tetrodotoxin and Pituitary-Neoplasms

1. Cells derived from a rat pituitary tumour (GC cell line) that continuously release growth hormone behave as endogenous pacemakers. In simultaneous patch clamp recordings and cytosolic Ca2+ concentration ([Ca2+]i) imaging, they displayed rhythmic action potentials (44.7 +/- 2.7 mV, 178 +/- 40 ms, 0.30 +/- 0.04 Hz) and concomitant [Ca2+]i transients (374 +/- 57 nM, 1.0 +/- 0.2 s, 0.27 +/- 0.03 Hz). 2. Action potentials and [Ca2+]i transients were reversibly blocked by removal of external Ca2+, addition of nifedipine (1 microM) or Ni2+ (40 microM), but were insensitive to TTX (1 microM). An L-type Ca2+ current activated at -33.6 +/- 0.4 mV (holding potential (Vh), -40 mV), peaked at -1.8 +/- 1.3 mV, was reduced by nifedipine and enhanced by S-(+)-SDZ 202 791. A T/R-type Ca2+ current activated at -41.7 +/- 2.7 mV (Vh, -80 or -60 mV), peaked at -9.2 +/- 3.0 mV, was reduced by low concentrations of Ni2+ (40 microM) or Cd2+ (10 microM) and was toxin resistant. Parallel experiments revealed the expression of the class E calcium channel alpha1-subunit mRNA. 3. The K+ channel blockers TEA (25 mM) and charybdotoxin (10-100 nM) enhanced spike amplitude and/or duration. Apamin (100 nM) also strongly reduced the after-spike hyperpolarization. The outward K+ tail current evoked by a depolarizing step that mimicked an action potential reversed at -69. 8 +/- 0.3 mV, presented two components, lasted 2-3 s and was totally blocked by Cd2+ (400 microM). 4. The slow pacemaker depolarization (3.5 +/- 0.4 s) that separated consecutive spikes corresponded to a 2- to 3-fold increase in membrane resistance, was strongly Na+ sensitive but TTX insensitive. 5. Computer simulations showed that pacemaker activity can be reproduced by a minimum of six currents: an L-type Ca2+ current underlies the rising phase of action potentials that are repolarized by a delayed rectifier and Ca2+-activated K+ currents. In between spikes, the decay of Ca2+-activated K+ currents and a persistent inward cationic current depolarize the membrane, activate the T/R-type Ca2+ current and initiate a new cycle.

Topics: Action Potentials; Animals; Antisense Elements (Genetics); Apamin; Barium; Biological Clocks; Cadmium; Calcium; Calcium Channel Blockers; Calcium Channels; Charybdotoxin; Computer Simulation; Cytosol; Dihydropyridines; Growth Hormone; Growth Hormone-Releasing Hormone; Nickel; Nifedipine; omega-Agatoxin IVA; omega-Conotoxin GVIA; Patch-Clamp Techniques; Peptides; Pituitary Neoplasms; Potassium; Rats; Ryanodine Receptor Calcium Release Channel; Sodium; Spider Venoms; Tetraethylammonium; Tetrodotoxin; Tumor Cells, Cultured

Sapecin B is structurally homologous to charybdotoxin (CTX), which is found in scorpion venom. This study investigated the effects of sapecin B on the Ca(2+)-activated K+ currents [IK(Ca)] and the rapidly inactivating K+ currents in clonal rat GH3 pituitary cells with whole cell voltage-clamp methods. Sapecin B (20 nM) reversibly blocked the CTX-sensitive Ix(Ca) (the BK current) in a dose-dependent manner, with a half-maximal inhibitory concentration of approximately 0.9 nM, comparable to that of 0.08-0.4 nM for CTX. The Ca2+ currents in GH3 cells, however, were not affected by sapecin B (40 nM), indicating that the blockade of IK(Ca) by sapecin B is not a secondary effect of Ca2+ current inhibition. The effect of sapecin B on IK(Ca) resembled that of CTX, as expected from the structural similarities shared by CTX and sapecin B. We also found that sapecin B largely inhibited the 4-aminopyridine-sensitive, rapidly inactivating K+ currents in a dose-dependent manner, with a half-maximal inhibitory concentration of approximately 40 nM, whereas CTX had little effect on this current in GH3 cells. Sapecin B may thus provide a useful tool, complementary to CTX, for probing the functional role of molecular domains in the BK channels and the structural similarities common to the BK and the rapidly inactivating A-type K+ channels.

