble ER (from 141.1 +/- 12.6 to 69.2 +/- 8.8 fmol/mg protein) with a concomitant increase in nuclear ER (from 58.2 +/- 8.6 to 129.2 +/- 11.6 fmol/100 micrograms DNA) 1 h after the injection. However, soluble ER was rapidly replenished, which was accompanied by nuclear ER reduction, and both values returned to the pre-injection levels at 4 h after the injection. An administration of 150 micrograms cycloheximide, that effectively blocked protein synthesis in the uterus of the E2-implanted rats, completely inhibited the replenishment of soluble ER induced by 5 micrograms E2. These findings, combined with our previous findings that replenishment of ER following a single E2 administration in the pituitary of chronically estrogenized rats was inhibited by cycloheximide, suggest that replenishment of ER is entirely dependent on protein synthesis in chronically estrogenized rats.     

Systemic availability and abortion-inducing effects of nocloprost after intravenous, subcutaneous and intragastric administration to pregnant guinea pigs

Nocloprost was administered to 3 groups of 4 pregnant guinea pigs intravenously and subcutaneously in a dose of 30 micrograms/kg and intragastrically in a dose of 100 micrograms/kg. Plasma nocloprost levels were measured at defined times up to 24 h p. adm. with a specific radioimmunoassay and induction of abortion monitored simultaneously. In one animal per group uterus pressure was recorded continuously up to 8 hours p.adm. Animals were sacrificed 7 days p.adm. and the number and state of fetuses in utero evaluated. Systemic availability of unchanged drug was 100% after intravenous (AUCi.v. = 8.6 +/- 2.0 ng h/ml) and subcutaneous (AUCs.c. = 11.5 +/- 1.2 ng h/ml) administration and approximately 30% after intragastric administration (AUCi.g. = 8.9 +/- 2.0 ng h/ml). The incidence of abortion after intragastric administration corresponded to that after subcutaneous administration. After intravenous injection the abortion rate was somewhat less, indicating that equal AUC-values do not necessarily indicate identical pharmacological effects.     

The effect of inhibitors in vivo on protein synthesis and the amino acid pool in the sheep blowfly, Lucilia cuprina.

1. The rates of detoxification of cycloheximide (33 mug/g fresh wt.), puromycin (167 mug/g fresh wt.) and actinomycin D (1 mug/g fresh wt.) were assessed in vivo on the basis of acid-insoluble [14C]leucine incorporation in the sheep blowfly, Lucilla cuprina; these were compared with quantitative estimates which took account not only of incorporation data but also of leucine pool size and turnover. Quantitatively, cycloheximide and puromycin were still at least 50% effective in inhibiting protein synthesis after 6.5 and 24.5h of exposure respectively, whereas values based only on incorporation data suggested that cycloheximide was 83% effective and puromycin completely ineffective after the respective periods. Quantitative estimates also showed that actinomycin D effectiveness increased with increasing exposure over 24.5h, in contrast with values based only on incorporation data, which suggested that it was completely ineffective after 24h.2. All inhibitors affected the dynamic state of the amino acid pool; there was a marked decrease in the rate of leucine-pool turnover as well as an increase in the half-life of leucine in the pool. 3. Inhibition of protein synthesis resulted in changes in leucine-pool size; the most pronounced increase occurred with cycloheximide and puromycin and the most pronounced decreases with actinomycin D. 4. Evidence is presented which suggests that proteolysis is functionally linked to protein synthesis, which determines its rate indirectly.     

Neutrophil chemotactic factors promote leukocytosis. A common mechanism for cellular recruitment from bone marrow.

