Effect of reducing agents-antioxidants on rat uterine aromatase activity in vitro

The effect of different reductors-antioxidants (ascorbic acid, cysteine, glutathione-SH and hydroquinone) on purified rat uterine aromatase activity was studied. It was established that all these compounds were aromatase activators. The possible mechanisms of this activation were discussed.     

Galanin stimulates rat pituitary growth hormone secretion in vitro

The effect of galanin on growth hormone (GH) secretion was investigated in monolayer cultures of rat anterior pituitary cells. Galanin caused a gradual increase in GH concentrations into the culture medium that was maximal at 90 minutes and sustained after 180 minutes. The ED50 for galanin-stimulated GH secretion was approximately 200 nM compared to an ED50 for rat GH-releasing factor (rGRF)-stimulated GH secretion of 10pM. Galanin and rGRF were additive in increasing GH release into the incubation medium. These data indicate that porcine-derived galanin has a direct effect on pituitary GH secretion in vitro.     

Contractile properties of the shortening rat diaphragm in vitro

Diaphragmatic fatigue has been defined in terms of the failure of the muscle to continue to generate a given level of tension. Appropriate shortening of the diaphragm is, however, just as important for adequate ventilation. In this study we have examined in vitro the contractile properties of the rat diaphragm under afterloaded isotonic conditions and the effect of fatigue on the ability of the diaphragm to shorten. Shortening of the muscle strips was found to depend on size of afterload, frequency of stimulation, duration of stimulation, and initial length of the muscle. The afterloaded isotonic length-tension relationship coincided with the relationship between length and active isometric tension only for relatively small afterloads. Fatigue of the muscle strips, induced by isometric or afterloaded isotonic contractions, was associated with a decline in the extent of shortening as well as a decrease in active isometric tension. Ability to shorten and ability to develop isometric tension did not decrease to the same extent under all conditions. We conclude that active shortening, as well as active isometric tension, is decreased by muscular fatigue and that changes in these properties can be different depending on experimental conditions. The results suggest that the definition of diaphragmatic fatigue should be expanded to include the ability of the muscle to shorten by an appropriate amount. The results also suggest that measurement of isometric performance may not provide a complete estimate of the overall performance of the fatigued diaphragm.     

Peripheral nervous control of cold-induced reduction in the respiratory quotient of the rat

Cold-exposed rats show a reduction in the respiratory quotient which is indicative of a relative shift from carbohydrates to lipids as substrates for oxidative metabolism. In the present study, the effects of food deprivation and cold exposure on the respiratory quotient were observed. In addition, the involvement of the three main branches of the peripheral nervous system (sympathetic, parasympathetic, and somatic) was investigated by means of synaptic blockade with propranolol, atropine, and quinine, respectively. Both propranolol and quinine blocked the cold-induced decrease in respiratory quotient and increase in heat production, whereas atropine had only minor and very brief effects. It is concluded that both the sympathetic and somatic branches are involved in the metabolic changes associated with cold-induced thermogenesis and that the increase in metabolic heat production involves a shift from carbohydrate to lipid utilization irrespective of which of the two branches is activated.     

The release of alanine by rat diaphragm muscle in vitro.

Z discs were isolated from Lethocerus flight muscle by removing the contractile proteins from myofibrils with a solution of high ionic strength. The protein composition of the Z discs was analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the major proteins were alpha-actinin, actin and tropomyosin. Z lines were selectively removed from intact myofibrils by digestion with crude lipase and chymotrypsin, but not by purified lipase.     

Refolding of recombinant porcine growth hormone in a reducing environment limits in vitro aggregate formation

Recombinant porcine growth hormone (rPGH) solubilized from bacterial inclusion bodies (IBs) using a cationic surfactant was oxidized to form disulphide bonds in a simple buffer solution containing 2-mercaptoethanol within an empirically derived optimal molar ratio of 2-mercaptoethanol:protein. A final yield of 55% monomeric rPGH was achieved at protein concentrations of up to 5 mg/ml without the need for removal of the 2-mercaptoethanol or the use of chaotrophic agents. In the absence of 2-mercaptoethanol only 15% monomeric rPGH was obtained, with the majority forming higher molecular weight aggregates. Using the procedure derived for porcine growth hormone, it may be possible to obtain high yields of native protein and overcome the need for using low protein concentrations and chaotrophic agents during in vitro refolding of other disulphide bonded recombinant proteins.     

Induction of responsiveness of rat diaphragm to ovine growth hormone after administration of reserpine.

The diaphragm of the pituitary intact rat is insensitive to the insulin-like effects of growth hormone unless weanling animals are used, and even then these effects are not achieved reliably. We report here that an intraperitoneal injection of reserpine is able to induce consistent responsiveness to ovine growth horomone (oGH) in hemidiaphragms from 20-27 day old rats as assessed by stimulation of 3H-AIB transport and 14C-phenylalanine incorporation into protein. Maximal stimulation of 3H-AIB transport (approximately 40%) can be elicited by addition of oGH (5 micrograms/ml) to hemidiaphragms after a 2 mg/kg injection of reserpine given 5 h prior to sacrifice. The degree of stimulation does not alter significantly if the rats are sacrificed 3, 5 or 12 h after administration of reserpine, although it decreases by 24 h. Administration of reserpine 3 h before sacrifice also leads to a 50% increase in 14C-phenylalanine incorporation into protein in rat diaphragms in response to the addition of oGH (5 micrograms/ml). The induced sensitivity to oGH is not due to inhibition of GH secretion by reserpine as demonstrated by RIA of plasma GH. Addition of a monoclonal antibody to the GH receptor (MAb263) did not result in a stimulation or inhibition of 3H-AIB uptake or stimulation of protein synthesis in reserpinized rat hemidiaphragms. These results suggest that reserpine can induce tissue responsiveness in rats 20-27 d.o. independent of plasma GH levels. Our results also imply that the type 1 GH receptor of Barnard, Bundesen, Rylatt and Waters (1985) does not mediate the insulin like actions of GH on rat diaphragm.     

The in vitro effects of benzodiazepines on the rat diaphragm. Structure-activity relationships

The effect of three different subgroups of benzodiazepines on the indirectly evoked twitch tension was investigated in the in vitro rat phrenic nerve - hemidiaphragm preparation. Two effects were observed: an initial increase in twitch tension at lower concentrations with some benzodiazepines, and a concentration-dependent depression at higher concentrations with all benzodiazepines. Significant differences for these effects were observed among the three subgroups of benzodiazepines and additionally within the subgroup of the 1,4-benzodiazepine-2-ketones. Structural requirements for both effects were different. For the increase of twitch tension a --CH3 substitution at R1 and a --F substitution at R2' were beneficial. For the twitch depression an --OH substitution at R3 and a --C1 substitution at R2' were optimal. An interaction between substituents at different substitution sites occurred. The potency of twitch depression showed a good correlation with literature reports of pKa values and a poor-to-inverse correlation with lipophilicity indices. A benzodiazepines antagonist, Ro 15-1788, caused no change in twitch tension in the concentration range of the investigated benzodiazepines nor did it prevent the twitch depression caused by benzodiazepines.