3-day Pretreatment Research Articles

Activin modulates the intracellular signaling system activated by gonadotropin-releasing hormone: dual effect on calcium messenger system and protein kinase-C pathway.

This study was performed to assess the effects of activin on intracellular mechanisms involved in GnRH action. When rat pituitary cell cultures were pretreated with activin-A (5-80 ng/ml) for 3 days, subsequent FSH and LH release (percentage of total cellular FSH and LH released during 4 h) in response to GnRH (10(-10)-10(-6) M) was not significantly different from that in cells pretreated with medium alone. In contrast, activin pretreatment increased the potency of both A23187 (Ca2+ ionophore) and phorbol 12-myristate 13-acetate [a protein kinase-C (PKC) activator] as secretagogues for FSH and LH release. FSH or LH release in response to another Ca(2+)-mobilizing secretagogue, maitotoxin (an activator of the GnRH receptor-associated Ca2+ channel), was not increased by activin. Although PKC is capable of influencing the actions of Ca2+, which is believed to be the second messenger for GnRH action, neither GnRH- nor maitotoxin-stimulated gonadotropin release was increased by activin even when the influence of activin on PKC was eliminated by the addition of a PKC inhibitor (staurosporine; 100 nM) during the final 30 min of the 3-day pretreatment period. These results indicate that although activin does not influence GnRH action with regard to gonadotropin release, it increases the sensitivity of the system regulating gonadotropin release to increases in cytosolic Ca2+ concentrations and PKC activation. Furthermore, activin appears to exhibit an inhibitory effect(s) at some point(s) in GnRH action in a PKC-independent manner, which could be responsible for opposing the increased sensitivity of the gonadotrope to Ca2+. The differential effects of activin on gonadotropin release in response to Ca(2+)-mobilizing secretagogues (ionophore and maitotoxin) raise the possibility that the activity of the GnRH receptor-associated Ca2+ channel may be suppressed by activin.

Ischemia/reperfusion-induced feline intestinal dysfunction: Importance of granulocyte recruitment

Although previous studies have reported that neutrophils play an important role in mediating the microvascular injury observed after reperfusion of ischemic intestine, the contribution of these phagocytic cells to the mucosal dysfunction remains unclear. Three series of experiments consisting of an untreated group, a short-term monoclonal antibody (MAb) IB4 treatment group (MAb IB4 given on the day of the experiment), and a long-term MAb IB4 treatment group (3-day pretreatment with MAb IB4) were performed using autoperfused segments of cat ileum exposed to 3 hours of ischemia followed by 1 hour of reperfusion. Mucosal myeloperoxidase activity, an index of mucosal granulocyte levels, increased from 12 to 25 U/g wet wt in the untreated group. In the short-term MAb IB4 experiments, baseline values were very similar to those of the untreated group but no increase in myeloperoxidase activity was observed after ischemia/reperfusion. Long-term pretreatment with MAb IB4 reduced baseline values of myeloperoxidase activity to approximately 1 U/g wet wt; the values remained at this level throughout the experiment. The permeability of the mucosal barrier was quantitated by measuring blood-to-lumen clearance to 51Cr-ethylenediaminetetraacetic acid (EDTA). The water absorptive capacity of the intestine was also measured. In the untreated group, mucosal permeability to 51Cr-EDTA increased sixfold and water absorption was abolished after reperfusion. Both short-term and long-term administration of MAb IB4 prevented the net fluid loss into the lumen, but only long-term administration of MAb IB4 blunted the increased mucosal permeability induced by ischemia/reperfusion. These data suggest that interstitial granulocytes contribute significantly to the mucosal dysfunction associated with reperfusion of the ischemic intestine.

Prednisone inhibits the appearance of inflammatory mediators and the influx of eosinophils and basophils associated with the cutaneous late-phase response to allergen.

