Accumulation of cAMP and calcium in S49 mouse lymphoma cells following hyposmotic swelling

Swelling of S49 "wild type" mouse lymphoma cells in hyposmolar medium was used to examine the effects of cellular deformation on cAMP metabolism. In S49 wild type mouse lymphoma cells incubated in a defined medium, progressive reductions in medium osmolarity of 5-50% resulted in proportionate expansion of cell volume. Increases in cell volume were accompanied by incremental increases in intracellular cAMP and calcium. These responses in S49 cells occurred rapidly, with increases in calcium concentration and cAMP content occurring within 1-2 min. Swelling of S49 cells in the absence of ions (hyposmolar versus normosmolar sorbitol) resulted in a significant accumulation of cAMP. Inclusion of papaverine or isobutyl methylxanthine amplified cAMP accumulation, and omission of calcium, sodium, or magnesium from the medium attenuated, but did not prevent accumulation of cAMP in S49 cells in response to swelling. Exposure to propranolol or nadolol attenuated the ability of swelling to increase cAMP concentration, while treatment with 2',5'-dideoxyadenosine or phentolamine had no effect on swelling-induced cAMP accumulation. It is concluded that cellular deformation of S49 wild type mouse lymphoma cells stimulates rapid accumulation of intracellular calcium and cAMP.     

Different effect of propranolol and verapamil on isoprenaline-induced changes in the chick embryonic heart

Isoprenaline (IPRO) has been reported to cause pathological lesions of the embryonic heart. The purpose of the present study was to ascertain whether the development of IPRO-induced changes can be reduced--similarly as in adults--by beta blockade or calcium antagonists. IPRO was administered to 10-day-old chick embryos intraamnially (i.a.) in a dose of 2 X 10 mg.kg-1 per 48 h; propranolol (Inderal) and verapamil (Isoptin) were injected i.a. in a dose of 1.0 or 10.0 mg.kg-1 before each injection of IPRO. It was found that propranolol completely blocked the cardiac IPRO-induced changes, i.e. cardiomegaly, avascular areas and elevation of cAMP. On the other hand, verapamil was found to have no protective effect in any dose used. Furthermore, it increased the mortality of experimental embryos. This fact support the hypothesis that cardiac sensitivity to calcium antagonists may differ during prenatal development.     

The participation of serotonin in regulating cyclic nucleotide levels in early sea urchin embryos

Abnormal cleavage, decrease in the intracellular cAMP and cGMP content and a trend for increase of extracellular cAMP content were observed in sea urchin embryos incubated with KIuR-14 serotoninolytic substance. The addition of serotonin leads to normalization of cleavage and cAMP and cGMP content. It suggests serotonin-specific effect of KIuR-14 and functional relations between serotonin and cyclic nucleotides.     

The effect of oxidized isoprenaline on the chick embryonic heart

Intra-amnial administration of isoprenaline (IPRO) to chick embryos induces a number of myocardial lesions. The purpose of the present study was to investigate whether similar changes may also be induced after injection of spontaneously oxidized isoprenaline and commercially obtained adrenochrome. Cardiotoxicity of these substances has been demonstrated in adult animals. IPRO, oxidized IPRO, or adrenochrome were administered intra-amnially to 10-day-old chick embryos at doses of 0.1, 1.0, 10.0, and 100.0 mg X kg-1. Parallel experimental groups received propranolol at a dose of 1 mg X kg-1, 15 s before injection of IPRO or oxidized IPRO. The cAMP level in the heart was determined by radioimmunoassay 2 and 30 min after administration of IPRO, oxidized IPRO, or adrenochrome at a single dose of 10.0 mg X kg-1. It has been found that in embryos the effect of IPRO and oxidized IPRO is dose dependent. The rise in mortality and development of cardiomegaly together with increased hydration and disturbances of the development of coronary vascularization were highly significant starting from the dose of 10 mg X kg-1. Furthermore, both drugs significantly increased cAMP levels in the embryonic heart. On the other hand, the administration of adrenochrome was without any effect. The changes induced by IPRO were prevented by the administration of the beta-blocking agent propranolol; the lesions induced by spontaneously oxidized IPRO were, however, prevented only partially.     