Topics: 4-Aminopyridine; Animals; Charybdotoxin; Diptera; Insect Proteins; Kinetics; Large-Conductance Calcium-Activated Potassium Channels; Patch-Clamp Techniques; Pituitary Gland, Anterior; Pituitary Neoplasms; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Calcium-Activated; Rats; Scorpion Venoms; Tetrodotoxin; Time Factors; Tumor Cells, Cultured

We have evaluated the inhibitory effect of dopamine on PRL secretion induced by blocking K+ channels. Tumor-derived GH4C1 cells and collagenase-dispersed normal anterior pituitary (AP) cells from young adult male rats were perifused with Krebs-Ringer Hepes medium. In both cell types blocking K+ channels with tetraethylammonium (TEA) induced PRL secretion but did not stimulate cyclic AMP generation. Blocking Na+ channels with 1 microM tetrodotoxin had no effect on basal or TEA-induced PRL secretion. Dopamine inhibited the TEA-induced rise in [Ca2+]i in GH4C1 cells expressing dopamine D2 short receptors. In normal AP cells, 1-100 nM dopamine blocked PRL secretion induced by 20 mM TEA in a log-linear concentration-dependent fashion, with a plateau at > 100 nM dopamine (IC50 30 nM). The D2 dopaminergic receptor agonist, quinpirole, at 100 nM completely blocked PRL secretion induced by 20 mM TEA. The D2 dopaminergic receptor antagonist, sulpiride, at 10 microM reversed the inhibitory effect of 10 microM dopamine on PRL secretion induced by 20 mM TEA. Pretreatment of cells with 100 ng/ml pertussis toxin (PTX) for 24 h prevented 100 nM dopamine inhibition of PRL secretion induced by 20 mM TEA. The data indicate that in both normal lactotroph cells and in tumor-derived cells expressing D2 receptors, PRL secretion stimulated by blocking K+ channels is inhibited by dopamine binding to D2 receptors on the plasma membrane. This inhibition involves interaction with PTX-sensitive Gi protein.

Topics: Adenylyl Cyclases; Animals; Cells, Cultured; Dopamine; Male; Pertussis Toxin; Pituitary Gland, Anterior; Pituitary Neoplasms; Potassium Channels; Prolactin; Rats; Rats, Sprague-Dawley; Receptors, Dopamine D2; Sodium Channels; Tetraethylammonium; Tetraethylammonium Compounds; Tetrodotoxin; Tumor Cells, Cultured; Virulence Factors, Bordetella

Electrophysiology of human growth hormone secreting tumour cells and its modification by hGRF has been studied using on-cell and Nystatin-perforated whole-cell recording configurations. Local application of hGRF (10 nM) produced an increase in the frequency of action potentials. Ca2+ currents were isolated by a ramp depolarizing pulse from -120 mV to +60 mV in the presence of tetrodotoxin (1 microM). Human GRF increased the Ca2+ currents which could be blocked by Ni+ (300 microM). We conclude that an increase in Ca2+ current is integral to the action of hGRF on these cells.

Topics: Action Potentials; Adenoma; Calcium Channels; Growth Hormone; Growth Hormone-Releasing Hormone; Humans; Membrane Potentials; Pituitary Neoplasms; Sodium Channels; Tetrodotoxin; Tumor Cells, Cultured

The mechanism of the inhibitory effect of local anesthetics on hormone secretion was studied in the GH4C1 line of rat pituitary tumor-derived cells. Lidocaine between 0.1 and 5 mM exerted significant dose-dependent inhibition on the increment in cytosol Ca2+ concentration ([Ca2+]i) and prolactin (PRL) secretion induced by 30 mM K+. For both effects the IC50 was 0.25 mM and maximal inhibition occurred at 5 mM. A normal response returned within 20 min after removal of lidocaine from the incubation medium. 1 microM tetrodotoxin had no effect on the 30 mM K+ induced [Ca2+]i transient or PRL secretion, indicating that Na+ channels are not involved in the inhibitory effect of lidocaine. Lidocaine similarly inhibited the [Ca2+]i increment and PRL secretion induced by 30% medium hyposmolarity and 1 microM Bay K 8644. Lidocaine was much less effective in inhibiting secretion induced by 1 microM phorbol 12-myristate 13-acetate (TPA) or 5 microM forskolin. 5 mM procaine produced effects similar to those of lidocaine. Our data suggest that in GH4C1 cells local anesthetics depress secretagogue-induced PRL secretion primarily by blocking Ca2+ influx, probably through L-type Ca2+ channels.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adenoma; Animals; Calcium; Colforsin; Lidocaine; Pituitary Neoplasms; Potassium; Procaine; Prolactin; Rats; Tetradecanoylphorbol Acetate; Tetrodotoxin; Tumor Cells, Cultured

