Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0043167 (pertussis)
 19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The somatostatin (SS) analog octreotide has been successfully used in the treatment of (neuro)endocrine tumors. The mechanism of action of the tumor (growth) inhibitory action by octreotide is not fully understood. We have investigated the effect of octreotide on 7315b rat pituitary tumor cell growth, PRL release, and intracellular PRL concentrations in vitro. When cultured in medium with 10% fetal calf serum, the number of high affinity SS receptors increased with increasing culture time. On days 7, 14, and 21 of culture, the number of SS receptors amounted to 978 +/- 217, 3588 +/- 705, and 5865 +/- 3332 fmol/mg protein, respectively, whereas they were not measurable on day 0. From days 0-7, 7-14, and 14-21 of culture, octreotide (1 pM to 1 microM) inhibited PRL release and the intracellular PRL concentration, with IC50 values in the nanomolar range. However, no inhibition of cell growth was observed by these octreotide concentrations from day 0-7 of culture, while octreotide inhibited cell growth in a dose-dependent fashion from days 7-14 and 14-21 of culture (maximal inhibition by 25% and 26%, respectively). In a series of nine consecutive experiments we found a significant positive correlation between the percent inhibition of cell growth induced by 1 microM octreotide and the number of SS receptors on 7315b cells (r = 0.7865; P = 0.012). Inhibition of PRL release did not correlate with SS receptor numbers. Octreotide (1 microM) inhibited forskolin (0.5 microM)-stimulated cell growth and intracellular PRL concentrations, while in the presence of a high concentration of forskolin (10 microM), octreotide had no effect on forskolin-stimulated cell growth and intracellular PRL concentrations. In addition, its PRL release inhibitory effect was significantly lower in forskolin-stimulated cultures. Pretreatment of the cells with pertussis toxin (10 micrograms/liter) completely prevented the inhibition of cell growth by octreotide and diminished the inhibitory effect of octreotide on PRL release. Finally, 1 microM octreotide significantly inhibited forskolin-stimulated cAMP production (by 29% and 53% on days 7 and 14 of culture, respectively). We conclude that 1) octreotide inhibits 7315b rat pituitary tumor cell proliferation via a pertussis toxin-sensitive GTP-binding protein- and adenylate cyclase-dependent mechanism; and 2) the number of SS receptors on 7315b pituitary tumor cells may determine whether octreotide exerts a direct antiproliferative effect, whereas its antihormonal effect occurs in the presence of relatively low numbers of SS receptors. This suggests a dissociation of the antiproliferative and antihormonal effects induced by octreotide.
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PMID:Dissociation of antiproliferative and antihormonal effects of the somatostatin analog octreotide on 7315b pituitary tumor cells. 132 74

When applied to rat anterior pituitary cells, angiotensin-II (AII) exerted two opposite effects on adenylate cyclase (AC) activity: a pertussis toxin (PTX)-sensitive inhibition of the enzyme with a maximal effect of -42 +/- 2% in crude cell membrane preparations, and, in contrast, a non-PTX-sensitive stimulation of cAMP production (maximal effect = 38 +/- 3%) in intact cells. The apparent affinity of both effects was equal to 1.8 nM. The stimulation of cAMP formation parallels the stimulation of PRL release. Under the same conditions, dopamine (DA) inhibited both membrane AC activity and cAMP formation in intact cells by a PTX-sensitive mechanism. After separation of pituitary cell types by sedimentation at unit gravity, the effects of AII and DA on intracellular cAMP and membrane AC activity coincided in the same fractions (those enriched in PRL cells). The stimulatory effect of AII on cAMP formation was about 5 times weaker than that of peptides positively coupled to AC as vasoactive intestinal peptide in total as well as in PRL-enriched cells. Since the AII receptor is also coupled to phospholipase-C (PLC) in a non-PTX-sensitive manner, we investigated whether protein kinase-C (PKC) could indirectly account for the positive effect of AII on cAMP formation. 12-O-Tetradecanolylphorbol 13-acetate (TPA), a stimulator of PKC was indeed able to increase intracellular cAMP; this effect was not additive with that of AII. conversely, application of the PKC inhibitors H7 [1-(5-isoquinolylsulfonyl)2-methyl-piperazine] and staurosporine or desensitization of PKC by long exposure of the cells to TPA abolished the cAMP response to TPA as well as that to AII. In addition, thyreoliberin, another activator of the PLC pathway, was able to stimulate cAMP formation in a PKC-dependent manner. DA inhibition of intracellular cAMP was not affected by any PKC inhibition. We conclude that in lactotroph cells, 1) the AII inhibitory coupling to AC observed in membrane preparations does not exist in intact cells, at least under basal conditions; and 2) the AII intracellular cAMP stimulation observed is not accounted for by a direct coupling with AC; it is due to a cross-talk of the PLC pathway mediated by PKC, an effect that might be shared by other PLC-stimulating mediators and may participate in the regulation of PRL release.
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PMID:Involvement of protein kinase-C in the effect of angiotensin-II on adenosine 3',5'-monophosphate production in lactotroph cells. 165 95

