Cyclic AMP response to recombinant human relaxin by cultured human endometrial cells—A specific and high throughput in vitro bioassay

IIn previous studies, we had shown the synergistic effect of 10-5 M forskolin (FSK) on the detection threshold of the cyclic AMP response to Luteinizing Hormones (LH) and Chorionic Gonadotropins (CG) from various species in the mouse Leydig Tumor cell (mLTC) cell line. Indepedently, we had started to study the effect of 10-12 – 10-6 M OXT also on the cyclic AMP response to LH and CG preparations on these same cells and found an amplifying effect on the luminescence response caused by gonadotropins. The aim was then to explore the effects of 10-12 – 10-6 M OXT on the gonadotropin-induced cAMP response, in the presence or absence of 10 µM FSK to optimize the assay down to a sensitivity compatible with the detection of the circulating concentrations of these hormones in various species. Finally the optimization relies on three independent phenomena: 1/ the inhibition of nucleotide phosphodiesterase by IBMX to avoid cAMP degradation, 2/ the strong synergy of 10 µM forskolin with low concentrations of LH or CG during the 1-hour luminescence measurement and, 3/ the stimulatory effect of 10-8M oxytocin on the amplitude of transfected cAMP-sensitive luciferase response. By doing this, the detectable concentrations are at the 1-10 pg/well (pM range) for the LHs and CGs from various species. The bioactivities of circulating LHs and CGs in blood or urine are therefore expected to be measurable in 10µL-plasma samples from mammalian species and maybe others.

Download Full-text

Cytochemical Evidence for Presynatic β-Adrenergic Regulation of Secretion in the Developing Rat Submandibular Gland

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100079590 ◽

1977 ◽

Vol 35 ◽

pp. 430-431

Author(s):

L.S. Cutler

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Submandibular Gland ◽

Neonatal Rat ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Parotid Glands ◽

Adrenergic Agonist ◽

Rat Submandibular Gland

Many studies previously have shown that the B-adrenergic agonist isoproterenol and the a-adrenergic agonist norepinephrine will stimulate secretion by the adult rat submandibular (SMG) and parotid glands. Recent data from several laboratories indicates that adrenergic agonists bind to specific receptors on the secretory cell surface and stimulate membrane associated adenylate cyclase activity which generates cyclic AMP. The production of cyclic AMP apparently initiates a cascade of events which culminates in exocytosis. During recent studies in our laboratory it was observed that the adenylate cyclase activity in plasma membrane fractions derived from the prenatal and early neonatal rat submandibular gland was retractile to stimulation by isoproterenol but was stimulated by norepinephrine. In addition, in vitro secretion studies indicated that these prenatal and neonatal glands would not secrete peroxidase in response to isoproterenol but would secrete in response to norepinephrine. In contrast to these in vitro observations, it has been shown that the injection of isoproterenol into the living newborn rat results in secretion of peroxidase by the SMG (1).

Download Full-text

The effects of auxin and antiauxin in an in vitro bioassay of flower regulatory activity in leaf exudate from tobacco plants

Physiologia Plantarum ◽

10.1034/j.1399-3054.1991.810101.x ◽

1991 ◽

Vol 81 (1) ◽

pp. 1-6

Author(s):

M. Hatori ◽

Y. Sakagami ◽

S. Marumo

Keyword(s):

Regulatory Activity ◽

In Vitro Bioassay ◽

Tobacco Plants ◽

Leaf Exudate

Download Full-text

In Vitro Effects of Ethanol on Rabbit Platelet Aggregation, Secretion of Granule Contents, and Cyclic AMP Levels in the Presence of Prostacyclin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646570 ◽

1989 ◽

Vol 61 (02) ◽

pp. 254-258 ◽

Cited By ~ 21

Author(s):

Margaret L Rand ◽

Peter L Gross ◽

Donna M Jakowec ◽

Marian A Packham ◽

J Fraser Mustard

Keyword(s):

Cyclic Amp ◽

Inhibitory Effect ◽

Rabbit Platelet ◽

Inhibitory Effects ◽

Low Concentrations ◽

Rabbit Platelets ◽

High Concentrations ◽

In Vitro Effects ◽

Aggregation Of Platelets

SummaryEthanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin- treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.

Download Full-text

Effect of Cyclic AMP and Cyclic GMP on Thromboplastin (Factor III) Synthesis in Human Monocytes In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0038-1665317 ◽

1983 ◽

Vol 50 (04) ◽

pp. 804-809 ◽

Cited By ~ 16

Author(s):

Torstein Lyberg

Keyword(s):

Cyclic Amp ◽

Cyclic Gmp ◽

Immune Complexes ◽

Phosphodiesterase Inhibitors ◽

Inhibitory Effect ◽

Intracellular Camp ◽

Human Monocytes ◽

Purified Protein Derivative ◽

A 23187

SummaryHuman monocytes in vitro respond to various agents (immune complexes, lectins, endotoxin, the divalent ionophore A 23187, 12-0-tetradecanoyl-phorbol 13-acetate [TPA], purified protein derivative [PPD] of Bacille Calmette-Guerin) with an increased synthesis of the protein component of thromboplastin. The effect of cyclic AMP and cyclic GMP on this response has been studied. Dibutyryl-cyclic AMP, prostaglandin E1 and the phosphodiesterase inhibitors 3-butyl-1-methyl-xanthine (MIX) and rac -4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 201724), separately and in combination have a pronounced inhibitory effect on the response to immune complexes and PPD, and a moderate effect on the response to endotoxin and lectins. The effect on TPA response and on the response to A 23187 was slight. Dibutyryl-cyclic GMP (1 mM) gave a slight inhibition of the TPA arid IC response, but had essentially no effect on the response to other inducers. The intracellular cAMP level increased when monocytes were incubated with IC, TPA or A 23187 followed by a decrease to basal levels within 1-2 hr, whereas lectin (PHA) and PPD did not induce such changes. The cAMP response to endotoxin varied. Stimulation with IC induced an increase in monocyte cGMP levels, whereas the other stimulants did not cause such changes.

Download Full-text