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Mechanisms of signal transduction at the dopaminergic D2 receptor
No full textD2 dopamine receptor activation induces inhibition of adenylate cyclase, with a rapid decrease of cAMP levels, and an ensuing blockade of IP3-dependent release of Ca2+ from intracellular stores. K+ channels are concomitantly activated and Ca2+ channels are possibly also inhibited. The increased K+ conductance causes hyperpolarization, which may be responsible for the abolition of Ca2+ action potentials and [Ca2+], fluctuations occurring both at rest and after activation of receptors coupled to PIP2 hydrolysis. Lucia Vallar and Jacopo Meldolesi analyse this spectrum of intracellular signals which might be sufficient to sustain inhibition of secretion in pituitary lactotroph cells and possibly the other effects of D2 receptors in other cell systems
alpha Latrotoxin of black widow spider venom binds to a specific receptor coupled to phosphoinositide breakdown in PC12 cells
No full textalpha Latrotoxin of black widow spider is known to bind with high affinity to surface sites of rat pheochromocytoma (PC12) cells, thereby causing depolarization, calcium influx and massive neurotransmitter release. We show here that the toxin causes the accumulation of inositol phosphates, the products of phosphoinositide breakdown. Inositol 1,4,5, trisphosphate was predominantly accumulated shortly after toxin application. Phosphoinositide breakdown appears to be a direct consequence of toxin binding because high K+ and ionophores (which induce depolarization, calcium influx and transmitter release by different mechanisms) were without such effect. Phosphoinositide breakdown is known as an event coupled to the activation of receptors of various hormones and transmitters. We suggest therefore that the alpha latrotoxin binding site is a receptor coupled across the membrane to the phosphoinositide hydrolysing system
Protein kinase C-mediated feed back inhibition of the Ca2+ response at the EGF receptor
No full textActivation of the EGF receptor in A431 cells induces the hydrolysis of phosphoinositides and a transient rise of the cytosolic Ca2+ concentration, [Ca2+]i, which are completely inhibited by acute pretreatment with activators of protein kinase C, such as phorbol esters. Down regulation of the enzyme (by long-term pretreatment of the cells with phorbol esters) causes the [Ca2+]i response to EGF to increase in magnitude and, especially, to become much more persistent (average t1/2 of [Ca2+]i decline 9 min with respect to 2.3 min in controls). These results demonstrate that the activation of protein kinase C induced by EGF in intact A431 cells is sufficient to trigger a feed back, autolimitative regulation of the EGF receptor that might play a prominent physiological role in the definition of the mitogenic activity of the growth factor
Inhibition of inositol phosphate production is a late, Ca2+-dependent effect of D2 dopaminergic receptor activation in rat lactotroph cells
No full textThe effect of dopamine, working through the activation of D2 receptors, on inositol phosphate production induced by thyrotropin-releasing hormone (TRH) was investigated in rat pituitary lactotroph cells. Dopamine (10 microM) did not modify the initial rapid stimulation of inositol 1,4,5-triphosphate and inositol bisphosphate observed within the first 15 s after TRH addition, but progressively inhibited the later inositol phosphate production induced by the neurohormone. This kinetics of inhibition was independent of dopamine preincubation time (from 2 to 10 min). The effect was still visible when dopamine was added after TRH. It was sensitive to pertussis toxin, was unchanged by increasing cellular cAMP levels with 8-Br-cAMP, but was greatly affected by treatments that modify the cytosolic free Ca2+ concentration. Specifically, the dopamine-induced inhibition was prevented by treatment of the cells with the Ca2+ ionophore ionomycin (100-200 nM) and was mimicked either by withdrawal of Ca2+ from the incubation medium or by blockade of voltage-gated Ca2+ channels with verapamil. The dopamine treatment did not decrease the cellular levels of the various phosphoinositides, strongly suggesting that the inhibition of inositol phosphate production is not due to precursor depletion. In isolated membranes, however, dopamine was unable to counteract the inositol phosphate accumulation triggered by TRH. Taken together, the data indicate that inhibition of inositol phosphate production is not a primary event triggered by D2 receptor activation, but is a late consequence, due to the previously demonstrated (Malgaroli, A., Vallar, L., Reza Elahi, F., Pozzan, T., Spada, A., and Meldolesi, J. (1987) J. Biol. Chem. 262, 13920-13927) inhibition by dopamine of the prolonged cytosolic free Ca2+ concentration increase induced by TRH via the activation of voltage-gated Ca2+ channels. These results are inconsistent with the possibility of a direct inhibitory coupling of D2 receptors to phospholipase C in rat pituitary lactotroph cells
Regolazione dell’omeostasi del calcio intracellulare
No full textRegolazione della concentrazione intracellulare dello ione calcio e sua modulazione farmacologic
Astrocytes, from brain glue to communication elements: the revolution continues
No full textFor decades, astrocytes have been considered to be non-excitable support cells of the brain. However, this view has changed radically during the past twenty years. The recent recognition that they are organized in separate territories and possess active properties--notably a competence for the regulated release of 'gliotransmitters', including glutamate--has enabled us to develop an understanding of previously unknown functions for astrocytes. Today, astrocytes are seen as local communication elements of the brain that can generate various regulatory signals and bridge structures (from neuronal to vascular) and networks that are otherwise disconnected from each other. Examples of their specific and essential roles in normal physiological processes have begun to accumulate, and the number of diseases known to involve defective astrocytes is increasing
Sorting of three secretory proteins to distinct secretory granules in acidophilic cells of cow anterior pituitary.