In the rat pituitary cell line GH3, carbachol inhibits PRL secretion in a pertussis toxin-sensitive manner. For elucidation of the underlying mechanisms, we studied the effect of carbachol on voltage-dependent Ca2+ currents. Under voltage-clamp conditions, carbachol inhibited whole-cell Ca2+ currents by about 25%. This inhibitory action of carbachol was not observed in cells treated with pertussis toxin, indicating the involvement of a pertussis toxin-sensitive G-protein. In membranes of GH3 cells, carbachol stimulated a pertussis toxin-sensitive high-affinity GTPase. In immunoblot experiments with peptide antisera, we identified two forms of the Gi alpha-subunit (41 and 40 kDa) and two forms of the Go alpha-subunit (40 and 39 kDa). The 40-kDa Gi alpha-subunit was recognized by an antibody specific for the Gi2 alpha-subunit, and the 39-kDa Go alpha-subunit was detected by an antibody specific for the Go2 alpha-subunit. Incubation of membranes with the photoreactive GTP analog [alpha-32P]GTP azidoanilide resulted in photo-labelling of 40- and 39-kDa pertussis toxin substrates comigrating with G-protein alpha-subunits of the corresponding molecular masses. Carbachol dose-dependently stimulated incorporation of the photoreactive GTP analog into the 39-kDa pertussis toxin substrate and, to a lesser extent, into 40-kDa pertussis toxin substrates. The data indicate that muscarinic receptors of GH3 cells couple preferentially to Go, which is likely to be involved in the inhibition of secretion, possibly by conferring an inhibitory effect to voltage-dependent Ca2+ channels.
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PMID:Inhibition of voltage-dependent Ca2+ currents and activation of pertussis toxin-sensitive G-proteins via muscarinic receptors in GH3 cells. 165 34

The reverse hemolytic plaque assay (RHPA) was used in this study to further characterize the mechanism whereby low concentrations of dopamine (DA) stimulate PRL secretion in vitro. Female Sprague-Dawley rats were used as a source of anterior pituitary cells for the RHPA. Pituitary cells were infused into Cunningham chambers along with a suspension of protein-A-coated ovine red blood cells. Excess cells were rinsed from the chambers leaving a monolayer of cells attached to the glass. The cells were then incubated with solutions containing PRL antiserum (1:40) and various concentrations of DA. After 4 h, a solution containing guinea pig complement (1:60) was infused into the chambers. Thirty minutes later, the cells were fixed and plaques (zones of hemolysis) surrounding PRL-producing cells (lactotrophs) were measured and used as an index of the amount of PRL secreted. Control cells that received no DA had a mean plaque area of 8,000 microns 2 and two distinct subpopulations of plaque sizes. This biphasic population of cells consisted of a small and a large plaque producing population. The mean plaque area surrounding lactotrophs was significantly (P less than 0.05) decreased if 1 microM or 10 microM DA was present (4,500 microns 2 and 3,500 microns 2, respectively). These cells which received inhibitory concentrations of DA demonstrated a monophasic distribution of plaque-forming cells. On the other hand, mean plaque area was significantly (P less than 0.05) increased if 0.1 nM or 1 nM DA was presented to the cells (15,000 microns 2 and 14,500 microns 2, respectively). These cells receiving stimulatory doses of DA exhibited a multiphasic distribution of plaque-forming cells. The possibility that the two physiological opposing actions of DA on PRL secretion might be mediated by different GTP binding proteins was also examined using cholera toxin (CTX) and pertussis toxin (PTX). Anterior pituitary cells were pretreated with either CTX (50 micrograms/ml) or PTX (5 micrograms/ml) for 1 h before initiation of the RHPA. In the RHPA, cells received no DA, a stimulatory dose of DA (0.1 nM), or a inhibitory dose of DA (10 microM). The effects of toxin pretreatment on mean plaque area of DA-treated cells was determined. PTX pretreatment significantly attenuated the inhibitory effects of DA while having no effect on the stimulatory effects of DA on PRL secretion. CTX significantly (P less than 0.05) potentiated the stimulatory effects of DA on PRL secretion and had no effect on inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The stimulatory and inhibitory effects of dopamine on prolactin secretion involve different G-proteins. 173 34

