Calcitonin inhibits the rise of intracellular calcium induced by thyrotropin-releasing hormone in GH3 cells

Both human and salmon calcitonins markedly inhibit the TRH-stimulated rise in intracellular [Ca2+] in GH3 cells. Calcitonin also inhibits prolactin release from these cells. Both [Ala] salmon calcitonin and salmon calcitonin (1-23) peptide amide also inhibit this rise in [Ca2+] and also inhibit TRH-stimulated prolactin release from GH3 cells as well as from primary pituitary cell cultures. It is likely that calcitonin inhibits prolactin release in the pituitary by decreasing the extent of the rise of intracellular calcium concentration. Neither an intact disulfide bond at the amino terminus nor residues 24-32 of the carboxyl terminus of salmon calcitonin are required for this inhibition.     

Pertussis toxin pretreatment abolishes dihydropyridine inhibition of calcium flux in the 235-1 pituitary cell line

In the present study we used 235-1 cells, a prolactin secreting clone derived from a pituitary tumor. In these cells maitotoxin, a calcium channels activator, likely acting on voltage sensitive calcium channels, increases intracellular free calcium measured by Quin 2 technique. Maitotoxin stimulation of calcium flux was inhibited both by nicardipine and verapamil in a dose dependent manner. Pertussis toxin pretreatment does not modify maitotoxin activation of calcium channels, while completely abolishes nicardipine inhibition of maitotoxin induced voltage sensitive calcium channels activation, without affecting verapamil effect. These results suggest a possible involvement of a pertussis toxin sensitive G protein in dihydropyridine inhibition of voltage sensitive calcium channels.     

Pertussis toxin abolishes angiotensin II-induced phosphoinositide hydrolysis and prostaglandin synthesis in rat renal mesangial cells.

Incubation of rat renal mesangial cells with angiotensin II (0.1 microM) resulted in transient breakdown of phosphatidylinositol 4,5-bisphosphate, rapid generation of diacylglycerol and phosphatidic acid, increased 45Ca2+ influx, increased intracellular [Ca2+] as measured by quin 2, and increased prostaglandin E2 synthesis. All of these processes were markedly inhibited time- and dose-dependently by prior exposure of cells to pertussis toxin. In contrast, the effects of the ionophore A23187 on 45Ca2+ influx and prostaglandin E2 synthesis were not altered by the exposure of the cells to pertussis toxin. The action of the toxin was not associated with alterations in cellular concentrations of cyclic AMP. Incubation of membrane fraction of mesangial cells with pertussis toxin resulted in ADP-ribosylation of Mr-42,000 protein. From all these results, it is likely that a G protein is involved in receptor-mediated signal transduction in renal mesangial cells.     

Stimulation of single L-type calcium channels in rat pituitary GH3 cells by thyrotropin-releasing hormone.

Hormonal stimulation of voltage-dependent Ca2+ channels in pituitary cells is thought to contribute to the sustained phase of Ca2+ entry and secretion induced by secretion stimulating hormones and has been suggested as a mechanism for refilling the Ca2+ stores. Using the cell-attached patch-clamp technique, we studied the stimulation of single Ca2+ channels by thyrotropin-releasing hormone (TRH) in rat GH3 cells. We show that TRH applied from the bath switched the activity of single L-type Ca2+ channels from a gating mode with very low open probability (po) to a gating mode with slightly smaller conductance but 10 times higher po. Interconversions between these two gating modes were also observed under basal conditions, where the equilibrium was shifted towards the low po mode. TRH applied from the pipette had no effect, indicating the involvement of a cytosolic compound in the stimulatory pathway. We show that TRH does not potentiate all the L-type Ca2+ channels in a given membrane patch and report evidence for co-expression of two functionally different L-type Ca2+ channels. Our results uncover the biophysical mechanism of hormonal stimulation of voltage-dependent Ca2+ channels in GH3 cells and are consistent with differential modulation of different subtypes of dihydropyridine-sensitive Ca2+ channels.     