Positive inotropic action of acetylcholine on the heart ventricles of the chick embryo

Acetylcholine increased twitch tension in the whole ventricle or in ventricular strips from 2-10-day chick embryos. The effect of acetylcholine was mediated by muscarinic receptors, since it was prevented by atropine, but not by tubocurarine or propranolol. Prostigmine significantly increased the sensitivity to acetylcholine in the strips from 7-day embryos, being almost ineffective in the strips from 3-day embryos. The decrease in acetylcholine sensitivity in the developing chick embryo is presumably associated with the increase in cholinesterase activity of the myocardial tissue.     

Studies on the alpha-andrenergic activation of hepatic glucose output. II. Investigation of the roles of adenosine 3':5'-monophosphate and adenosine 3':5'-monophosphate-dependent protein kinase in the actions of phenylephrine in isolated hepatocytes.

The effects of the alpha-adrenergic agonist phenylephrine on the levels of adenosine 3':5'-monophosphate (cAMP) and the activity of the cAMP-dependent protein kinase in isolated rat liver parenchymal cells were studied. Cyclic AMP was very slightly (5 to 13%) increased in cells incubated with phenylephrine at a concentration (10(-5) M) which was maximally effective on glycogenolysis and gluconeogenesis. However, the increase was significant only at 5 min. Cyclic AMP levels with 10(-5) M phenylephrine measured at this time were reduced by the beta-adrenergic antagonist propranolol, but were unaffected by the alpha-blocker phenoxybenzamine, indicating that the elevation was due to weak beta activity of the agonist. When doses of glucagon, epinephrine, and phenylephrine which produced the same stimulation of glycogenolysis or gluconeogenesis were added to the same batches of cells, there were marked rises in cAMP with glucagon, minimal increases with epinephrine, and little or no changes with phenylephrine, indicating that the two catecholamine stimulated these processes largely by mechanisms not involving cAMP accumulation. DEAE-cellulose chromatography of homogenates of liver cells revealed two major peaks of cAMP-dependent protein kinase activity. These eluted at similar salt concentrations as the type I and II isozymes from rat heart. Optimal conditions for preservation of hormone effects on the activity of the enzyme in the cells were determined. High concentrations of phenylephrine (10(-5) M and 10(-4) M) produced a small increase (10 tp 16%) in the activity ratio (-cAMP/+cAMP) of the enzyme. This was abolished by propranolol, but not by phenoxybenzamine, indicating that it was due to weak beta activity of the agonist. The increase in the activity ratio of the kinase with 10(-5) M phenylephrine was much smaller than that produced by a glycogenolytically equivalent dose of glucagon. The changes in protein kinase induced by phenylephrine and the blockers and by glucagon were thus consistent with those in cAMP. Theophylline and 1-methyl-3-isobutylxanthine, which inhibit cAMP phosphodiesterase, potentiated the effects of phenylephrine on glycogenolysis and gluconeogenesis. The potentiations were blocked by phenoxybenzamine, but not by propranolol. Methylisobutylxanthine increased the levels of cAMP and enhanced the activation of protein kinase in cells incubated with phenylephrine. These effects were diminished or abolished by propanolol, but were unaffected by phenoxybenzamine. It is concluded from these data that alpha-adrenergic activation of glycogenolysis and gluconeogenesis in isolated rat liver parenchymal cells occurs by mechanisms not involving an increase in total cellular cAMP or activation of the cAMP-dependent protein kinase. The results also show that phosphodiesterase inhibitors potentiate alpha-adrenergic actions in hepatocytes mainly by a mechanism(s) not involving a rise in cAMP.     

Influence of cAMP and calcium on epinephrine-mediated hepatic glycogenolysis in rainbow trout,Oncorhynchus mykiss

1. The role of cAMP and of calcium in mediating epinephrine-stimulated glycogenolysis was studied by incubating rainbow trout liver in vitro.2. Epinephrine significantly stimulates glucose release from liver pieces incubated in either calciumcontaining or calcium-free medium. However, the development of the glycogenolytic profile occurred more rapidly in the presence of calcium.3. The β-antagonist, propranolol, inhibited epinephrine-stimulated glucose release from liver pieces incubated in either calcium-containing or calcium-free medium.4. Calcium ionophore, A3187, stimulated glucose release from liver pieces incubated in calciumcontaining medium. Verapamil, a putative calcium channel blocker, had no effect on A23187-stimulated glycogenolysis. However, verapamil completely inhibited epinephrine-stimulated glycogenolysis.5. Dibutyryl cAMP and IBMX, singly or together, stimulated glucose release from liver pieces. cAMP-mediated glycogenolysis was more pronounced in liver pieces incubated in calcium-containing medium.6. These results indicate that epinephrine-stimulated hepatic glycogenolysis in rainbow trout proceeds through the activation of β-adrenergic receptors and that both cAMP and calcium are involved in the post-receptor signal transduction process.     