Different bacterial toxins capable of modifying specific alpha-subunits of G-proteins were used to characterize the guanine nucleotide-binding protein (G-protein) dependency of the effects of endothelins (ETs) on PRL, LH, and FSH secretion. Primary cultures of anterior pituitary cells obtained from female rats were preincubated for 24 h with 20 ng/ml pertussis toxin (PTX) or 2 micrograms/ml cholera toxin (CTX) before challenge with ETs. Both ET-1 and ET-3 elicited a concentration-dependent inhibition of PRL secretion and stimulated the release of LH and FSH secretion on pituitary cells not treated with toxins. Based on the calculated ration of the half-maximal effective concentrations (EC50) of ET-1 and ET-3, ET-1 showed 7800, 20, and 14 times greater potency than ET-3 on PRL, LH, and FSH secretion, respectively. PTX, a selective inhibitor of Gi and several other G proteins, increased the basal secretion of PRL and completely eliminated the responsiveness of lactotroph cells to ET-1 and ET-3. Pretreatment with PTX caused a markedly different effect on LH and FSH secretion: while basal LH release was slightly increased, FSH secretion was markedly depressed by PTX. Moreover, while ET-induced LH secretion was enhanced by PTX, the effectiveness of ETs on FSH release was completely abolished. CTX, known as an activator of Gs proteins, decreased the basal secretory activity of lactotrophs but did not influence the ET-induced decrease of PRL release. CTX pretreatment (like PTX before) elicited a strikingly different effect on LH and FSH: while basal LH secretion was enhanced, basal FSH secretion was markedly inhibited by CTX. Moreover, while the effectiveness of ETs on LH secretion was not changed significantly, the stimulatory effect of ETs on FSH secretion was diminished after CTX pretreatment. Thus, the inhibition of PRL secretion by ETs requires a PTX-sensitive G protein while the ET-induced stimulation of FSH secretion involves both PTX- and CTX-sensitive elements. The fact that pretreatments with PTX or CTX influenced basal secretion of PRL, LH, and FSH suggests that PTX- and/or CTX-sensitive G proteins are directly involved in the process of exocytosis. Additionally, these findings might indicate an active paracrine/autocrine regulation of pituitary cells in culture that are impaired or enhanced by the bacterial toxins employed. Though the broad substrate specificity of PTX and CTX and the multiplicity of G protein families did not allow us to identify the specific G protein(s) involved, these data reveal the diversity of ET-induced intracellular signaling mechanisms in lactotrophs and gonadotrophs.
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PMID:The effects of endothelins on the secretion of prolactin, luteinizing hormone, and follicle-stimulating hormone are mediated by different guanine nucleotide-binding proteins. 193 91

