Atanasio Pandiella

EGF raises cytosolic Ca2+ in A431 and Swiss 3T3 cells by a dual mechanism: Redistribution from intracellular stores and stimulated influx

The 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.

Plasma membrane hyperpolarization and [Ca2+]i increase induced by fibroblast growth factor in NIH-3T3 fibroblasts: Resemblance to early signals generated by platelet-derived growth factor

The effects of different substances on [Ca2+]i and membrane potential (measured by fura-2 and bis-oxonol fluorescence techniques, respectively) were studied in wild-type and NIH-3T3 fibroblasts transfected with the cDNA encoding the human epidermal growth factor receptor. Application of partially purified PDGF or FGF induced, after a lag (0.5-1 min), a [Ca2+]i increase composed by an initial, slow peak, sustained primarily by intracellular Ca2+ release followed by a plateau, sustained by Ca2+ influx from the medium. The [Ca2+]i changes were paralleled by plasma membrane hyperpolarization mainly due to the activation of a K+ efflux, since raising the extracellular K+ concentration progressively reversed the effect of both growth factors. These responses were much slower than those induced by other agents (bradykinin, extracellular ATP, and EGF). The close resemblance between PDGF- and FGF-induced early signals (time-course and insensitivity to phorbol esters) suggests similar transmembrane signalling mechanisms at the cognate receptor.

Protein kinase C-mediated feed back inhibition of the Ca2+ response at the EGF receptor

Activation 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.

Thyrotrophin-Releasing Hormone Raises Cytosolic Free Calcium Concentration in Human Adenomatous Somatotrophs and Corticotrophs; Comparison with in vivo Responsiveness to Thyrotrophin-Releasing Hormone in Patients with Acromegaly or Cushing's Disease.

The effect of thyrotrophin‐releasing hormone (TRH) on intracellular free Ca2+ concentration, [Ca2+)i, was investigated with the fluorescent dye fura‐2 in cell suspensions obtained from 13 human growth hormone‐secreting adenomas and 6 adrenocorticotrophin‐secreting adenomas. Preoperatively, 9 out of 13 acromegalic patients showed a positive growth hormone response to TRH administration while none of the 6 patients with Cushings disease had a plasma adrenocorticotrophin increase after TRH injection. In all the growth hormone‐secreting adenomas the addition of TRH (100 nM) caused a significant rise in [Ca2+]i (from a resting level of 133±40 (±SD) to a value of 284±119 nM at 100 nM TRH, n = 42; P<0.001). The transient induced by TRH was found to have a dual origin, one due to Ca2+ mobilization from intracellular stores which was maintained in presence of EGTA (3mM) and verapamil (10 μM) and a plateau phase due to Ca2+ influx from the extracellular media. Somatostatin (0.1 μM) lowered both resting [Ca2+]i and TRH‐induced transients. The effect of gonadotrophin‐releasing hormone on [Ca2+]i was evaluated on cell suspensions obtained from 6 growth hormone‐secreting adenomas. Gonadotrophin‐releasing hormone (100 nM) caused a marked rise in [Ca2+]i (from 179±25 to 283±15nM) on the cell suspension obtained from the only in vivo responsive adenoma while it was ineffective in the remaining 5. Although TRH was ineffective in modifying plasma adrenocorticotrophin levels in all patients with Cushings disease, in 5 out of 6 tumors the addition of 100 nM TRH caused a significant rise in [Ca2+]i (from 102.5 ± 36 to 163±66 nM, n = 22; P < 0.005). However, the effect of TRH on [Ca2+]i was significantly lower than that caused by arginine vasopressin, a physiological stimulator of adrenocorticotrophin release ([Ca2+]i values; 145±78 nM at 100 nM TRH versus 300±140 at 10 nM arginine vasopressin, n = 15; P<0.05). Moreover, the effect of arginine vasopressin on [Ca2+]i was detectable at concentrations as low as 0.1 nM while TRH was effective at concentrations higher than 1 nM. By contrast, gonadotrophin‐releasing hormone was ineffective in increasing [Ca2]i in all the adrenocorticotrophin‐secreting adenomas studied. Collectively, these data indicate that sensitivity to TRH is present in almost all the growth hormone‐ and adrenocorticotrophin‐secreting adenomas independently of the responsiveness of the individual patients to the peptide.

Mechanisms of Action of Growth Factors

Understanding the mechanisms that control cell growth is a central problem in cell biology today. During the last few years, it has become increasingly clear that one or more defects in the physiological mechanisms that regulate cell proliferation can be the cause of tumor cell growth. The study of normal cell division is therefore important not only per se, but especially in relation to its pathological implications.

Alpha-1-Adrenergic Stimulation of Cytosolic Ca2+ Concentration and Growth Hormone (GH) in Rat Somatotrophs

It is well established that the α-adrenergic system plays a primary role in the regulation of growth hormone (GH) release in humans and animals (1–3). As far as the site of action of α-adrenergic agents is concerned, the high levels of epinephrine in the portal circulation (4) as well as the identification of a α1-adrenergic receptors in the pituitary gland, point to the possible direct effect of adrenergic agents on hormone release. However, studies with isolated anterior pituitary cells have yielded rather conflicting results: α-adrenergic drugs have been reported either to inhibit, stimulate or not to influence GH release from pituitary cells in culture (3). With respect to the mechanisms of action of α-adrenergic agents, in all the systems so far studied, such as vascular smooth muscle cells, hepatocytes and myocardial cells, α1 adrenergic agents have been demonstrated to increase cytosolic free Ca2+ concentrations (5–7).