The role of ion channels in the secretion of gonadotropins from anterior pituitary gonadotropes has been difficult to study at the single cell level because the cells are difficult to distinguish from other pituitary cell types. Recently, a cell line, alpha T3-1, has been generated that makes and secretes the alpha-subunit of gonadotropins. These cells have GnRH receptors, but not TRH receptors, and are, thus, specific to the gonadotrope lineage. We have used the patch clamp technique to investigate the types of ion channels expressed in alpha T3-1 cells and to test for electrophysiological responses to GnRH and a phorbol ester. These cells express TTX-sensitive sodium channels with rapid kinetics, several types of potassium channels, including Ca2(+)-sensitive ones, and two types of calcium channels. The currents through calcium channels are augmented by application of 100 nM GnRH or 10 nM phorbol 12-myristate 13-acetate, a phorbol ester. The augmentation by GnRH and phorbol 12-myristate 13-acetate is consistent with other reports that a portion of stimulated gonadotropin release is dependent on external calcium and sensitive to block by dihydropyridine antagonists. Thus, this cell line may be useful for studies of mechanisms underlying responses to GnRH.

Topics: Animals; Calcium Channels; Cell Line; Electrophysiology; Gonadotropin-Releasing Hormone; Membrane Potentials; Pituitary Neoplasms; Potassium Channels; Rats; Sodium Channels; Tetradecanoylphorbol Acetate; Tetrodotoxin; Thyrotropin-Releasing Hormone

Spontaneous and CRF-stimulated changes in the cytosolic free calcium concentration ([Ca2+]i) were studied in two types of corticotrophs: 1) cultured human ACTH-secreting pituitary adenoma cells (hACTH cells), and 2) identified small ovoid corticotrophs cultured from normal rat pituitaries. [Ca2+]i was monitored in individual corticotrophs by dual emission microspectrofluorimetry using indo-1 as the intracellular fluorescent Ca2+ probe. In hACTH cells, [Ca2+]i measurements were carried out in combination with electrophysiological recordings obtained using whole cell patch-clamp techniques. It was shown that a single spontaneous Ca(2+)-dependent action potential led to a marked transient increase in [Ca2+]i in human tumoral corticotrophs. Spontaneous fluctuations in [Ca2+]i were also observed in unpatched corticotrophs whether derived from human pituitary tumors or normal rat tissue. Based on their striking kinetic features and their sensitivity to external Ca2+, we suggest that these spontaneous [Ca2+]i transients were the consequence of action potential firing. Under separate voltage-clamp (patch-clamp) conditions, tumor corticotrophs showed two Ca2+ current components: a low threshold, rapidly inactivating (T-type) current, and a higher threshold, slowly inactivating (L-type) current. The dihydropyridine Ca2+ channel blocker PN 200-110 (100 nM) abolished the L-type current without affecting the T-type current, while the inorganic Ca2+ channel blocker Cd2+ (200 microM) suppressed both Ca2+ currents. The Na+ channel blocker tetrodotoxin (5 microM) did not affect inward currents in tumor corticotrophs. Both L- and T-type voltage-gated Ca2+ channels were involved in controlling [Ca2+]i transients in both tumor and normal corticotrophs, inasmuch as Cd2+ (200 microM) abolished [Ca2+]i) transients, while PN 200-110 (100 nM) greatly diminished, but did not completely abolish, [Ca2+]i transients. The latter did not appear to depend on a voltage-dependent Na+ influx, since they were unaffected by tetrodotoxin (5 microM). Corticotrophs generate [Ca2+]i transients in response to the hypothalamic secretagogue CRF by acting on their membrane excitability. Indeed, we demonstrated in combined fluorescent and electrophysiological experiments that CRF (100 nM) had a coordinate action on human tumoral corticotrophs comprised of a modest depolarization and an increase in the frequency of both action potentials and subsequent [Ca2+]i transients. A coincident increase in the

Topics: Action Potentials; Adenoma; Adrenocorticotropic Hormone; Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Corticotropin-Releasing Hormone; Cytosol; Egtazic Acid; Female; Humans; Isradipine; Membrane Potentials; Oxadiazoles; Pituitary Gland; Pituitary Neoplasms; Rats; Rats, Inbred Strains; Spectrometry, Fluorescence; Tetrodotoxin; Tumor Cells, Cultured

The sensitivity of voltage-dependent sodium current to the sodium channel blocker tetrodotoxin (TTX) is altered by transfection of a c-Ha-ras oncogene into an excitable cell line. Control AtT-20 cells, a cell line derived from a mouse anterior pituitary tumor, were found to express both a TTX-sensitive and a TTX-resistant sodium current. AtT-20 cells transfected with the c-Ha-ras gene expressed only a TTX-sensitive current. Properties of TTX-sensitive and -resistant currents were also examined. No differences in voltage dependence of activation or inactivation between the TTX-sensitive and -resistant currents were observed. The rate of inactivation of the TTX-resistant current in control cells was slower, than that of the TTX-sensitive current in either control or ras-transfected AtT-20 cells.