The effect of TRH on cytosolic free calcium concentrations, [Ca2+]i, was evaluated on cell suspensions obtained from 6 human PRL secreting pituitary adenomas. In these cells resting [Ca2+]i levels were variable (mean +/- SE; 103.8 +/- 6.5, n = 25); the addition of 100 nM TRH caused a marked [Ca2+]i rise within 20 sec., the peak values ranging from 200 to 437 nM (285 +/- 10.8 nM, n = 10). The transients induced by TRH were composed by a rapid increase, due to mobilization of calcium from intracellular stores, followed within a few seconds by a lower plateau which was due to stimulated influx from the extracellular space. In fact, when EGTA and verapamil were applied after TRH they caused the Ca2+ plateau to dissipate rapidly. The addition of 1 microM dopamine (DA) caused a substantial decrease of resting [Ca2+]i (about 10-30%) as well as an inhibition of the plateau phase induced by TRH. The effect of DA completely depended on extracellular Ca2+. The TRH-induced transients observed in adenomatous cells were quite similar in size and time course to those recorded in normal rat lactotrophs. As previously observed in rat lactotrophs, in adenomatous cells treatment with pertussis toxin (PTx, 1 microgram/ml for 4 h) was unable to affect the [Ca2+]i transients induced by TRH while completely abolished the effect of DA. The effects of TRH on in vivo and in vitro PRL secretion were also evaluated. Before surgery, no patient showed a positive response to the iv administration of 200 micrograms TRH (serum PRL levels: 95 +/- 62 ng/ml in basal conditions vs 124 +/- 92 after TRH, P = NS).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:TRH raises cytosolic Ca2+ in human adenomatous lactotrophs. 210 97

The expression of guanine nucleotide-binding proteins (G-proteins) was compared in two clonal lines of rat Nb2 node lymphoma cells, the lactogen-dependent Nb2-11C line and the lactogen-independent Nb2-Sp (spontaneous) line. Both cell lines expressed mRNA transcripts for the G-protein species Gs alpha [1.85 kilobases (kb)], Gi2 alpha (2.35 kb), Go alpha (4.1-4.5 kb), and Gi3 alpha (3.5 kb). Gi1 alpha was not detected. ADP ribosylation in the presence of activated cholera or pertussis toxins and [32P]NAD demonstrated the presence of G-proteins in the membrane fractions of both lines. The cholera toxin substrates consisted of two proteins (mol wt, 46.5 and 43.5 kD), while a single protein (mol wt, 41.5 kD) was ADP ribosylated by pertussis toxin. Surprisingly, the cholera toxin-sensitive proteins (Gs) were at least 20-fold less abundant in the Nb2-Sp cells than in the Nb2-11C cells. Since Gs and Gi2 are associated with the adenylate cyclase system and the regulation of intracellular cAMP, the effects of the cAMP analog, (Bu)2cAMP (dbcAMP), on Nb2-11C and Nb2-Sp cell growth were examined. dbcAMP (100 microM) completely inhibited the growth of lactogen-dependent Nb2-11C cells. The inhibitory effect of dbcAMP was exerted at an early point in the cell cycle, as it also inhibited PRL-stimulated c-myc expression measured 3 h after addition of the mitogen. In contrast, dbcAMP had only minor inhibitory effects on lactogen-independent Nb2-Sp cells, increasing their doubling time from 20 to 30 h and slightly reducing their density at confluence. The inhibitory effect of dbcAMP on both cell lines was reversible. Nb2-11C cells resumed growth after a lag period of approximately 3 days. The recovered cells did not arise from selection of a cAMP-resistant subpopulation, since both they and normal untreated Nb2-11C cells remained equally sensitive to dbcAMP. Similarly, Nb2-Sp cells resumed their normal doubling time upon removal of dbcAMP. The observation that the lactogen-independent Nb2-Sp cell line contained 20-fold less cholera toxin-sensitive Gs protein provides circumstantial evidence that dysfunction of the adenylate cyclase system may be implicated in the autonomous growth of these cells. This possibility is strengthened by the observation that Nb2-Sp cells are markedly less sensitive than the Nb2-11C clone to the growth inhibitory effects of an exogenous cAMP analog.
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PMID:The role of G-proteins in the mitogenesis of rat lactogen-dependent and lactogen-independent Nb2 lymphoma cells. 215 99