Fluctuating temperatures have different effects on embryonic sensitivity to ABA in Sorghum varieties with contrasting pre-harvest sprouting susceptibility

The effect of fluctuating temperatures on the germination ofimmature caryopses of two Sorghum varieties presenting contrastingsusceptibility to pre-harvest sprouting was investigated. Fluctuatingtemperatures were able to stimulate germination of immaturecaryopses of both varieties from early stages of development(i.e. 15 d after pollination). Isolated embryos from both varietiesgerminated well in water irrespective of the thermal regimeof incubation. However, the ability of ABA to block germinationin Redland B2 (sproutingsusceptible) isolated embryos was significantlyreduced when embryos were incubated under fluctuating temperaturesfrom 23 DAP onwards. No such effect was found in IS 9530 (sprouting-resistant)embryos. No differences in the pattern with which embryonicABA content decreased during whole grain incubation were foundin 25 and 35 DAP grains from both varieties incubated underconstant or fluctuating temperatures. Therefore, these resultsindicate that alternating temperatures can promote germinationthrough different mechanisms. One of them is the decrease inembryo sensitivity to ABA inhibition which appears to be actingin Redland B2 caryopses from 23 DAP onwards; the other one seemsto be independent of ABA level and sensitivity and is activeat very early stages of development in one variety (RedlandB2) and throughout seed development in the other (IS 9530). Key words: Germination, dormancy, fluctuating temperatures, abscisic acid, seed development, Sorghum bicolor     

Accumulation and leakage of abscisic acid during embryo development and seed dormancy in wheat

Seed dormancy develops latein embryogenesis after a period of potential prematuregermination and has been associated with levels ofabscisic acid (ABA) in, and sensitivity to, ABA ofembryos. In wheat (Triticum aestivum L.)embryos, there are two peaks in levels of ABA duringdevelopment: the first occurs 25 days afterpollination (DAP) and the second from 35 to 40 DAP. The first peak of ABA appears to be associated withthe development of the embryo's sensitivity to ABAsince such sensitivity was altered in seeds on earsthat were incubated in a solution of ABA from 15 and20 DAP. In the embryos of Kitakei wheat, a line thatexhibits dormancy, the second peak, at around 35 DAP,was more prolonged in comparison to Chihoku, anon-dormant line. The results support the proposedinvolvement of ABA in the formation and maintenance ofseed dormancy during middle and late embryogenesis. When developing embryos were incubated in water,embryonic ABA leaked out from the embryos, inparticular between 30 and 40 DAP. Prematuregermination observed between 30 and 40 DAP might berelated to such leakage of ABA from embryos.     

Effect of beta-adrenoceptor antagonists and some related drugs on maximal electroshock seizures in mice

(+/-) Propranolol (1-50 mg/kg), (+) propranolol (50 mg/kg) and pindolol (10-50 mg/kg) exhibited significant protective effects against MES (maximum electroshock seizures), whereas, timolol (1 mg/kg), the propranolol analog, UM-272 (1 and 10 mg/kg), and the beta-agonist, terbutaline (1 and 10 mg/kg) were ineffective. Cholinergic agents, physostigmine (0.01-1.0 mg/kg), and atropine (1 and 10 mg/kg), the serotonin antagonist, cyproheptadine (0.05 mg/kg), and the prostaglandin synthesis inhibitor, indomethacin (10 mg/kg), were also without effect on the MES extensor phase. Further, pretreatment of mice with terbutaline, atropine, cyproheptadine or indomethacin did not influence the anti-MES effect of propranolol to any significant extent. The results indicate that the observed anticonvulsant effects of beta-adrenoceptor antagonists are unrelated to noradrenergic or other central neurotransmitter systems and that a non-specific mechanism, probably a membrane stabilizing effect is involved.     

