Absence Of Receptor Activation Research Articles

Oxidative stress in acidic conditions increases the production of inositol phosphates in chick retinal cells in culture

The effect of oxidative stress on the production of [ 3H]inositol phosphates (InsP) by retinal cells in culture was analyzed. The process of oxidation was induced by incubating the cells with ascorbic acid and ferrous sulphate, and increased extent of oxidation was obtained by varying the pH from neutral to moderate acidosis (pH 6.5). The oxidative process significantly reduced cell viability (about 15%) by decreasing the capacity of mitochondria dehydrogenases to reduce tetrazolium salts, but had no effect on the leakage of lactate dehydrogenase. The production of [ 3H] InsP, in the absence of receptor activation, was increased dose dependently by oxidative stress. Maximal increases to 189 ± 7%, 197 ± 13%, and 329 ± 22% were observed, respectively, for inositol monophosphates (InsP 1), inositol bisphosphates (InsP 2), and inositol trisphosphates (InsP 3), at 2.5 nmol thiobarbituric acid reactive substances (TBARS)/mg protein. The response to cholinergic receptor activation was slightly decreased in cells oxidized in acidic conditions. Antagonists of glutamate receptors failed to inhibit the enhancement in InsP that occurred upon cellular oxidation, suggesting that the effect was not mediated by activation of glutamate receptors. Cellular oxidation increased by about two fold the uptake of 45Ca 2+ in the absence of agonist stimulation. However, stimulation of phospholipase C by Ca 2+ did not mediate the increase in [ 3H]InsP upon cell oxidation in acidic conditions, because the addition of 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1- H-pyrrole-2,5-dione (U-73122), an inhibitor of phospholipase C-dependent processes, did not affect the production of [ 3H]InsP in oxidized cells. Nevertheless, U-73122 significantly inhibited carbachol- and K +-stimulated accumulation of [ 3H]InsP. Furthermore, the enhancement of [ 3H]InsP induced by ascorbate/Fe 2+ was still observed in the absence of external Ca 2+. This increase in the production of InsP did not substantially induce the release of Ca 2+ from internal stores. The results suggest that both Ca 2+-dependent and Ca 2+-independent pathways are involved in oxidative stress-mediated InsP increment, and that the enzymes of the InsP metabolism may be affected by oxidation.

The effect of Ca 2+ on the translocation of protein kinase C in bovine tracheal smooth muscle

The role of calcium in protein kinase C redistribution was studied in bovine tracheal smooth muscle preparations contracted by methacholine. Previous results have shown that, in the presence of normal extracellular Ca 2+, 10 μM methacholine produced a sustained contraction and a sustained translocation of protein kinase C from the cytosol to the membrane. In the present study, when tissues were preincubated in Ca 2+-free buffer containing 1 mM EGTA, methacholine produced a rapid but transient elevation in membrane-associated protein kinase C activity which was detected at 30 s and had returned to basal within 20 min. The redistribution of protein kinase C from the cytosol to the membrane induced by 1 μM methacholine in normal Ca 2+ was reversed by removal of the extracellular Ca 2+ and addition of 2 mM EGTA during agonist stimulation. Removal of the Ca 2+ caused approximately 50% relaxation after 10 min. Verapamil (30 μM) partially reversed the methacholine-induced protein kinase C redistribution and caused approximately 40% relaxation after 15 min. Sodium nitroprusside (10 μM) caused a rapid relaxation and complete reversal of the protein kinase C redistribution induced by methacholine. High K + (60 mM) also induced a sustained contraction and redistribution of protein kinase C from the cytosol to the membrane. Suitable antagonists were added to the bathing medium to block the effects of endogenous mediators which could be released by KCl-induced depolarization. Thus, translocation of protein kinase C is obtained in the absence of receptor activation. These observations indicate that alterations in [Ca 2+] i may be responsible for the changes in protein kinase C redistribution observed during agonist-induced contractions of bovine tracheal smooth muscle.

Activation of D1 receptors stimulates accumulation of gamma-aminobutyric acid in slices of the pars reticulata of 6-hydroxydopamine-lesioned rats.

D1 dopamine receptors are present on terminals of striatal neurons to the pars reticulata of the substantia nigra in the rat. Here we have studied the effect of the activation of these receptors on the synthesis of gamma-aminobutyric acid (GABA) in slices of the pars reticulata of the substantia nigra isolated from 6-hydroxydopamine-lesioned rats. The synthesis was judged by the accumulation of GABA after inhibiting GABA transaminase with aminooxyacetic acid. Both dopamine and SCH 23390, a D1 agonist, stimulated the synthesis. The effect of both compounds was blocked by SCH 23390, a D1 antagonist, but not by sulpiride, a D2 antagonist. In the absence of receptor activation, the synthesis was very slow. The results suggest a trophic influence of dopamine upon the synthesis of GABA via D1 receptors.

