dihydropyridines 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

Ca2+ influx via voltage-dependent Ca2+ channels is known to be elicited during action potentials but possibly also occurs at the resting potential. The steady-state current through voltage-dependent Ca2+ channels and its role for the electrical activity was, therefore, investigated in pituitary GH3 cells. Applying the recently developed 'nystatin-modification' of the patch-clamp technique, most GH3 cells (18 out of 23 cells) fired spontaneous action potentials from a baseline membrane potential of 43.7 +/- 4.6 mV (mean +/- s.d., n = 23). The frequency of action potentials was stimulated about twofold by Bay K 8644 (100 nM), a Ca(2+)-channel stimulator, and action potentials were completely suppressed by the Ca(2+)-channel blocker PN 200-110 (100 nM). Voltage clamping GH3 cells at fixed potentials for several minutes and with 1 mM Ba2+ as divalent charge carrier, we observed steady-state Ca(2+)-channel currents that were dihydropyridine-sensitive and displayed a U-shaped current-voltage relation. The results strongly suggest that the observed long lasting, dihydropyridine-sensitive Ca(2+)-channel currents provide a steady-state conductivity for Ca2+ at the resting potential and are essential for the generation of action potentials in GH3 pituitary cells.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Action Potentials; Animals; Calcium Channel Blockers; Calcium Channels; Cell Line; Dihydropyridines; Electric Conductivity; Electrophysiology; Isradipine; Membrane Potentials; Pituitary Neoplasms; Rats

The stimulation of PRL gene transcription by TRH involves the two branches of the phosphatidyl inositol pathway as shown by pharmacological mobilization of intracellular Ca2+ stores and activation of protein kinase C. However, TRH receptor occupancy also results in the activation of voltage-dependent Ca2+ channels. Thus, we attempted to determine whether a specific class of voltage-dependent Ca2+ channels, the dihydropyridine (DHP)-sensitive Ca2+ channels, might also be involved in the transcriptional action of TRH. This was studied in rat pituitary tumor GH3B6 cells by runoff assay and measurement of mRNA levels, using two DHPs, BAY K8644 which increases and PN 200-110 which decreases the influx of Ca2+. We show that the PRL mRNA levels and the rate of PRL gene transcription were stimulated by BAY K8644 and inhibited by PN 200-110 in a dose-dependent manner indicating that DHP-sensitive Ca2+ channels can control the expression of the PRL gene. Furthermore, PN 200-110 abolished the BAY K8644-induced stimulations. By contrast, the stimulations of the PRL gene expression induced by TRH or by the phorbol ester TPA were not abolished by the calcium channel antagonist PN 200-110 whereas treatments combining TRH or TPA with BAY K8644 revealed the absence of any additive effect. Altogether these observations suggest that TRH, and TPA, might activate pathway(s) interacting with those triggered by the Ca2+ channel agonist for regulating PRL gene transcription but they do not support the hypothesis of a necessary implication of DHP-sensitive calcium channels in the regulation of PRL gene transcription by TRH.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium Channel Blockers; Calcium Channels; Dihydropyridines; Gene Expression; Isradipine; Kinetics; Oxadiazoles; Pituitary Neoplasms; Prolactin; Rats; RNA, Messenger; Tetradecanoylphorbol Acetate; Thyrotropin-Releasing Hormone; Tumor Cells, Cultured

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.

Topics: Animals; Calcium; Dihydropyridines; Ion Channels; Marine Toxins; Nicardipine; Oxocins; Pertussis Toxin; Pituitary Neoplasms; Prolactin; Tumor Cells, Cultured; Verapamil; Virulence Factors, Bordetella

PRL synthesis by GH cells in culture has previously been shown to increase when calcium is added to cultures grown in calcium-depleted medium or when cultures are treated for 18 h or longer with the dihydropyridine calcium channel agonist BAY K8644, whereas the antagonist nimodipine inhibits PRL. The experiments described here were designed to test whether differences in PRL synthesis caused by the dihydropyridines are due to changes in PRL mRNA levels, whether structurally different classes of calcium channel blockers alter PRL production, and whether long term treatment with calcium channel agonists and antagonists alters intracellular free calcium, [Ca2+]i. PRL synthesis and PRL mRNA levels were increased similarly by BAY K8644 and decreased in parallel by the dihydropyridine antagonist nimodipine, while overall protein and RNA synthesis were not changed by either the agonist or antagonist. Two calcium channel blockers which act at different sites on L-type channels than the dihydropyridines also inhibited PRL synthesis without affecting GH; 5 microM verapamil reduced PRL by 64% and 15 microM diltiazem by 89%. Partial depolarization with 5-25 mM KCl increased PRL synthesis up to 2-fold. The intracellular free calcium ion concentration was estimated by Quin 2 and averaged 142 nM for control cultures in normal medium, and 128 and 168 nM for cultures treated 72 h with nimodipine or BAY K8644, respectively. Nimodipine totally prevented the calcium rise obtained upon depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Dihydropyridines; Pituitary Gland; Pituitary Neoplasms; Prolactin; Rats; RNA, Messenger; Tumor Cells, Cultured