Propranolol inhibits cyclic AMP accumulation and amylase secretion in parotid acinar cells stimulated by isobutylmethylxanthine and forskolin.

Propranolol inhibited cyclic AMP (cAMP) accumulation stimulated by 3-isobutyl-1-methylxanthine (IBMX) or forskolin in rat parotid acinar cells. The inhibition by propranolol was highly potent; 10(-7) M propranolol was sufficient for the maximum inhibition (approx. 50% at 5 min). The inhibitory effect was observed in both intact and saponin-permeabilized parotid cells, but the effect was more prominent in permeabilized cells than in intact cells. Other beta-blockers, like alprenolol and atenolol, were as effective as propranolol, but butoxamine (beta 2-selective) was slightly less effective. The inhibition by propranolol was similarly detected in the cells prepared from pertussis-toxin-pretreated rats, suggesting that inhibitory guanine nucleotide regulatory protein (Gi) is not involved in the inhibitory mechanism. Propranolol also inhibited the exocytosis of amylase stimulated by IBMX or forskolin. In the presence of propranolol and IBMX, the responsiveness of saponin-permeabilized cells to exogenous cAMP was markedly increased, indicating that propranolol neither promotes the degradation of cAMP nor prevents the inhibitory effect of IBMX on cAMP phosphodiesterase.     

3': 5'-Nucleotide phosphodiesterase in the superior cervical ganglion of the rat. Evidence for multiple forms and influence of the beta-adrenergic receptor system in the electrophoretic pattern of the enzyme.

Two forms of a cAMP phosphodiesterase were found in the superior cervical ganglia of the rat. The influence of various detergents on solubility and activity of the enzyme was studied. Two Km values were determined, one of 0.9mum and the other 270mum cAMP. Separation by polyacrylamide disc electrophoresis revealed four distinct peaks of enzyme activity. One of these activities was increased when ganglia were incubated with adrenaline for at least 4 h. The increase caused by adrenaline was unaffected when ganglia were pre-incubated with cycloheximide, but was blocked in the presence of propranolol, a beta-adrenergic antagonist. Incubation with dibutyryl cAMP for 4 h mimicked the effect of adrenaline. Brief incubation with adrenaline was without effect. Electrophoresis in the presence of 0.1% Lubrol W, a non-ionic detergent, showed that one band of activity disappeared, while the main peak of enzyme activity increased in size. Two-dimensional starch gel electrophoresis confirmed the results of the disc electrophoresis.     

Effect of drugs influencing central serotonergic mechanisms on pentazocine-induced catalepsy in mice

Cataleptic effect of pentazocine in mice was affected by pretreatment with dexfenfluramine, fluoxetine, buspirone, p-chlorophenylalanine, cyproheptadine, mianserin, cisapride, ondansetron, pindolol and propranolol. The results suggest that drugs which influence the activity of central serotonergic systems do modulate pentazocine-induced catalepsy in mice.     

Hypothalamic cyclic AMP after the injection of ovarian steroids into ovariectomized rats

The effect of ovarian steroids on the concentration of adenosine 3',5'-cyclic monophosphate (cAMP) in the hypothalamus was studied in ovariectomized rats. Ovariectomized rats exhibited a lower cAMP concentration than intact rats. The administration of a single dose of estradiol benzoate (50 micrograms/kg body weight) resulted 3 days later in a rise of cAMP values, but levels did not reach those observed in estrous rats. Progesterone (2 mg/rat) injected 3 days after the priming dose of estradiol benzoate produced 4 h later no further changes in hypothalamic cAMP. The changes in hypothalamic cAMP concentration induced by estrogen treatment depend, at least in part, on noradrenergic inputs, since they were prevented by the injection of the norepinephrine synthesis inhibitor, diethyldithiocarbamate. In addition, administration of the beta-blocking agent, propranolol, to estradiol- and estradiol-progesterone-treated rats lowered the concentration of cAMP in the hypothalamus in a dose-dependent manner. In contrast, the administration of an alpha-blocking agent, phenoxybenzamine, had no effect at the tested concentration. The results of this study indicate that estrogen increases cAMP concentration in the hypothalamus by a noradrenergic mechanism involving beta-receptors. Moreover, the findings suggest that estrogen induces an increase in the number of beta-receptor sites, whereas progesterone increases the apparent propranolol sensitivity for these receptor sites.     