To better define the effect of systemic glucocorticoids on the cutaneous early and late phase response (LPR), nine atopic subjects were examined in a double-blind cross-over study using skin chambers fixed over denuded skin blisters. A challenge was carried out by placing allergen in the chamber for 60 min in subjects who received either a 3-day pretreatment with 60 mg/day of prednisone or placebo. Skin chamber cell counts and inflammatory mediators (histamine, PGD2, and leukotriene C4 (LTC4)) were measured at hourly intervals for 12 h. Prednisone pretreatment did not alter the immediate skin erythema or release of histamine but ablated the late secondary erythema and rise in histamine. The median histamine values during h 10, 11, and 12 in the placebo and prednisone pretreatment visits were 3.73 and 0.22 ng/ml, respectively (p less than or equal to 0.02). Prednisone did not alter PGD2 production; however, LTC4 production was suppressed during the LPR. The cumulative median LTC4 values during h 7, 8, and 9 were 5.6-fold (p less than or equal to 0.05) more after placebo than after prednisone pretreatment. Prednisone altered cellular traffic more dramatically than it did inflammatory mediators. The influx of eosinophils, which peaked during the 9th and 10th h in placebo-treated patients, was completely blocked by prednisone (p less than or equal to 0.02) for every h from 6 through 12. The influx of basophils, which started during the 9th h and peaked during the 12th h in placebo-treated patients, was suppressed at all time points (p less than or equal to 0.02) in prednisone-treated patients. There was no significant alteration in neutrophil transit into the skin chambers induced by prednisone. We suggest that the selective blockade of eosinophil and basophil influx by prednisone and the associated decrease in inflammatory mediators may contribute to the blockade of the clinical expression of the cutaneous LPR.

Modulation of endothelial cell shape and growth by retinoids

Physiologic concentrations of retinol (1 × 10 −6 M) caused capillary and aortic endothelial cells (EC) to undergo a morphologic change, characterized by a rounder cell body, increased refractility at cell edges, and longer cytoplasmic processes distributed in a bipolar fashion. Computer image analysis of retinoid-treated EC revealed that both retinoic acid and retinol affected cellular area. Twenty-four hours following retinoic acid treatment, EC occupied a greater area than control ( P < 0.03) or retinol-treated EC ( P < 0.02). By Day 7, however, retinoic acid-treated EC occupied equivalent cellular areas as compared to control cells ( P = 0.8). In contrast, by Day 7, retinol-treated EC occupied a smaller cellular area than control ( P < 0.002) or retinoic acid-treated EC ( P < 0.001). Proliferation studies revealed that within the first 72 hr of retinol treatment, basal EC growth was inhibited by 33% and the cells exhibited a lowered responsiveness to basic fibroblast growth factor (bFGF). In contrast, EC treated with retinoic acid and pericytes treated with each of the retinoids were not inhibited. The inhibitory effect of the 72 hr retinol treatment was reversible. Following 3 days exposure to retinol, EC given fresh media without retinoid underwent a population doubling in a subsequent 3-day period. However, in the continued presence of retinol, EC were 100% growth-inhibited. After a 3-day pretreatment with retinol, with or without continued retinol treatment, EC were refractile to the mitogenic action of bFGF in a subsequent 3-day period. These results demonstrate that retinol inhibits the basal and growth factor-stimulated growth of EC and causes a significant shape alteration of EC, supporting our hypothesis that vitamin A may be one of the signals that modify the growth and phenotype of EC.

Cultured pituitary cell GtH response to GnRH at different stages of rainbow trout oogenesis and influence of steroid hormones

In rainbow trout, a variable in vivo pituitary sensitivity to GnRH has been previously observed, depending on the stage of oogenesis. The purpose of the present work was to study, in vitro, the role of oestradiol (E2) and 17α-hydroxy,20β-dihydroprogesterone (17α20βP), respectively, involved in vitellogenesis and in oocyte maturation, upon this variability. The study was performed using primary cultures of whole pituitary cells from animals at different stages of oogenesis and subjected to increasing doses of salmon GnRH (sGnRH) after a 3-day pretreatment with control medium or medium supplemented with the steroid at levels corresponding to those circulating at the time of particular events of the sexual cycle (maturation and vitellogenesis). In control cultures, pituitary GtH responsiveness to sGnRH was maximal at ovulation, since at this time the gonadotrophs were able to respond to 10 −9 M sGnRH, whereas during vitellogenesis and preovulatory stages, the minimal effective dose of sGnRH ranged between 10 −6 and −8 M. We have demonstrated that 17α20βP has a positive or negative effect by acting directly on pituitary cell responsiveness to sGnRH, depending on the stages at which it is applied; its effect is positive during early vitellogenic and preovulatory stages whereas it is negative at the time of ovulation. E2 also increased pituitary responsiveness to sGnRH when applied during early vitellogenesis at low doses, corresponding to circulating levels at the time of ovulation; higher levels of E2, corresponding to circulating levels found during the last stages of vitellogenesis, did not modify pituitary responsiveness but increased cell GtH content. Results obtained are discussed in relation to hormonal changes (gonadotropin and steroid hormones) observed by different authors during the sexual cycle.