Modulation of thyroid hormone nuclear receptors by cholera toxin in cultured GH1 cells

The cellular actions of the thyroid hormones L-thyroxine and L-triiodothyronine are mediated by the association of hormone with a chromatin-associated receptor. In cultured GH1 cells, a hormone-responsive rat pituitary cell line, thyroid hormone decreases the concentration of its receptor at early incubation times by reducing the accumulation of newly synthesized receptor. In this study, we demonstrate that cholera toxin also reduces the amount of nuclear receptor in GH1 cells in a time- and dose-dependent fashion, without altering the affinity of the receptor for hormone. The reduction of receptor mediated by cholera toxin is not secondary to a generalized inhibition of cell protein synthesis or cell replication rates and this effect can be abolished by pretreatment of the cholera toxin with soluble ganglioside II3-alpha-N- acetylneuraminosylgangliotetraosylceramide . This effect requires an intact cholera toxin molecule and does not occur at similar concentrations of the membrane-binding B subunit of cholera toxin. In order to study the influence of cholera toxin on thyroid hormone receptor turnover, we have used a dense amino acid-labeling technique. The results indicate that cholera toxin does not change the half-life of receptor, but decreases the rate of appearance of newly synthesized receptor. This decreased rate completely accounts for the lowered steady state receptor levels. The extent of cAMP stimulation by cholera toxin does not correlate with the extent of receptor reduction and forskolin, which stimulates cAMP 25- to 500-fold, does not decrease thyroid hormone receptor abundance. These studies suggest that cholera toxin modulates receptor levels by a mechanism(s) that is not mediated by cAMP in GH1 cells.     

Pertussis toxin blocks the inhibitory effect of muscarinic cholinergic agonists on cyclic AMP accumulation and prolactin secretion in GH3 anterior-pituitary tumour cells.

The inhibition of prolactin secretion and cyclic AMP accumulation in GH3 cells by muscarinic agonists was blocked by preincubation of the cells with pertussis toxin (islet-activating protein). There was a lag of approx. 80 min in the onset of the effect on secretion. These results suggest that muscarinic agonists decrease prolactin secretion by inhibiting adenylate cyclase activity.     

Pertussis toxin reduces the number of splenic Foxp3+ regulatory T cells

Pertussis toxin (PTx) is a bacterial toxin used to enhance the severity of experimental autoimmune diseases such as experimental autoimmune encephalomyelitis. It is known to promote permeabilization of the blood-brain barrier, maturation of APC, activation of autoreactive lymphocytes and alteration of lymphocyte migration. In this study, we show that i.v. injection of PTx in mice induces a decrease in the number of splenic CD4(+)CD25(+) regulatory T cells (Treg cells). Furthermore, PTx not only induces a depletion of the dominant CD4(+)CD25(+)Foxp3(+) subpopulation of splenic Treg cells, but also reduces to a similar extent the CD4(+)CD25(-)Foxp3(+) subpopulation. On a per cell basis, the suppressive properties of the remaining Treg cells are not modified by PTx treatment. The reduction in splenic Treg cells is associated with preferential migration of these cells to the liver. Additionally, Treg cells exhibit a high sensitivity to PTx-mediated apoptosis in vitro. Finally, in vivo depletion of Treg cells by injection of an anti-CD25 Ab, and PTx treatment, present synergistic experimental autoimmune encephalomyelitis exacerbating effects. Therefore, we identify a new effect of PTx and provide an additional illustration of the influence of microbial components on the immune system affecting the balance between tolerance, inflammation and autoimmunity.     

Stimulation of calcium intake by growth-hormone-releasing-hormone in GH3 cells]

The effect of GH-RH in the intra-cytosolic free Ca2+ concentration was studied in GH3 cells. To this end, we have used microspectrofluorimetry performed on single cells. We show that 60% of cells respond to a brief application of 100 nM GH-RH by an increase of their [Ca2+]i (mean increase 100% over basal values). This response which is blocked by calcium channel inhibitors results from an increased influx of Ca2+ ions from the external medium.     

Pertussis toxin blocks the inhibitory effects of somatostatin on cAMP-dependent vasoactive intestinal peptide and cAMP-independent thyrotropin releasing hormone-stimulated prolactin secretion of GH3 cells

It was shown that somatostatin (SRIF) inhibited cAMP-dependent vasoactive intestinal peptide (VIP)-stimulated prolactin (PRL) release by a GH3 clonal strain of rat pituitary tumor cells and decreased basal PRL secretion and inhibited PRL release in response to thyrotropin releasing hormone (TRH) whose action was independent of prior synthesis of cAMP. Pretreatment of these cells with pertussis toxin prevented SRIF's inhibitory effects on basal and TRH-stimulated hormone secretion as well as its VIP-stimulated responses. The blockade of SRIF's inhibitory effect on the actions of TRH or VIP was dependent on both the duration of preincubation and concentration of the toxin and was correlated with the ability of the toxin to catalyze the ADP-ribosylation of the 39,000-Da membrane protein. It is likely that this pertussis toxin substrate is involved in signal transduction of SRIF on cAMP-dependent actions of VIP and cAMP-independent action of TRH. However, the mechanism of SRIF's action on TRH is not clear, since SRIF did not affect the intracellular responses by TRH, neither intracellular Ca2+ mobilization nor the increase of 1,2-diacylglycerol formation following the breakdown of polyphosphoinositides.     