Effect of adenylyl cyclase activation on intracellular and extracellular cAMP and cGMP in preimplantation cattle blastocysts

The effects of direct and indirect activation of adenylyl cyclase on the production of intracellular and extracellular cAMP and cGMP by 13- to 16-day-old cattle embryos were determined. Embryos were incubated for 2 h in a Krebs Ringer bicarbonate medium containing the phosphodiesterase inhibitor isobutyl-methylxanthine, to which stimulating agents forskolin (100 mumol l-1), cholera toxin (2 micrograms ml-1), or both were added. Total (intra- and extracellular) basal cAMP and cGMP concentrations ranged from 6.65 +/- 0.895 to 3.4 +/- 0.708 fmol microgram-1 protein in 13-day-old embryos and from 4.05 +/- 1.151 to 0.19 +/- 0.041 fmol microgram-1 protein in 16-day-old embryos. Forskolin induced an increase (P < 0.001) in cAMP that ranged from 5.4-fold on day 13 to 2.7-fold on day 16, whereas cholera toxin induced an increase (P < 0.001) that ranged from 30-fold at day 13 to 21-fold at day 16, similar to the effect of forskolin and cholera toxin combined. Individually, forskolin and cholera toxin had no effect on cGMP concentrations, but together they induced an increase (P < 0.05). cAMP (P < 0.01) and cGMP (P < 0.001) concentrations decreased with embryo age from day 13 to day 16 for all treatments; the decrease was greater for cGMP than cAMP (5-24-fold versus 1.6-3.3-fold, respectively). It is concluded that inducible adenylyl cyclase is present in 13- to 16-day-old cattle embryos and that the embryos secrete cAMP and cGMP into the incubation medium. In addition, basal and inducible concentrations of cAMP and cGMP decrease with embryo age from day 13 to day 16. These observations indicate that cAMP and cGMP may have a role in the rapid embryonic cell proliferation that occurs at this time or in signalling to the endometrium.     

Effect of monoamine receptor agonists and antagonists on cyclic AMP accumulation in human cerebral cortex slices.

In human cerebral cortex slices noradrenaline, isoproterenol (a beta-adrenergic agonist), dopamine, apomorphine (a dopaminergic agonist), and serotonin stimulated cyclic AMP formation: noradrenaline greater than or equal to isoproterenol greater than dopamine = apomorphine = serotonin. Clonidine (and alpha-adrenergic agonist) was ineffective in stimulating cyclic AMP formation in temporal cortex slices. The stimulatory effect of noradrenaline and isoproterenol was blocked by propranolol (a beta-adrenergic blocker) but not by phentolamine (an alpha-adrenergic blocker). Pimozide (a selective dopaminergic antagonist) inhibited the increase of cyclic AMP formation induced by dopamine or apomorphine but not that induced by noradrenaline, isoproterenol, or serotonin. Neither propranolol or phentolamine had any effect on dopamine- or serotonin-stimulated cyclic AMP formation. Chlorpromazine blocked the increase of cyclic AMP formation induced by noradrenaline, dopamine or serotonin, while cyproheptadine, a putative central serotonergic antagonist, was ineffective. These observations suggest that there may be at least two monoamine-sensitive adenylate cyclases in human cerebral cortex which have the characteristics of a beta-adrenergic and a dopaminergic receptor, respectively, and also possibly a serotonergic receptor.     

5-Carboxamidotryptamine: a potent agonist mediating relaxation and elevation of cyclic AMP in the isolated neonatal porcine vena cava

5-Carboxamidotryptamine (5-CT) caused concentration dependent relaxation of isolated rings from the porcine vena cava contracted with either prostaglandin F2 alpha, histamine or alpha-methyl 5-hydroxytryptamine. Relaxation was not inhibited by propranolol (l microM), atropine (1 microM), indomethacin (3 microM), mepyramine (1 microM), cimetidine (1 microM), or cocaine (10 microM). Methysergide, but not cyproheptadine, was a competitive antagonist of the relaxant effect of 5-CT with a pA2 value of 7.88. 5-Carboxamidotryptamine also increased the intracellular levels of cyclic AMP, an effect which was antagonised by methysergide (apparent pA2: 7.95) but not cyproheptadine. The alpha-methyl analogue of 5-hydroxytryptamine did not cause relaxation or elevate cyclic AMP. These results suggest that 5-CT causes relaxation and elevation of cyclic AMP by interaction with a specific 5-hydroxytryptamine receptor which is '5-HT1-like'.     