Mechanism of Action of 2-Hydroxyestradiol on Steroidogenesis in Ovarian Granulosa Cells: Interactions with Catecholamines and Gonadotropins Involve Cyclic Adenosine Monophosphate1

Previously we have shown that 2-hydroxyestradiol (2-OH-E2) synergizes with catecholamines to enhance progesterone production by porcine granulosa cells in vitro. The present studies were undertaken to determine if the synergistic effects of 2-OH-E2 and catecholamines were 1) modulated by gonadotropins, 2) unique to catecholamines, and 3) mediated by cyclic adenosine monophosphate (cAMP). Undifferentiated granulosa cells from 1- to 3-mm porcine follicles were cultured in serum-free medium for periods of 6-9 days. A 3-day pretreatment plus a 4-day cotreatment versus a 4-day cotreatment of granulosa cell cultures with follicle-stimulating hormone (FSH) did not significantly alter progesterone production stimulated by a saturating concentration of epinephrine (EPI; 2 micrograms/ml) but significantly reduced the effect of 4 micrograms/ml 2-OH-E2 on Day 7 of culture. Four-day cotreatment of either FSH or luteinizing hormone (LH) from Day 3 to 7 of culture dramatically enhanced progesterone production stimulated by 2-OH-E2 and estradiol (E2) but not by EPI when measured on Day 7 of culture. Progesterone production (expressed as "-fold of controls") stimulated by 4-day treatment of EPI, 2-OH-E2, or EPI-plus-2-OH-E2 was 1.4 +/- 0.2, 8.2 +/- 2.2, and 10.7 +/- 1.0, respectively, in the presence of LH (n = 5 experiments), and 1.9 +/- 0.1, 7.8 +/- 1.4, and 10.6 +/- 1.8, respectively, in the presence of FSH (n = 3 experiments). Similar to E2, 2-OH-E2 significantly enhanced the stimulating effect of the cAMP analog 8-bromo-cAMP (0.5 mM) on progesterone production.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of 31 kDa bovine inhibin on FSH and LH in rat pituitary cells in vitro: antagonism of gonadotrophin-releasing hormone agonists.

The effects of 31 kDa bovine inhibin on the release of FSH and LH stimulated by gonadotrophin-releasing hormone (GnRH) or its agonist analogue buserelin have been studied using 5-day-old cultures of pituitary cells prepared from adult male Sprague-Dawley rats. Exposure of cultures to increasing concentrations of inhibin for 3 days before and during a 4-h stimulation with GnRH resulted in the progressive suppression of both basal and stimulated gonadotrophin release. At the highest inhibin concentrations FSH release was abolished (inhibin median inhibitory concentration (IC50) = 0.15 U/ml) whereas LH release was suppressed by 75% (IC50 = 0.93 U/ml). To correct for the reduced size of the FSH pool resulting from inhibin pretreatment, the amount of FSH or LH released by an agonist was expressed as a proportion of the total hormone available for release in each case. Following this adjustment, concentrations of inhibin producing maximal effects increased the GnRH median effective concentration for FSH release 4.1-fold and that for LH release 2.2-fold, with inhibin IC50 values of 0.45 and 0.32 U/ml respectively. Inhibin also suppressed the maximum proportion of both FSH and LH that excess GnRH released in 4 h by 36%, with IC50 values of 0.53 and 0.76 U/ml respectively. These effects were not changed by reduction of the inhibin pretreatment period from 3 days to 1 day or by exclusion of inhibin during the stimulation period. After a 3-day pretreatment, inhibin inhibited gonadotrophin release by buserelin less effectively than that by GnRH, but the pattern of antagonism was the same.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of hexobarbital-induced sleep by lysine and its metabolites