Calcium-dependent inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells

The inactivation of calcium channels in mammalian pituitary tumor cells (GH3) was studied with patch electrodes under voltage clamp in cell-free membrane patches and in dialyzed cells. The calcium current elicited by depolarization from a holding potential of -40 mV passed predominantly through one class of channels previously shown to be modulated by dihydropyridines and cAMP-dependent phosphorylation (Armstrong and Eckert, 1987). When exogenous calcium buffers were omitted from the pipette solution, the macroscopic calcium current through those channels inactivated with a half time of approximately 10 ms to a steady state level 40-75% smaller than the peak. Inactivation was also measured as the reduction in peak current during a test pulse that closely followed a prepulse. Inactivation was largely reduced or eliminated by (a) buffering free calcium in the pipette solution to less than 10(-8) M; (b) replacing extracellular calcium with barium; (c) increasing the prepulse voltage from +10 to +60 mV; or (d) increasing the intracellular concentration of cAMP, either 'directly' with dibutyryl-cAMP or indirectly by activating adenylate cyclase with forskolin or vasoactive intestinal peptide. Thus, inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells only occurs when membrane depolarization leads to calcium ion entry and intracellular accumulation.     

Nuclear calcium signaling by inositol trisphosphate in GH3 pituitary cells

It has been proposed that nuclear and cytosolic Ca(2+) ([Ca(2+)](N) and [Ca(2+)](C)) may be regulated independently. We address here the issue of whether inositol trisphosphate (IP(3)) can, bypassing changes of [Ca(2+)](C), produce direct release of Ca(2+) into the nucleoplasm. We have used targeted aequorins to selectively measure and compare the changes in [Ca(2+)](C) and [Ca(2+)](N) induced by IP(3) in GH(3) pituitary cells. Heparin, an IP(3) inhibitor that does not permeate the nuclear pores, abolished the [Ca(2+)](C) peaks but inhibited only partly the [Ca(2+)](N) peaks. The permeant inhibitor 2-aminoethoxy-diphenyl-borate (2-APB) blocked both responses. Removal of ATP also inhibited more strongly the [Ca(2+)](C) than [Ca(2+)](N) peak. The [Ca(2+)](N) and [Ca(2+)](C) responses differed also in their sensitivity to IP(3), the nuclear response showing higher affinity. Among IP(3) receptors, type 2 (IP(3)R2) has a higher affinity for IP(3) and is not inactivated by ATP removal. We find that IP(3)R2 immunoreactivity is present inside the nucleus whereas the other IP(3)R subtypes are detected only in the cytoplasm. The nuclear envelope (NE) of GH(3) cells showed deep invaginations into the nucleoplasm, with cytosol and cytoplasmic organella inside. These results indicate that GH(3) pituitary cells possess mechanisms able to produce selective increases of [Ca(2+)](N).     

Calcium movements and intracellular calcium distribution in neoplastic GH3 cells

Experiments were carried out to investigate the nature of the calcium homeostatic mechanisms in neoplastic GH3 rat pituitary cells. GH3 cells grown and maintained in Ham's F10 culture medium contained 35 nmoles calcium/mg cell protein. When stimulated by thyrotropin releasing hormone (TRH) or elevated K+ concentrations, only the latter caused cell calcium levels to rise although both resulted in hormone release. When exposed to EGTA, the GH3 cells lost calcium. When the temperature was lowered to 4 degrees C, the cells gained calcium and when rewarmed were able to extrude the previously accumulated calcium. The increased cell calcium following cold exposure could be blocked by prior treatment with rotenone. If rotenone was added subsequent to the cold exposure, it did not block the extrusion seen upon rewarming. In the absence of glucose in the medium, the GH3 cells took up more calcium upon exposure to 4 degrees C, and upon rewarming the cells could not return to their previous low levels. There are thus significant differences in calcium homeostasis between the neoplastic GH3 cells and their normal pituitary counterparts. When intracellular calcium was localized with the potassium pyroantimonate technique, there was calcium found in/on mitochondria, membrane bound vesicles and plasma membrane. Nuclear staining was sparse, and nucleolar staining was virtually absent. Upon stimulation with TRH, there was a decrease in mitochondrial calcium along with increases in both plasma membrane and nucleolar calcium levels. Since total calcium is unchanged, this indicates a significant calcium redistribution in response to TRH. The increased nucleolar calcium may reflect a calcium dependent increase in mRNA synthesis as has been reported. Since TRH presumably acts at a surface receptor, the increased plasma membrane calcium might be functionally related to receptor activation.     