The effect of different wavelengths of light during incubation on the development of rhythmic pineal melatonin biosynthesis in chick embryos

Rhythmic pineal melatonin biosynthesis develops in chick embryos incubated under a light (L)-dark (D) cycle of polychromatic white light. The spectral sensitivity of the embryonic pineal gland is not known and was investigated in this study. Broiler breeder eggs (Ross 308, n=450) were incubated under white, red, green or blue light under the 12L : 12D cycle. Melatonin was measured in extracts of pineal glands by radioimmunoassay. The daily rhythm of pineal melatonin levels in 20-day-old chick embryos was confirmed during the final stages of embryonic life under all four wavelengths of light with expected higher concentrations during dark- than light-times. The highest pineal melatonin levels were determined in chick embryos incubated under red and white light and lower levels under green light. The incubation under blue light resulted in the lowest melatonin biosynthesis. Pineal melatonin concentrations increased substantially on post-hatching day two compared with pre-hatching levels and we did not find differences between birds incubated and kept in either white or green light. Our results demonstrate a selective sensitivity of the chick embryo pineal gland to different wavelengths of light. Rhythmic melatonin production is suggested as a possible mechanism, which transfers information about the quality of ambient light to the developing avian embryo.     

EGF and TGF-alpha supplementation enhances development of cloned mouse embryos

In this study, we sought to determine the extent to which mitogenic growth factors affect the survival and development of cloned mouse embryos in vitro. Cloned embryos derived by intracytoplasmic nuclear injection (ICNI) of cumulus cell nuclei into enucleated oocytes were incubated in culture media supplemented with EGF and/or TGF-alpha for 4 days. Compared to control, treatment with either growth factor significantly increased the blastocyst formation rate, the total number of cells per blastocyst, the cell ratio of the inner cell mass and the trophectoderm (ICM:TE ratio), and EGF-R protein expression in cloned embryos. In most instances these effects were enhanced in cloned embryos when EGF and TGF-alpha were combined. Although fewer blastocysts developed from cloned than from fertilized one-cell stage embryos, growth factor treatment appeared to have the greatest effect on cloned embryos. These results demonstrate that mitogenic growth factors significantly enhance survival and promote the preimplantation development of cloned mouse embryos.     

Improved felid embryo development by group culture is maintained with heterospecific companions

Domestic cat embryos of excellent quality appear to improve development of conspecific embryos when cultured together, providing an avenue for improving development of embryos from valuable species or individuals. To have relevance to rare species, it would be useful to understand if this advantage could be conferred by heterospecific companions because there usually are severely limited numbers of conspecific embryos available from wildlife donors. In the first study, we incubated single test cat embryos alone (controls) or with 10 cat embryos or 10 or 20 mouse embryos under similar regimented conditions (each group shared 20 microl medium). In the second study, single test cat embryos were cultured alone, with 10 conspecific or 20 mouse embryos or 10 cattle embryos (each group shared 20 microl medium). Single test embryos in all treatment groups achieved similar (P>0.05) stages of compaction and blastocyst development. In the first study, only the test embryos incubated with 10 cat or 20 mouse companion embryos achieved blastocyst expansion. The average total cell number within test embryos incubated with 10 cat or 20 mouse companions was greater (P<0.05) than controls or those placed with 10 mouse embryos. In the second study, test embryos in all groups achieved blastocyst expansion and had more (P<0.05) total cells per embryo than the solitary controls. Inner cell mass to trophoblast cell ratio did not differ among treatments in either study. Thus, companion mouse and cattle embryos selected for excellent quality confer a benefit to singleton cat embryos, although the number of companions necessary to grant an advantage may be species dependent. If this phenomenon can be extrapolated across species, this may be an avenue for 'common animal embryos' to improve developmental potential of embryos from rare, unrelated taxa.