The effects of dopamine (DA) on voltage-dependent Ca2+ currents were investigated in cultured rat lactotroph cells using the patch clamp recording technique. Each recorded cell was identified by the reverse hemolytic plaque assay. In the whole-cell configuration, two types of Ca2+ currents, L and T, were characterized on the basis of their kinetics, voltage sensitivity, and pharmacology. The L component had a threshold of -25 mV, showed little inactivation during a 150-msec voltage step, and was maximal at +10 mV. Cadmium ions (100 microM) significantly reduced its amplitude (75%). The T component was activated at a membrane potential close to -50 mV, was maximal at -10 mV, and showed a voltage-dependent inactivation between -90 and -30 mV. It was quickly inactivated during a maintained depolarization (time constant, 27 ms at -30 mV) and was strongly reduced (80%) by nickel ions (100 microM). Bath application of DA (10 nM) caused a markedly general depression of inward Ca2+ currents, acting differently on the T- and L-type currents. DA application shifted the voltage-dependence of the L-type current activation toward depolarization values (8 mV) without modifying its time- and voltage-dependent inactivation. In contrast, DA enhanced the inactivation of the T-type current by accelerating its time-dependent inactivation (25% decrease in the time constant of inactivation) and by shifting the voltage-dependence of the T-type current inactivation toward hyperpolarizing values (-63 mV in control vs. -77 mV in the presence of DA). These effects of DA were dose-dependent and involved the activation of a D2 receptor type. They were mimicked by bromocriptine application (10 nM), whereas sulpiride (100 nM) blocked the DA-evoked response. The D1 antagonist SCH 23390 was ineffective up to 100 microM. All of these DA-induced modifications in Ca2+ currents were abolished using a GTP-free pipette solution or after pretreatment of cells with pertussis toxin, suggesting that DA can regulate the function of Ca2+ channels through GTP-binding proteins (G-proteins). Our results show that DA acts simultaneously by reducing both voltage-dependent Ca2+ currents on lactotroph cells. Thus, DA reduces the entry of Ca2+ ions across the surface membrane and thereby influences electrical activity and the cytosolic free Ca2+ concentration involved in both basal and evoked PRL release.
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PMID:Dopamine inhibits two characterized voltage-dependent calcium currents in identified rat lactotroph cells. 216 20

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.
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PMID:Dopamine inhibits prolactin secretion stimulated by the calcium channel agonist Bay-K-8644 through a pertussis toxin-sensitive G protein in anterior pituitary cells. 245 6

The hypothalamic peptide somatostatin (SRIF) suppresses secretory activity in phenotypically distinct pituitary endocrine cells. We have used tight-seal whole-cell recording techniques to study the peptide's effects on the electrical properties of tumor pituitary cells derived from rat (GH3/B6) and human adenomas that secrete human PRL in a SRIF-sensitive manner. Both cell types exhibited qualitatively similar electrophysiological properties and electrical responses to SRIF. Under the experimental conditions employed the majority of cells spontaneously generated Ca2+-dependent actions potentials. The actions of the peptide on cellular excitability were markedly affected by the presence of horse and fetal calf sera. Without these additives the electrical responses faded and could not be studied in detail. Therefore, recordings were conducted in media containing sera. In the presence of sera almost all cells spontaneously generated Ca2+ action potentials, and peptide-induced changes in excitability were well preserved. SRIF depressed spontaneous and evoked action potential activity in a dose-dependent manner at concentrations that reduced intracellular free calcium ([Ca2+]i) and suppressed basal PRL release. Current and voltage clamp experiments revealed coordinate actions of the peptide on excitable membrane properties. SRIF (1 nM) enhanced a depolarization-activated, rapidly inactivating outward K+ current, thereby effectively reducing the rate at which action potentials occurred. Over the 10-1000 nM range SRIF slowly activated a virtually noninactivating K+ conductance over a wide range of membrane potential. This effectively hyperpolarized cells away from the threshold for triggering Ca2+-dependent action potentials and shunted the membrane. The peptide induced K+ conductance activated at the level of the resting potential was progressively lost during the intracellular dialysis of whole-cell recording. Dilute aqueous lysates of cells included in the patch pipette prevented much of the rundown of this SRIF-induced electrical response while inclusion of an ATP-regenerating system preserved some of the peptide action. Over the 10-100 nM concentration range SRIF also reduced voltage-dependent Ca2+ current. Furthermore, pretreatment of cells with pertussis toxin abolished SRIF action on cellular excitability, suggesting that SRIF can regulate the function of ionic channels through GTP-binding proteins (G proteins). The results demonstrate that SRIF acts coordinately on the primary conductances expressed in tumor PRL cells to attenuate or block Ca2+ action potential generation and thus Ga2+ entry from extracellular sources.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Somatostatin blocks Ca2+ action potential activity in prolactin-secreting pituitary tumor cells through coordinate actions on K+ and Ca2+ conductances. 245 3


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