Dephosphorylated oligoadenylates modulate high voltage-activated calcium currents in GH3 cells

Effects of different forms of C2-5A (2,5ApApA; 2,5ApApepoxyA; and 3,5ApApA) on high voltage-activated (HVA) calcium currents in GH3 cells were studied using the whole-cell patch-clamp recording technique. Addition of 10 µM 2,5ApApA, a core (dephosphorylated) oligoadenylate, to the pipette solution induced an increase in HVA calcium current. Ten minutes after the whole-cell configuration was established, the current magnitude was enhanced about twofold compared with that observed at 2 min. High concentration of Mg²⁺ (5 µM) in the pipette solution blocked this effect. 2,5ApA and 3,5ApApA oligoadenylates, and products of 2,5A hydrolysis, adenosine and AMP, did not change the value of HVA current. A chemically modified analog of 2,5ApApA (2,5ApApepoxyA) has been the oligoadenylate most stable under phosphodiesterase action. Addition of 2,5ApApepoxyA to the pipette solution under the same conditions caused a smaller effect than 2,5ApApA did.Neirofiziologiya/Neurophysiology, Vol. 26, No. 6, pp. 405–408, November–December, 1994.     

Multiple conductance levels of the dihydropyridine-sensitive calcium channel in GH3 cells

Summary Calcium channels in GH₃ cells exhibit at least five conductance levels when examined in cell-attached or outside-out patches. These channels resemble the high threshold Ca²⁺ current in their range of activation and inactivation, and in their sensitivity to dihydropyridines (DHP). Mean open times for the five levels were brief (<1 msec) in control solutions but increased in the presence of BAY K 8644. In 100mm Ba²⁺ and BAY K 8644, the five predominant slope conductances were 8–9, 12–13, 16–18, 23–24, and 28 pS. The present study is the first report of multiple levels of the DHP-sensitive Ca²⁺ channel occurring with high frequency in native membranes. The range of conductance levels that we observed encompasses the range of conductances found for two other different types of Ca²⁺ channels and indicates that unit conductance should be used with caution as a distinguishing characteristic for identification of different channel types.     

Thyrotropin-releasing hormone-induced rise in cytosolic calcium and activation of outward K+ current monitored simultaneously in individual GH3B6 pituitary cells

Thyrotropin-releasing hormone (TRH) acts on pituitary cells to raise the cytosolic free Ca2+ concentration ([Ca2+]i) and causes simultaneously a transient hyperpolarization of the plasma membrane. The combination of the microfluorimetric monitoring of [Ca2+]i with electrophysiological recordings obtained using the patch clamp technique in its whole cell configuration, allows the analysis of the correlation between changes in [Ca2+]i and the alterations in ionic currents at the plasma membrane. It was shown that in the absence of hormone stimulation, a depolarization-induced change in steady state [Ca2+]i, as well as the internal perfusion with Ca2+ at microM levels at constant membrane potential led to the activation of outward K+ current. TRH stimulation resulted in a marked but transient rise in [Ca2+]i; concomitantly, there was an increase in membrane conductance and an enhancement of outward current. During the time course of an individual response, an excellent correlation between the changes in [Ca2+]i and those in conductance or current was observed. The relative changes of current and conductance during the TRH response were consistent with the activation of a single type of ionic current, the apparent reversal potential of which coincided with the equilibrium potential for K+. A strong correlation between the TRH-induced changes in [Ca2+]i and K+, conductance was demonstrated in a large number of cells with varied kinetic features: significant correlation coefficients were found both for the transition time from basal to maximal values (r = 0.85, p less than 0.001) as well as for the total duration of the responses (r = 0.68, p less than 0.002). It is concluded that during the early phase of TRH action, the hormone-induced rise in [Ca2+]i is the principal cause of enhanced K+ channel activation.     

Cholera toxin affects nuclear ADP-ribosylation in GH1 cells

Incubation of GH1 cells with cholera toxin for 24 h inhibits [32P]ADP-ribose incorporation into histones and non-histone nuclear proteins by more than 50%. The toxin produces a generalized decrease of incorporation into all protein acceptors and into the poly(ADP-ribosyl)ated components excised from chromatin after micrococcal nuclease digestion. The cellular levels of NAD were also decreased (40 to 80%) after treatment with cholera toxin. The inhibition of poly(ADP-ribosyl)ation is preceded by an increase of [32P]ADP-ribose incorporation, since incubation with the toxin for 3 h caused an increase instead of a decrease of incorporation. Incubation with dibutyryl cyclic AMP for 24 h also inhibited nuclear poly(ADP-ribosyl)ation, thus showing that the effect of cholera toxin might be mediated by cyclic AMP.