We investigated cellular responses in a rabbit to i.v. administration of five established chemotactic factors (leukotriene B4 (LTB4), platelet-activating factor (PAF), C5a, N-Formyl-Met-Leu-Phe (F-MLF), and IL-8), and each exerted a characteristic effect on circulating white blood cell levels. All five factors induced a rapid and transient leukopenia. The blood was nearly devoid of circulating neutrophils 5 min after administration of each chemotactic factor. Other leukocytes were also variably depleted during the leukopenic phase, including eosinophils, basophils, monocytes, and lymphocytes. The lymphocyte numbers remained significantly depressed (approximately 30%) for as long as 3 h after administration of PAF or f-MLF. Each chemotactic factor produced a marked neutrophilia (i.e., 250-400% of baseline levels) after the initial leukopenia. Eosinophil numbers were elevated along with the neutrophil response in the C5a- and LTB4-treated animals. Basophil levels were significantly elevated only in LTB4-treated animals. The cellular response to PAF, f-MLF, and IL-8 appeared to be specific for the neutrophils. The kinetic profiles of the neutrophilia induced by PAF (10 micrograms/kg) or f-MLF (2.5 micrograms/kg) were similar, with maximal responses occurring 3 to 4 h after administration. In contrast, LTB4 (10 micrograms/kg), IL-8 (2.5 micrograms/kg), and C5a (5 micrograms/kg) induced a more rapid neutrophilia, with peak responses occurring 1 to 1.5 h after injection, and remaining elevated for 3 to 4 h. In all animals the neutrophilia was accompanied by a relative increase in the number of nonsegmented neutrophils (bands), suggesting that a major component of leukocytosis is caused by the release of bone marrow reserves. Phenidone (10 mg/kg), a dual cyclooxygenase/5-lipoxygenase inhibitor, affected neither the neutropenia nor the neutrophilia induced by C5a, f-MLF, or PAF. The protein synthesis inhibitor actinomycin D also failed to suppress neutrophil responses induced by either C5a or PAF. These results suggest that leukocytosis is a common response induced by all neutrophil chemotactic factors. Leukocytosis appears to be a direct result of the dynamic adaptive response of neutrophils to chemotactic factor stimulation without involvement of a secondary mediator system.     

Protein synthesis inhibitors, like growth factors, may render resting 3T3 cells competent for DNA synthesis: a radioautographic and cell fusion study

Serum-deprived (0.1-0.2%) resting NIH 3T3 mouse fibroblasts pre-incubated with cycloheximide (7.5 micrograms/ml), or puromycin (10 micrograms/ml), were fused with stimulated cells taken 10 h after changing the medium to one containing 10% serum, and DNA synthesis was investigated in the nuclei of monokaryons, homodikaryons and heterodikaryons using radioautography with the double-labelling technique. Pre-incubation of resting cells with inhibitors of protein synthesis for 1-4 h abolished their ability to suppress DNA synthesis in stimulated nuclei in heterokaryons. Three hours after the removal of cycloheximide from the medium, the resting cells acquired once again the inhibitory capacity for entry of stimulated nuclei into the S period. This inhibitory influence disappeared also in the case of post-fusion cycloheximide application as well as following an 8-12 h pre-treatment of resting cells with actinomycin D (1 microgram/ml) prior to fusion. Pre-incubation of resting cells for 12 h with PDGF (1 u/ml-1) followed by an 8-48 h incubation in serum-free medium stimulated the onset of DNA synthesis. A brief exposure (45 min) of resting cells to cycloheximide (7.5 micrograms/ml), or puromycin (7.5 micrograms/ml), exerted a similar effect, inducing by itself the entry of cells into the S period. The results support the assumption that acquirement, by resting cells, of competence for DNA replication includes as a necessary step the down-regulation of intracellular growth inhibitors whose formation depends on protein synthesis.     

Collagen proline hydroxylase activity in the uterus of the rat during rapid collagen synthesis in vivo

The activity of collagen proline hydroxylase in the 27,000g supernatant of the uterus was compared in the normal 20-day-old rat and in the adult rat 21 days after ovariectomy. The cofactor requirements of this enzyme were shown to be qualitatively the same as the enzyme from rat liver and skin. The specific activity of collagen proline hydroxylase in the uterus of the immature rat is approximately 250% higher than that of the ovariectomized animal. Although the total protein of the uterus of the ovariectomized rat is much greater, the total activity of this enzyme is 50% higher in the uterus of the immature rat. The daily administration of 5 μg estradiol-17β for 4 consecutive days to either animal results in a significant increase in the activity of collagen proline hydroxylase. Enzyme activity increases significantly 24 hr after the first dose of estradiol-17β and remains elevated in a reproducible pattern throughout the experimental period. Other estrogens including estriol, estrone, diethylstilbestrol, and ethynylestradiol-3-methyl ether also increase significantly the activity of collagen proline hydroxylase in the uterus of the immature rat. The activity of collagen proline hydroxylase was compared in the 27,000g supernatant of uterus of the immature and ovariectomized rat in a dose-response study with estradiol-17β and there appears to be little, if any, difference in total enzyme capacity. These results suggest that the failure of collagen to accumulate in the uterus of the ovariectomized rat administered estradiol-17β is unrelated to a low activity of collagen proline hydroxylase.     

