Respective EC50 Values Research Articles

A quantitative study of the actions of excitatory amino acids and antagonists in rat hippocampal slices.

1. A quantitative pharmacological investigation of the actions of excitatory amino acids on hippocampal CA1 neurones has been made using a new slice preparation developed for grease gap recording; d.c. potential was measured across a grease barrier placed between alvear fibres and the bathing medium. 2. In Mg2+-free perfusate, N-methyl-D-aspartate (NMDA, 1-100 microM), quisqualate (1-500 microM), kainate (1-200 microM) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 1-100 microM) caused dose-dependent depolarizations. 3. The dose-response relationships were fitted to logistic expressions. The maximum responses to AMPA, NMDA and kainate were similar; their respective EC50 values were 5, 13 and 23 microM. Quisqualate had a smaller maximum; its EC50 value was 10 microM. The slopes of the dose-response relationships were different for the 4 agonists; the order of steepness of the slopes was NMDA greater than AMPA greater than kainate greater than quisqualate. 4. Similar amino acid-induced depolarizations were observed in slices of just the CA1 region or in whole slices bathed in tetrodotoxin. Isolated alvear fibres, however, were insensitive to the excitatory amino acids. 5. D-2-Amino-5-phosphonovalerate (APV, 50 microM) selectively and reversibly antagonized responses induced by NMDA (apparent pA2 = 5.21). 6. Kynurenic acid (1 mM) reversibly depressed responses to the three agonists tested. The dose-ratios for antagonism of AMPA, kainate and quisqualate were 6.9, 5.6 and 4.6 respectively. 7. This preparation has a different sensitivity profile to agonists from those of previously reported preparations of spinal cord, neocortex and cerebellum. The greater sensitivity to NMDA may be due to the higher density of NMDA receptors in the hippocampus. The effects of the antagonists, APV and kynurenate, are similar to those found in other brain areas.

Dopamine Inhibits Prolactin Secretion Stimulated by the Calcium Channel Agonist Bay-K-8644 through a Pertussis Toxin-Sensitive G Protein in Anterior Pituitary Cells*

In primary culture of anterior pituitary cells, BAY-K-8644, a calcium channel agonist, stimulated PRL secretion by 83% with EC50 of 18 nM. This effect was blocked by nifedipine, a calcium channel antagonist. The stimulations of PRL secretion induced by potassium (50 mM) and BAY-K-8644 were additive. Dopamine inhibited basal as well as BAY-K-8644-stimulated PRL secretion by 64% and 75%, respectively, and with respective EC50 values of 4.5 and 0.6 nM. In the presence of 50 mM K+, dopamine only partially blocks the dose-dependent stimulation of PRL secretion induced by the calcium channel agonist. The inhibitory dopamine effect was blocked by (+)butaclamol, a specific dopamine receptor antagonist. The dopamine response was also blocked by 1-sulpiride, a specific dopamine D2 receptor antagonist, and mimicked by RU 24926, a specific dopamine D2 receptor agonist, suggesting that the dopamine effect on BAY-K-8644-stimulated PRL secretion was mediated through a D2 dopamine receptor. Although unknown, the mechanism by which dopamine inhibited the BAY-K-8644-stimulated PRL secretion involves a GTP binding protein sensitive to Bordetella pertussis toxin. In fact, the dopamine inhibition of PRL secretion induced by the calcium channel agonist was blocked by the pretreatment of cells with the toxin. These results suggest that dopamine D2 receptors in lactotroph cells modulate calcium influx through a GTP binding protein.

Beta-adrenergic stimulation of ion transport in primary cultures of avian salt glands.

Adrenergic stimulation of transmural ion transport was identified and characterized in primary cultures of avian salt gland. Adrenergic activation was mediated by beta-receptors since stimulation of the short-circuit current (Isc) was blocked by propranolol but not phentolamine. The Isc's elicited by isoproterenol, epinephrine, and norepinephrine were dose dependent, with respective EC50 values of 1.5 X 10(-8) M, 5.0 X 10(-6) M, and 1.1 X 10(-5) M. The apparent Ki for propranolol inhibition after isoproterenol stimulation was 7.5 X 10(-10) M. 8-Br cyclic AMP (8-Br cAMP) and forskolin-elicited Isc's that were insensitive to propranolol, were potentiated by theophylline, and inhibited by furosemide or ouabain. Isoproterenol also induced an increase in ouabain-sensitive respiration in acutely dispersed cells from salt-stressed juvenile or unstressed adult animals, but not in fully salt-stressed adults. The data indicate that, in addition to the well-established cholinergic receptors, beta-adrenergic receptors can control ion transport in these glands. Furthermore, the results suggest for the first time that an intracellular effector pathway involving cAMP is present.

Stimulation of growth hormone release and synthesis by estrogens in rat anterior pituitary cells in culture.