Lysine has been shown to be metabolized in the rat brain to pipecolic acid which is a precursor of piperidine. Lysine and its proposed metabolites in this pathway were studied for the first time for their effect on the sleeping time induced by hexobarbital in the rat. Only L -lysine and D -lysine were found to prolong sleeping time significantly without toxic effect. A 3-day pretreatment with L -lysine produced an even more profound sleep prolongation. In most cases sleep enhancement was accompanied by a significant shortening of the time of sleep onset. Quantification of brain hexobarbital levels in the control and treated rats indicates that prolongation of sleeping time was not produced by inhibition of hexobarbital metabolism. The sleep prolonging effect of lysine, therefore, may be a direct action of lysine, or the metabolite(s) derived in vivo from lysine, on the central nervous system.

The effects of wetting and drying on the subsequent germination of Rumex crispus

A period of 1 to 3 days imbibition followed by drying altered the subsequent germination responses of seeds of Rumex crispus L. When the imbibition took place under alternating light and temperature conditions subsequent germination was faster and more complete. In contrast, when the seeds imbibed at a constant temperature and in nearly continuous darkness subsequent germination was delayed and less complete. Subsidiary tests demonstrated that (a) within a 24-h drying period, the seeds lost virtually all water imbibed over 1 to 3 days; (b) a 3-day imbibition pretreatment was followed by faster, more complete germination in a 0.25 M mannitol solution; (c) the duration of the drying period (up to 29 days) had little effect on the outcome; and (d) seeds pretreated for 1 to 3 days under an alternating light and tempèrature regime germinated more rapidly than untreated seeds when sown in soil in a greenhouse. These results help to explain why seedlings of R. crispas appear rapidly each summer following a drought-ending downpour, whereas seeds of the same species buried deep in the soil acquire a secondary dormancy and may remain viable for up to 80 years.

Steroids: theoretical and experimental basis for utilization in prevention of contrast media reactions.

In vitro and in vivo studies were done to examine the effects of methylprednisolone on the adverse reactions induced by contrast media. At very high concentrations, the steroid potentiated the complement-activating effect produced in vitro by iodipamide, but inhibited the immune and nonimmune mechanisms of hemolysis. Rabbits pretreated for 3 days with intramuscular methylprednisolone (at high or low dosages) were significantly protected against an LD47 challenging dose of iodipamide. Those treated once with a low intravenous dose immediately prior to iodipamide challenge were protected to a lesser degree. Rabbits treated once with a very high intravenous dose of steroid evidenced no protection. A hyper-responsive dog was consistently protected against adverse reactions to injected sodium iothalamate by a 3-day steroid pretreatment.

Assimilate Movement in Lolium and Sorghum Leaves. I. Irradiance Effects on Photosynthesis, Export and the Distribution of Assimilates

At low light levels (20 W m-2 PAR), little difference was detectable in net photosynthetic rates between leaves of the C3 grass Lolium temulentum and the C4 grass Sorghum sudanense. At low light, both species also had similar rates of export of photosynthate per unit leaf area, although the rate of export of 14C, following the assimilation of 14CO2 by the uppermost fully expanded leaf, was much greater in Sorghum. As light levels were increased to 96 W m-2 PAR or greater, net photosynthesis rose more in Sorghum than in Lolium and a greater proportion of the assimilate was exported from the Sorghum leaf, while Lolium with the thicker leaf showed a much greater capacity for storage of photosynthate. High and low light pretreatments, which altered the amount of both soluble and insoluble dry matter in the leaf, had little effect on the percentage rate of export of 14C following the uptake of 14CO2, which suggests that the bulk of the stored material may be effectively separated from the pathway of transfer from the chloroplast to the vascular system within the leaf. The pattern of distribution of 14C-labelled assimilates to other parts of the plant was only slightly affected by reduced light during the 4-h translocation period' however, a 3-day pretreatment at low light significantly reduced the proportion of the assimilates moving to the roots.