Effect of aldosterone antagonists on aldosterone-induced activation of Mg2+ -HCO3- -ATPase and carbonic anhydrase in rat intestinal mucosa

In previous studies, Mg2+ -dependent, HCO3- -activated ATPase in the brush border and carbonic anhydrase in the cytoplasm of rat duodenal and jejunal mucosa decreased after adrenalectomy. Both enzyme activities increased to near normal levels 4 h after i.p. injection of aldosterone (40 micrograms/kg). These results suggest the possibility that both enzymes in the small intestinal mucosa may be mediators of the action of aldosterone. In the present studies, therefore, the effects of actinomycin D (500 micrograms/kg, i.p.), spironolactone (50 mg/kg, s.c.) and potassium canrenoate (50 mg/kg, s.c.) on aldosterone-induced activation of both enzymes in the upper small intestinal mucosa from adrenalectomized rats were examined to clarify the mechanism of action of aldosterone in enzyme levels. Actinomycin D inhibited carbonic anhydrase activity in small intestinal mucosa from normal rats 4 h after i.p. injection but had no effect on ATPase activity, while two other drugs had no effect on either enzyme activity in normal rats up to 4 h later. Pretreatment with these 3 drugs 1 h before aldosterone administration (40 micrograms/kg, i.p.) to adrenalectomized rats blocked the aldosterone-induced activation of ATPase and carbonic anhydrase in the upper small intestine. On the other hand, adrenalectomy and administration of aldosterone and its antagonists, alone or in combination, had no effect on kidney enzyme activities. These results confirm that Mg2+ -HCO3- -ATPase and carbonic anhydrase are mediators of the action of aldosterone in the upper small intestinal mucosa.     

Action of actinomycin D on glycosidase levels in the small intestine of hamsters

Actinomycin D affects a number of functions of the epithelial cells of the small intestine. Maltase, saccharase and lactase levels in the small intestine of hamsters treated with various dosages of actinomycin D over various periods of time, differed from those observed in control animals: administration of 0.25 micrograms/g body weight, gave rise to a statistically significant increase in the maltase and saccharase levels measured after 4 h and a statistically significant reduction in the lactase levels measured after 8 h; administration of 1.5 micrograms/g body weight reduced the activity of all three enzymes at all times post-administration, the decrease being statistically significant for maltase after 2 and 8 h.     

Is protein synthesis necessary for prostaglandin production by guinea-pig endometrium?

The outputs of prostaglandin (PG) F-2 alpha, PGE-2 and 6-keto-PGF-1 alpha from Day-7 and Day-15 guinea-pig endometrium in culture were reduced by the inclusion of actinomycin D, cycloheximide and puromycin in the culture medium, with the output of PGF-2 alpha from Day-15 endometrium being particularly affected during the first 6 h of culture. The intrauterine administration of actinomycin D on Day 10 decreased the outputs of PGF-2 alpha and PGE-2, but not of 6-keto-PGF-1 alpha, from Day-15 endometrium in culture without affecting PG output from Day-15 myometrium in culture. Actinomycin D, cycloheximide and puromycin did not reduce PG output when superfused over the Day-7 and Day-15 guinea-pig uterus in vitro for 20 min, indicating that these compounds do not have a rapid inhibitory effect on endometrial PG synthesis. In fact, they tended to stimulate PG output during this 20-min period, with cycloheximide having a pronounced effect on PGE-2 output. The synthesis of secreted proteins, but not of cellular proteins, was greater by Day-15 than by Day-7 endometrium in culture. Actinomycin D, cycloheximide and puromycin inhibited the synthesis of secreted and cellular proteins by Day-7 and Day-15 endometrium in culture. Protein synthesis and PG synthesis in the endometrium were both inhibited to a greater extent by cycloheximide and puromycin than by actinomycin D. The intrauterine administration of actinomycin D on Day 10 reduced the syntheses of secreted and cellular proteins by Day-15 endometrium in culture. These findings indicate that the endometrial synthesis of PGs, particularly of PGF-2 alpha towards the end of the oestrous cycle, is dependent upon endometrial protein synthesis.     

