Calcium Channel Agonist Bay K8644 Research Articles

Cadmium uptake and toxicity via voltage-sensitive calcium channels.

The mechanism of cellular uptake of cadmium, a highly toxic metal ion, is not known. We have studied cadmium uptake and toxicity in an established secretory cell line, GH4C1, which has well characterized calcium channels. Nimodipine, an antagonist of voltage-sensitive calcium channels, protected cells against cadmium toxicity by increasing the LD50 for CdCl2 from 15 to 45 microM, whereas the calcium channel agonist BAY K8644 decreased the LD50. Organic calcium channel blockers of three classes protected cells from cadmium toxicity at concentrations previously shown to block high K+-induced 45Ca2+ influx and secretion. Half-maximal protective effects were obtained at 20 nM nifedipine, 4 microM verapamil, and 7 microM diltiazem. Increasing the extracellular calcium concentration from 20 microM to 10 mM also protected cells from cadmium by causing a 5-fold increase in the LD50 for CdCl2. Neither the calcium channel antagonist nimodipine nor the agonist BAY K8644 altered intracellular metallothionein concentrations, while cadmium caused a 9-20-fold increase in metallothionein over 18 h. Cadmium was a potent blocker of depolarization-stimulated 45Ca2+ uptake (IC50 = 4 microM), and the net uptake of cadmium measured with 109Cd2+ was less than 0.3% that of calcium. Although the rate of cadmium uptake was low relative to that of calcium, entry via voltage-sensitive calcium channels appeared to account for a significant portion of cadmium uptake; 109Cd2+ uptake at 30 min was increased 57% by high K+/BAY K8644, which facilitates entry through channels. Furthermore, calcium channel blockade with 100 nM nimodipine decreased total cell 109Cd2+ accumulation after 24 h by 63%. These data indicate that flux of cadmium through dihydropyridine-sensitive, voltage-sensitive calcium channels is a major mechanism for cadmium uptake by GH4C1 cells, and that pharmacologic blockade of calcium channels can afford dramatic protection against cadmium toxicity.

Regulation of thyroid hormone receptors and responses by thyrotropin-releasing hormone in GH4C1 cells.

The present study was undertaken to test the effects of TRH on thyroid hormone receptors and responses in GH4C1 rat pituitary tumor cells. TRH caused a loss of up to 32% of specific nuclear thyroid hormone binding sites with an ED50 of approximately 1 nM, and this loss was additive to the receptor down-regulation caused by T3 itself. Scatchard analysis of nuclear T3 binding revealed that 10 nM TRH decreased the concentration of T3 receptors from Bmax (femtomoles per mg protein) of 110 to 50 while receptor affinity in serum-free medium changed from dissociation constant (Kd) 110 to 50 pM with TRH. TRH lowered the GH response to 0.5 nM T3 from 215% to 127% of control. The concentrations of TRH required to decrease T3 receptors and T3 responses were similar and indicated that these TRH effects are mediated by the TRH receptor. In the absence of added thyroid hormone TRH had little effect on the rate of GH synthesis. TRH did not affect the binding of 0.5 nM [125I]T3 to receptors during the first 8 h but reduced T3 receptor occupancy up to 25-50% in different experiments after 24 h. TRH blocked the induction of GH by T3 only after 48 h or longer. When cells were incubated for 2 weeks with or without 2 nM T3 and 10 nM TRH, the stimulation of cell growth by T3 was decreased by TRH (2- vs. 5-fold increase in cell number) as was stimulation of GH by T3 (5- vs. 13-fold). As expected, T3 blunted the PRL response to TRH from 19- to 3-fold. The effects of TRH on the density of thyroid hormone receptors could be mimicked by the calcium channel agonist BAY K8644 plus a protein kinase C-activating phorbol ester which together caused a 53% reduction in thyroid hormone binding. The dose-response and temporal relationships suggest a causal relationship between the TRH-mediated decrease in thyroid hormone receptors and the decrease in thyroid hormone responses in GH4C1 cells. It has previously been shown that thyroid hormones decrease the concentration of TRH receptors and TRH responsivity in pituitary cells. The results shown here for GH4C1 cells suggest that TRH regulation of T3 responses may also be important in feedback control at the pituitary level.