We have investigated the potential effect of estrogens in the control of GH secretion in rat anterior pituitary cells in primary culture. We have found that a 72-h preincubation with 17 beta-estradiol (E2) caused an approximately 2- to 3-fold stimulation of basal and GH-releasing factor (GRF)-induced GH release as well as cellular GH content at EC50 values of 44, 35, and 15 pM, respectively. Estrone and estriol also increased GH release at respective EC50 values of 100 and 250 pM. The stimulatory effects of these steroids on GH release and cellular GH content were competitively blocked by simultaneous incubation with the antiestrogen LY156758. In contrast to thyroid and glucocorticoid hormones, a 72-h pretreatment with E2 failed to potentiate GRF-induced cAMP accumulation or enhance the sensitivity of the GH response to GRF. However, E2 increased the stimulatory effect of submaximal concentrations of dexamethasone on spontaneous and GRF-induced GH release as well as on total GH, but did not further increase the effect of maximal dexamethasone concentrations. As determined by a 60-min pulse labeling with [35S]methionine performed after a 72-h preincubation with E2, GH and PRL synthesis were increased by about 50% above control values (P less than 0.005). The present data clearly indicate for the first time that E2, at physiological concentrations, exerts a stimulatory effect on spontaneous and GRF-induced GH release as well as on cellular GH content, probably resulting, at least in part, from stimulation of GH synthesis.

Molecular approach to the calcium channel.

Tritiated 1,4-dihydropyridines (nimodipine, nitrendipine, nifedipine, PN 200-110) and [3H]D-cis-diltiazem as well as [3H]verapamil were employed to directly identify calcium channels in membranes for excitable tissues. The channels, when probed with 1,4-dihydropyridines, exhibit the following properties: 1,4-Dihydropyridine calcium channel blockers bind in a temperature-dependent, reversible manner and with high affinity (dissociation constants 0.2-2 nM at 37 degrees C) to a finite number of sites. For chiral 1,4-dihydropyridines, the binding is stereoselective. Hill slopes of approximately 1.0 are observed. In brain, heart, and solubilized skeletal-muscle membranes, an absolute requirement for certain divalent cations exists in order to bind the ligands with high affinity. Cooperativity (negative and positive) between Me2+ and 1,4-dihydropyridine binding sites is observed. 1,4-Dihydropyridine binding sites are down-regulated in a complex manner by the optically pure enantiomers of D-600 and verapamil. These channel blockers induce, to a different extent, a low-affinity state of the 1,4-dihydropyridine binding site. It is postulated that this allosteric site, at which these blockers act, is closely coupled to the 1,4-dihydropyridine binding site and that a spectrum of compounds exists that differ in their affinity as well as their intrinsic activity to induce the down-regulation. The 1,4-dihydropyridine binding sites are up-regulated by D-cis-diltiazem and KB-944. The up-regulation is temperature-dependent and induces a high-affinity state for 1,4-dihydropyridine channel blockers, accompanied by distinct alterations of the kinetics as well as the pharmacological profile of the 1,4-dihydropyridine binding sites. Complex interactions exist between the channel blockers that induce up-regulation and those that induce down-regulation of the binding. For a given radiolabeled 1,4-dihydropyridine, a tissue-specific (but not species-specific) equilibrium binding dissociation constant is observed. Thus, all heart (human, rat, guinea pig, frog, bovine) have the same KD (0.25 nM at 37 degrees C) for, [3H]nimodipine. The same is observed for brain (KD = 0.5 nM) and for skeletal muscle (KD = 1-2 nM). Three subtypes of channels can be distinguished on the basis of the KD and the tissue-specific up-regulation by D-cis-diltiazem. Subtype-selective drugs exist; e.g., AQA 39 is an inhibitor of [3H]nimodipine binding at skeletal-muscle calcium channels, but not at brain channels. Despite their different pharmacological and kinetic profiles, calcium channels in skeletal muscle and brain have the same molecular size (Mr) when probed by radiation inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)

Energy-dependent extrusion of cyclic 3',5'-adenosine-monophosphate. A drug-sensitive regulatory mechanism for the intracellular nucleotide concentration in rat erythrocytes.

In reticulocyte-rich suspensions of red blood cells from rats extrusion of cAMP as a regulatory mechanism of intracellular cAMP was investigated. In response to isoprenaline and/or the phosphodiesterase inhibitors Ro 20-1724 and rolipram extrusion of cAMP increases dependent on the concentration of the drugs and time of exposure. However, these drugs exert their effects on the extrusion of cAMP only indirectly, i.e. via increased intracellular levels of cAMP, since the respective EC50-values of the drugs for intracellular accumulation and extrusion of cAMP are identical (isoprenaline: approximately 50 nM; rolipram: approximately 1 microM; Ro 20-1724: 15 microM). The dependence of the rate of extrusion on intracellular levels of cAMP is characterized by a typical concentration-effect relationship from which a maximal capacity of cAMP extrusion of 3-6 nmol/10 min/10(9) cells and a half maximal effective intracellular cAMP concentration of 40-50 nmol/10(9) cells can be derived. This relationship has been inferred from either kinetic or steady-state approaches. At rapidly changing intracellular levels of cAMP an apparent time lag of extracellular cAMP accumulation is obligatorily conditioned by this relationship. Vasodilating drugs which lower the ATP content of the cells as well as the uncoupler of oxidative phosphorylation, FCCP, inhibit the extrusive process (papaverine greater than FCCP greater than dipyridamole greater than dilazep much greater than hexobendine greater than or equal to carbocromene) leading to a 3-5-fold increase of the intrato extracellular concentration gradient of cAMP. It is concluded that extrusion of cAMP is a saturable and energy-dependent process which regulates the intracellular cAMP concentration independent of the activities of adenylate cyclase and phosphodiesterase.