Full text linkThe distribution of three proteins discharged by regulated exocytosis--growth hormone (GH), prolactin (PRL), and secretogranin II (SgII)--was investigated by double immunolabeling of ultrathin frozen sections in the acidophilic cells of the bovine pituitary. In mammotrophs, heavy PRL labeling was observed over secretory granule matrices (including the immature matrices at the trans Golgi surface) and also over Golgi cisternae. In contrast, in somatotrophs heavy GH labeling was restricted to the granule matrices; vesicles and tubules at the trans Golgi region showed some and the Golgi cisternae only sparse labeling. All somatotrophs and mammotrophs were heavily positive for GH and PRL, respectively, and were found to contain small amounts of the other hormone as well, which, however, was almost completely absent from granules, and was more concentrated in the Golgi complex, admixed with the predominant hormone. Mixed somatomammotrophs (approximately 26% of the acidophilic cells) were heavily positive for b oth GH and PRL. Although admixed within Golgi cisternae, the two hormones were stored separately within distinct granule types. A third type of granule was found to contain SgII. Spillage of small amounts of each of the three secretory proteins into granules containing predominantly another protein was common, but true intermixing (i.e., coexistence within single granules of comparable amounts of two proteins) was very rare. It is concluded that in the regulated pathway of acidophilic pituitary, cell mechanisms exist that cause sorting of the three secretory proteins investigated. Such mechanisms operate beyond the Golgi cisternae, possibly at the sites where condensation of secretion products into granule matrices takes place
EGF raises cytosolic Ca2+ in A431 and Swiss 3T3 cells by a dual mechanism. Redistribution from intracellular stores and stimulated influx
No full textThe changes in Ca2+ homeostasis and phosphoinositide hydrolysis induced by EGF were studied in human epidermoid carcinoma A431 cells both when attached to a substratum and after detachment and suspension. The cytosolic Ca2+ concentration was measured by the conventional fluorimetric technique, using the specific probe, quin2, as well as by a new microscopic technique in which single cells are investigated after loading with another probe, fura-2. EGF applied in the complete, Ca2+-containing medium caused a rapid rise in the cytosolic Ca2+ concentration, that remained elevated for several minutes. In Ca2+-free, EGTA-containing medium, part of this response persisted, as revealed by quin2 results in suspended cells and microscopic results with fura-2. The lack of Ca2+ rise seen in attached cells loaded with quin2 and treated with EGF in Ca2+-free medium was probably the result of a Ca2+ buffer artifact. Concomitantly to the Ca2+ signal, EGF induced phosphoinositide hydrolysis, with stimulated accumulation of inositol 1,3,4,trisphosphate and -1,3,4,5-tetrakisphosphate. These results, as well as additional microscopic fura-2 results in Swiss 3T3 fibroblasts, demonstrate that the Ca2+ signal elicited by EGF is due to two components: redistribution from an intracellular store (possibly mediated by generation of inositol trisphosphate) and stimulated influx across the plasmalemma. This latter process was not detected in 3T3 cells treated with either PDGF or bombesin (growth factors that cause much greater phosphoinositide hydrolysis and Ca2+ redistribution responses than EGF). It is therefore suggested that the Ca2+ influx effect of EGF is under the control of a separate, as yet unidentified mechanism
Specific localization of the alpha-latrotoxin receptor in the nerve terminal plasma membrane
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