Spatial and temporal distribution of agonist-evoked cytoplasmic Ca2+ signals in exocrine acinar cells analysed by digital image microscopy.

High resolution digital video imaging has been employed to monitor the spatial and temporal development of agonist-induced cytosolic Ca2+ signals in fura 2-loaded exocrine acinar cells. Enzymatically isolated mouse pancreatic and lacrimal acinar cells or small acinar cell clusters were used. These retain their morphological polarity so that the secretory granules in individual cells are located at one pole, the secretory pole. In acinar cell clusters the granules are located centrally, oriented to surround what would be in situ referred to as the lumen. In pancreatic and lacrimal acinar cells inositol-1,4,5-triphosphate-generating agonists [acetylcholine (ACh) and cholecystokinin octapeptide (CCK) for the pancreas and ACh in the lacrimal gland] give rise to a rapidly spreading Ca2+ signal that is initiated at the secretory pole of the cells. The initial increase in [Ca2+]i in the luminal pole is independent of extracellular Ca2+ indicating that the earliest detectable intracellular Ca2+ release is specifically located at the secretory pole. In lacrimal acinar cells ATP acts as an extracellular agonist, independent of phosphoinositide metabolism to activate a receptor-operated calcium influx pathway which, as for ACh, gives rise firstly to an increase in intracellular Ca2+ concentration in the secretory pole. We propose that this polar rise in intracellular Ca2+ concentration is due to Ca(2+)-induced Ca2+ release. By contrast, when Ca2+ release and Ca2+ influx are induced in the absence of receptor activation by thapsigargin and ionomycin, the Ca2+ signal develops diffusely and slowly with no localization to the secretory pole.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the rat colonic aldosterone receptor and its activation process.

Aldosterone increases sodium absorption, short circuit current, and transmural potential difference in rat colon. We studied the rat colonic aldosterone receptor using the synthetic glucocorticoid, 11 beta, 17 beta-dihydroxy-17 alpha-propynylandrosta-1,4,6-triene-3-one, to prevent binding to the glucocorticoid receptor. Specific aldosterone binding was found in proximal and distal colon. Heating to 25 degrees C decreased binding within 15 min, but the protease inhibitor, phenylmethylsulfonyl fluoride, stabilized binding. Binding was highest in terminal distal colon. Competitive binding assay showed aldosterone specificity compared to other competitors was greater at 30 than at 4 degrees C, suggesting temperature-sensitive changes in receptor specificity. Scatchard analysis revealed a straight line with a KD of 2.5 nM at 0 degrees C and 4.1 nM at 30 degrees C. Bmax was higher in distal than in proximal colon (30 degrees C, 156 +/- 33 versus 65 +/- 9 fmol/mg protein) and increased by 36% in proximal and 180% in distal colon at 30 degrees C compared to 0 degrees C. DEAE-cellulose chromatography of unactivated receptor demonstrated a single peak eluting at 200-250 mM KCl. Heat, ATP, and gel filtration did not activate the receptor, whereas increasing cytosolic salt concentration to 300 mM KCl, raising the pH to 8, or adding EGTA and EDTA caused increased DNA-cellulose binding and a new peak eluting at 30-80 mM KCl on DEAE-cellulose chromatography. There is a specific aldosterone receptor in colon with increasing number of binding sites from proximal to most distal segments paralleling aldosterone's physiological effects. Absence of receptor activation with heat, gel filtration, or ATP suggests differences between activation of the aldosterone receptor and other steroid hormone receptors.

Muscarinic receptors on intact human fibroblasts. Absence of receptor activity in adult skin cells.

The intact human fibroblast has been used in clinical and basic research studies of receptor-mediated control of cell function, however there is little information about the relationship between muscarinic receptor density and the regulation of cyclic AMP (cAMP) accumulation. We have compared the muscarinic receptor characteristics of both lung and skin intact fibroblasts at fetal and adult stages of development using carbachol-mediated inhibition of cAMP accumulation and the binding of [3H]quinuclidinyl benzilate (QNB). Prostaglandin E1 (PGE1) stimulated cAMP accumulation in all four cell lines, while carbachol inhibited cAMP accumulation only in the fetal lung, adult lung, and to a lesser extent, the fetal skin. Adult skin fibroblasts did not display significant evidence of inhibitory muscarinic receptor activity. [3H]QNB binding was saturable for the fetal and adult lung, and the fetal skin, yielding Kd values of approximately 0.5 nM for these cell lines. Bmax values were 360 fmol/mg for fetal skin, 600 fmol/mg for adult lung, and 876 fmol/mg for the fetal lung. Specific binding of [3H]QNB to adult skin fibroblasts was found to be low and variable. Comparisons of the Bmax values and maximal inhibitory capacities showed that the receptor density paralleled receptor activity in all cell lines. The lack of muscarinic receptor activity in the adult skin fibroblast was confirmed in several different adult skin cell lines, indicating that the adult skin fibroblast may not be an appropriate model for muscarinic receptor activity in clinical investigations.