Topics: Dose-Response Relationship, Drug; Drug Resistance; Electric Conductivity; Genes, ras; Humans; Pituitary Gland, Anterior; Pituitary Neoplasms; Sodium; Tetrodotoxin; Transfection; Tumor Cells, Cultured

The mechanisms by which an activator of cyclic AMP-dependent protein kinase, corticotropin releasing factor (CRF) and the protein kinase C stimulant, phorbol myristate acetate (PMA) regulate the level of intracellular free calcium in the mouse anterior pituitary cell line AtT-20 were examined using the fluorescence probe Quin 2. The increase in cytosolic calcium in AtT-20 cells induced by CRF and PMA was blocked by calcium channel antagonists indicating that both agents stimulate calcium influx. The ability of PMA to raise cytosolic calcium levels was prevented by the sodium channel antagonist tetrodotoxin, suggesting that phorbol esters depolarize the cell membrane or increase action potential frequency to enhance calcium influx. The K+ channel antagonists, tetraethylammonium, cesium and 4-aminopyridine, inhibited PMA-stimulated calcium influx in AtT-20 cells. Thus, one mechanism by which protein kinase C activation may lead to a depolarization of the cell membrane is through a reduction in K+ currents. In contrast, neither tetraethylammonium or cesium reduced CRF-stimulated calcium influx into AtT-20 cells. The stimulation of calcium influx by CRF, therefore, appears to not involve changes in K+ currents in AtT-20 cells. CRF activates cyclic AMP-dependent protein kinase to stimulate calcium influx either by facilitating calcium conductance directly or by modifying the membrane potential or firing activity of AtT-20 cells.

Topics: Adrenocorticotropic Hormone; Animals; Calcium; Corticotropin-Releasing Hormone; Cyclic AMP; Mice; Pituitary Gland, Anterior; Pituitary Neoplasms; Potassium Channels; Tetradecanoylphorbol Acetate; Tetraethylammonium Compounds; Tetrodotoxin; Tumor Cells, Cultured

1. Voltage-clamp recordings were made from cultured AtT-20 pituitary cells using the whole-cell patch-clamp technique. Cells were perfused internally with Cs+ to block K+ currents and bathed externally with either 1 microM tetrodotoxin or with tetraethylammonium (TEA) as a Na+ substitute to block voltage-activated Na+ currents. 2. Depolarizing voltage steps from a holding potential of -80 mV to potentials positive to -30 mV evoked two currents: a fast inward current that activated between -30 and +70 mV and a slowly activating current (designated "slow step current") that was inward between -30 and near 0 mV (the Cl- equilibrium potential) and outward positive to about 0 mV. Repolarization to -80 mV revealed a slowly decaying, inward tail current, whose magnitude with respect to step potential closely matched the current-voltage relationship of the voltage-activated Ca2+ current. 3. Activation of the fast inward current, slow step current, and tail current, was prevented by extracellular application of Cd2+ or removal of extracellular Ca2+. Replacement of extracellular Ca2+ with Ba2+ potentiated the fast inward current but blocked the slow step and tail currents. Intracellular perfusion with greater than 1 mM of the Ca2+ chelators ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) or [1,2-bis(2)aminophenoxy]ethane N,N,N',N'-tetraacetic acid (BAPTA) prevented activation of the slow step and tail currents, but not the fast inward current. 4. The reversal potential of the slow inward current was sensitive to changes in the Cl- equilibrium potential but not to substitution of TEA for Na+. The slow step current, but not the fast inward current, was partially blocked by the Cl- channel blocker, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. 5. These data indicate that both the slow inward tail current and the slowly activating, reversible step current were a Ca2+-dependent Cl- current, similar to that described in other neuronal and nonneuronal cell types. The fast inward current was a voltage-activated Ca2+ current, described previously in these and other cells. 6. In the absence of intracellular EGTA, the tail current decayed with complex kinetics, its time course apparently dependent on the magnitude of the voltage-activated Ca2+ current. In the presence of 200 microM intracellular EGTA, the tail current decayed significantly faster and often decayed exponentially.