The effect of phenobarbital and sodium dehydrocholate on the biliary excretion of methylmercury in the rat

Phenobarbital pretreatment significantly increased the biliary excretion of methylmercury in rats. Methylmercury excretion between 1 and 4 hr after the administration of 1 mg Hg/kg, iv, as methylmercury, was 7.04 ± 0.32 (SE) μg/kg/hr in control rats; 9.68 ± 0.18 μg/kg/hr following 1-day phenobarbital pretreatment; 9.40 ± 0.11 μg/kg/hr following 3-day phenobarbital pretreatment. Compared with 1-day phenobarbital treatment, the 3-day treatment caused no additional increase in biliary methylmercury excretion although liver weight increased significantly. Liver methylmercury concentrations in phenobarbital-treated rats were not higher than in the controls. Bile flow was increased 25–36% by phenobarbital. Infusion of sodium dehydrocholate increased bile flow 2.6-fold but did not affect methylmercury excretion. It is considered likely that phenobarbital increased the biliary excretion of methylmercury by promoting the formation of methylmercury complex(es) or conjugate(s) suitable for biliary excretion.

Release of creatine phosphokinase from muscle—II: Effect of multiple doses of polymyxin B or compound 48/80 on increases in plasma enzyme levels

A single dose of Polymyxin B, 5 mg/kg, or Compound 48/80, 3 mg/kg, increases the skeletal muscle-type creatine phosphokinase (PCPK) activity in rat plasma. 3 After a 1- to 3-day period of daily treatments with increasing doses of Polymyxin B or Compound 48/80, the same doses of Polymyxin B or Compound 48/80, i.p., as cited above produce markedly smaller or no increases in PCPK, plasma aspartate aminotransferase (GOT) or plasma lactic dehydrogenase activities. However, administration of Polymyxin B, 10 mg/kg, i.p., or Compound 48/80, 6 mg/kg, i.p., on the day after completion of the 3-day pretreatment with the same drug did produce increased PCPK activities. The increase in PCPK activity produced by Polymyxin B, 5 mg/kg, or Compound 48/80, 3 mg/kg, was significantly smaller in rats pretreated with Compound 48/80 or Polymyxin B, respectively, for 3 days, than in naive rats. Ten days after completing the 3-day Polymyxin B pretreatment, Polymyxin B, 5 mg/kg, produced an increase in PCPK activities. The increases in PCPK activity produced by restraint stress or other pharmacological agents were not diminished by prior treatment with Polymyxin B for 3 days. The evidence suggests that tolerance to the toxic effects of Polymyxin B or Compound 48/80 on skeletal muscle and other tissues develop with two or more doses of Polymyxin B or Compound 48/80.

Effect of nicotine on cardiac necrosis induced by corticosteroid-electrolyte-stress

Myocardial necrosis was produced in rats by the following regimen: daily injections of dexamethasone and desoxycorticosterone acetate, and gavage with a solution of monobasic sodium phosphate for 5 days, followed by 7 1 2 hours of restraint on the sixth day. Separate groups given the above-described treatment were concomitantly administered 0.14, 0.57, and 2.28 mg. per kilogram per day of nicotine alkaloid in the drinking water. Either phenelzine, 1 mg. per kilogram, or mecamylamine, 12.5 mg. per kilogram, was administered daily to separate groups given the steroid-salt-stress treatment and to other groups also administered 2.28 mg. per kilogram per day of nicotine. Reserpine, 1 μg per animal per day, was injected alternately 3 or 7 days prior to, and also during, steroid-salt-stress treatment, both with and without the administration of 2.28 mg. per kilogram per day of nicotine. The two higher doses of nicotine significantly (P < 0.01) increased the average lesion severity, the per cent mortality, and the per cent incidence of lesions. Myocardial potassium was significantly decreased (P < 0.01 and 0.05). Treatment with mecamylamine or phenelzine did not reduce the response to the steroid-salt-stress treatment, but significantly (P < 0.01) reduced the severity of the response when nicotine was added to the regimen. Seven days of pretreatment with reserpine did not change the response to nicotine, whereas a 3-day pretreatment increased (P < 0.01) the severity of myocardial lesions.