Polyamine levels and diamine oxidase activity in hypertrophic heart of spontaneously hypertensive rats and of rats treated with isoproterenol

Polyamine levels and diamine oxidase (EC 1.4.3.6) activity were studied in hypertrophic heart of spontaneously hypertensive rats as well as in the heart of Wistar rats during the development and regression of cardiac hypertrophy induced by isoproterenol administration. In spontaneously hypertensive rats, putrescine content and diamine oxidase activity were higher than those found in normotensive Kyoto-Wistar control rats. During the development of cardiac hypertrophy induced by isoproterenol, there was an increase in polyamine content and diamine oxidase activity. The administration of cycloheximide or actinomycin D prevented the increase in diamine oxidase activity during the first 24 h after isoproterenol administration, demonstrating that the rise in diamine oxidase activity was due to synthesis of new enzyme. Following the cessation of isoproterenol treatment, cardiac hypertrophy regressed and polyamine levels and diamine oxidase activity diminished toward control values. The administration of aminoguanidine to isoproterenol-treated rats caused in the heart an inhibition of diamine oxidase activity that led to an increase in putrescine level beyond the values found in animals given isoproterenol alone. The results suggest that the enhancement of diamine oxidase activity plays a role in the regulation of putrescine level in hypertrophic heart.     

Altered tail-skin temperature responsiveness in streptozotocin-induced diabetic rats

We evaluated the tail-skin temperature response to administration of several doses of isoproterenol in streptozotocin-induced diabetic rats after 48 h and after 4 weeks of diabetes. Blood glucose concentrations were significantly increased over controls 48 hours after administration of streptozotocin (65 mg/kg, i.v.) and remained elevated to a similar degree in the 4-week group. Basal rectal temperature and tail-skin temperature (TST) were not different between controls and the diabetic groups and were not affected by administration of saline. However, administration of isoproterenol (25 micrograms/kg, s.c.) caused a significant rise in TST in the control group, but not in the rats diabetic for 4 weeks. A similar but exaggerated response was observed in the controls after subcutaneous administration of 40 micrograms/kg and 100 micrograms/kg of isoproterenol. The TST response in the 4-week diabetic rats still remained negligible with the two higher doses of isoproterenol. When the data were summarized as area under the TST curve, a dose-dependent increase was observed in the control groups and a significant absence of response was observed in the 4-week group. The rats studied 48 h after streptozotocin injection had a similar TST response to the control group after administration of 40 micrograms/kg of isoproterenol. Colonic temperatures did not significantly change between the two groups in any of the studies, although the colonic temperatures tended to rise in the control groups following administration of isoproterenol. We conclude from this study that the absence of a tail-skin temperature response in rats diabetic for 4 weeks results from either a reduced beta-adrenergic receptor mediated response or an altered neural thermoregulatory reflex response, or both. These changes are probably not due to streptozotocin treatment or increases in blood glucose.     

Effects of intranasal administration of atrial natriuretic hormone on spontaneously hypertensive rats

The effects of the intranasal administration of synthetic alpha-human atrial natriuretic polypeptide (alpha-hANP) were investigated in 14 anesthetized spontaneously hypertensive rats (SHR; Okamoto-Aoki strain). They were given intranasally synthetic alpha-hANP in distilled water at doses of 10 micrograms/kg, 50 micrograms/kg and 100 micrograms/kg. Intranasal application of 200 microliter of distilled water as a control was also performed in 3 anesthetized SHR. Sixteen anesthetized SHR were examined for the effects of intravenous administration of alpha-hANP at doses of 4 micrograms/kg, 10 micrograms/kg, 20 micrograms/kg and 40 micrograms/kg. Urinary volume and the urinary excretion of sodium increased 2- to 3-fold during the 50 minutes following intranasal administration of a single dose of 50 micrograms/kg or 100 micrograms/kg, although neither the urinary volume nor the urinary excretion of sodium increased after intranasal administration of 10 micrograms/kg of alpha-hANP or 200 microliter of distilled water. There were no significant changes in arterial pressure or heart rate after the intranasal administration of synthetic alpha-hANP or distilled water. In contrast, arterial pressure was decreased and urinary volume and urinary excretion of sodium were increased, in a dose dependent manner, within 5 minutes after intravenous bolus-injection of alpha-hANP and returned to their baseline levels within 20 minutes. These results indicate that intranasal administration of synthetic alpha-hANP exerts its diuretic effect without concomitant changes in arterial pressure or heart rate in SHR.