Topics: Animals; Barium; Calcium; Cell Line; Cesium; Chlorides; Ion Channels; Kinetics; Membrane Potentials; Pituitary Neoplasms; Sodium; Tetraethylammonium; Tetraethylammonium Compounds; Tetrodotoxin

The ionic conductance mechanisms underlying action potential behavior in GH3 and GH4/C1 rat pituitary tumor cell lines were identified and characterized using a patch electrode voltage-clamp technique. Voltage-dependent sodium, calcium, and potassium currents and calcium-activated potassium currents were present in the GH3 cells. GH4/C1 cells possess much less sodium current, less voltage-dependent potassium current, and comparable amounts of calcium current. Voltage-dependent inward sodium current activated and inactivated rapidly and was blocked by tetrodotoxin. A slower-activating voltage-dependent inward calcium current was blocked by cobalt, manganese, nickel, zinc, or cadmium. Barium was substituted for calcium as the inward current carrier. Calcium tail currents decay with two exponential components. The rate constant for the slower component is voltage dependent, while the faster rate constant is independent of voltage. An analysis of tail current envelopes under conditions of controlled ionic gradients suggests that much of the apparent decline of calcium currents arises from an opposing outward current of low cationic selectivity. Voltage-dependent outward potassium current activated rapidly and inactivated slowly. A second outward current, the calcium-activated potassium current, activated slowly and did not appear to reach steady state with 185-ms voltage pulses. This slowly activating outward current is sensitive to external cobalt and cadmium and to the internal concentration of calcium. Tetraethylammonium and 4-aminopyridine block the majority of these outward currents. Our studies reveal a variety of macroscopic ionic currents that could play a role in the initiation and short-term maintenance of hormone secretion, but suggest that sodium channels probably do not make a major contribution.

Topics: Action Potentials; Animals; Biological Transport, Active; Cations; Cell Line; Ion Channels; Pituitary Gland, Anterior; Pituitary Hormones, Anterior; Pituitary Neoplasms; Rats; Tetrodotoxin

Effects of verapamil on membrane electrical properties and prolactin release were studied in a rat anterior pituitary cell line GH3. Thyrotropin-releasing hormone (TRH), Ba2+, and high concentration of K+ enhance the release of prolactin from GH3 cells. These stimulatory actions on prolactin release were inhibited by adding 10(-4) M verapamil to the bathing mediums. The maximum rate of rise of the Ca action potential was reduced to 17% of the control by addition of 10(-4) M verapamil. Ba2+ caused a sustained membrane depolarization because Ba2+ goes through the Ca channels and blocks the development of the delayed rectification. This effect of Ba2+ was also inhibited by verapamil. Verapamil suppressed both the Na+ and outward K+ currents in addition to the Ca2+ current. The suppressive effect of verapamil on the voltage-sensitive Ca current is probably responsible for the inhibition of TRH- and high K+-stimulated prolactin release because the suppression of the Na+ and outward K+ currents does not inhibit the stimulatory actions of these secretagogues.

Topics: Action Potentials; Animals; Calcium; Cell Line; Ion Channels; Kinetics; Pituitary Neoplasms; Prolactin; Rats; Sodium; Tetrodotoxin; Verapamil

The electrical and secretory activities of mouse pituitary tumor cells (AtT-20/D-16v), which contain and release the ACTH/beta-endorphin family of peptides, were studied by means of intracellular recordings and radioimmunoassays. Injection of depolarizing current pulses evoked action potentials in all cells and the majority (82%) displayed spontaneous action potential activity. Action potentials were found to be calcium-dependent. Barium increased membrane resistance, action potential amplitude and duration, and release of ACTH and beta-endorphin immunoactivity. Isoproterenol increased both action potential frequency and hormone secretion. Raising the external calcium concentration increased the frequency and amplitude of the action potentials and stimulated secretion of ACTH and beta-endorphin immunoactivity. Thus, stimulation of secretory activity in AtT-20 cells was closely correlated with increased electrical activity. However, a complete blockade of action potential activity had no effect on basal hormone secretion in these cells. These results suggest that the mechanisms underlying stimulated hormone secretion are different from those responsible for basal secretory activity. It is proposed that the increased influx of calcium due to the increased action potential frequency initiates the stimulated release of hormone from these cells.

Topics: Action Potentials; Adrenocorticotropic Hormone; Animals; Barium; beta-Endorphin; Calcium; Cell Line; Cobalt; Endorphins; Isoproterenol; Mice; Norepinephrine; Pituitary Neoplasms; Tetrodotoxin