Opiate receptor mediated hyperthermic responses in rat following Ca++ channel antagonists

The effects of morphine sulfate on rectal temperature and on Ca++-stimulated Mg++ATPase activity in crude synaptosomal fraction (P2) of cortex, hypothalamus and cerebellum were investigated in rat. Morphine (3-15 mg/kg, SC) produced hyperthermia at 30-120 min after the drug administration. The Ca++/Mg++ ATPase activity in hypothalamus and cortex was decreased while there was no change in Mg++ ATPase activity. The enzyme activity in cerebellum was not affected. The opiate antagonist naloxone hydrochloride (5 mg/kg, SC) antagonized the effect of morphine on rectal temperature and Ca++/Mg++ ATPase activity. The effects of different calcium channel antagonists (nimodipine 1 mg/kg, verapamil 2.5 mg/kg and diltiazem 10 mg/kg, SC) on the changes induced by morphine were also investigated. These antagonists not only antagonized morphine hyperthermia, but also the inhibitory effect of morphine on Ca++/Mg++ ATPase activity in hypothalamus. The calcium channel agonist BAY K8644 (3 mg/kg, SC) produced hypothermia and also stimulation of Ca++/Mg++ ATPase activity in hypothalamus. Naloxone failed to alter these effects of BAY K8644. These studies demonstrate that Ca++ transport in hypothalamus, as indicated by Ca++/Mg++ ATPase activity, plays an important role in thermoregulation and thermoregulatory changes induced by opiates.

Calcium channel agonist BAY k8644 enhances anterior pituitary secretion in rat and monkey

A calcium channel agonist BAY k8644 was applied to anterior pituitary (AP) cells in vitro. BAY k8644 (0.1-10 microM) stimulated prolactin and growth hormone (GH) release from monolayer AP cultures; the calcium channel antagonist D-600 (1-10 microM) completely blocked this effect. By utilizing a perifusion system, we observed an immediate and sustained amplification of prolactin (2.9-fold), growth hormone (2.3-fold), and luteinizing hormone (LH, 1.6-fold) release during the 1-h application of BAY k8644 (3 microM). A hypophysiotrophic peptide pulse 4 h after the BAY k8644 was removed confirmed that the cells remained responsive to their natural secretagogues. In another perifusion study 10-3,000 nM BAY k8644 produced a graded increase in prolactin release that was maintained over the 30-min exposure period. Finally, individual primate mammotrophs and somatotrophs showed a marked enlargement of hemolytic plaque area, an index of hormone release, 1 h after BAY k8644 (1 microM). We conclude that this synthetic dihydropyridine enhances the rate of prolactin, GH, and LH release from AP cells of two species. Because this is the first synthetic calcium channel agonist, structure-function studies characterizing calcium channel activation and exocytosis are now feasible.

The effects of dihydropyridines of neurotransmitter release from cultured neuronal cells

Depolarizing stimuli increase the release of transmitter substances from cultured PC12 pheochromocytoma cells and reaggregate cultures of mouse mesencephalic dopamine neurones. We measured the stimulated release of ( 3H) norepinephrine and ( 3H) dopamine from these systems respectively. In the cultured mouse dopaminergic neurones, several organic calcium channel blockers including nitrendipine, D-600, verapamil and diltiazem were unable to inhibit potassium-evoked transmitter release. However, release was blocked by 3 mM cobalt. The novel dihydropyridine calcium channel agonist BAY K8644 also had no effect on basal or evoked dopamine release. In contrast, BAY K8644 greatly stimulated the potassium-evoked release of ( 3H) norepinephrine from PC12 cells. The BAY K8644 enhanced release could be blocked by the dihydropyridine antagonist nitrendipine. These results indicate that while stimulus-secretion coupling in the PC12 cell line involves dihydropyridine sensitive calcium channels, this is not the case in primary cultured neurones.

Accurate freehand injection into the lateral brain ventricle of the conscious mouse.

Accurate freehand injection into the lateral brain ventricle of the conscious mouse.