Rapid actions of calcitriol and its side chain analogues CB1093 and GS1500 on intracellular calcium levels in skeletal muscle cells: a comparative study.

1. The ability of synthetic analogues of the secosteroid hormone 1alpha,25-dihydroxy-vitamin-D3 [calcitriol, CT; 1,25(OH)2D3] to exert non-genomic (rapid) effects on target cells has been scarcely studied. To evaluate the pharmacological potential of the CT side-chain analogues CB1093 and GS1500, we compared their fast effects on intracellular calcium concentration ([Ca2+]i) in chick skeletal muscle cells with those elicited by the natural hormone. 2. Both analogues, similarly to CT, specifically induced rapid (30-60 s) and sustained rises in [Ca2+]i levels. CB1093 and GS1500 were more potent than the natural hormone at concentrations as low as 10(-13) M (4.5 fold stimulation) and 10(-12) M (2.5 fold), respectively, whereas higher concentrations (10(-9)- 10(-8) M) of CT were more effective than the analogues in elevating [Ca2+]i. Cyclic AMP was markedly increased by both analogues pointing for a role of this messenger in the fast actions of the synthetic compounds. 3. In Ca2+ free medium CT and analogues elicited a transient elevation in [Ca2+]i. The PLC inhibitors U73122 (2 microM) and neomycin (0.5 mM), as well as depletion of intracellular stores with thapsigargin (1 microM), completely prevented CB1093/GS1500-dependent changes in [Ca2+]i suggesting that, similarly to CT, these analogues mobilized Ca2+ from an IP3/thapsigargin-sensitive store. 4. The voltage-dependent calcium channel (VDCC) blocker nifedipine (2 microM) reduced by 50-60% the influx phase of the [Ca2+]i response to CB1093 and GS1500, indicating that VDCC contributed partially to Ca2+ entry. The Ca2+ readdition protocol suggested that analogue-dependent activation of a SOC entry pathway accounted, to the same extent as for CT, for the remaining non-VDCC mediated Ca2+ influx.     

20-epi-vitamin D3 analogues: a novel class of potent regulators of cell growth and immune responses.

The 20-epi-vitamin D3 analogues are a novel class of vitamin D3 derivatives, structurally related to 1 alpha,25-dihydroxycholecalciferol (1 alpha,25(OH)2D3). They are characterized by an altered stereochemistry at carbon 20 in the side-chain. In vitro, these new analogues were found to be considerably more potent as regulators of growth and differentiation in the human histiocytic lymphoma cell line U 937 than 1 alpha,25(OH)2D3, despite a practically unchanged calcemic activity in vivo. The most potent analogue, KH 1060, inhibited cell proliferation by 50% at 10(-12) M (14,000 times more active than 1 alpha,25(OH)2D3). At the same time, KH 1060 induced cell differentiation at concentrations as low as 10(-14)M. In addition, the 20-epi-vitamin D3 analogues were found to be very potent inhibitors of T-lymphocyte proliferation induced by interleukin-1 or alloantigen. In this respect, they were several orders of magnitude more active than the potent immunosuppressive agent cyclosporin A (CyA). KH 1060, the most potent analogue, inhibited interleukin-1-induced mouse thymocyte proliferation by 50% at 3 x 10(-16) M and allogeneic stimulation of mouse spleen lymphocytes at 5 x 10(15) M. These effects were considered to be mediated by inhibition of interleukin-2 release from activated T-lymphocytes. The new analogues are of potential interest in the prevention of graft rejection and in the treatment of psoriasis, cancer and auto-immune diseases.     

Syntheses and differentiating action of vitamin D endoperoxides. Singlet oxygen adducts of vitamin D derivatives in human myeloid leukemia cells (HL-60)

Singlet oxygen adducts of various vitamin D derivatives, 6,19-dihydro-6,19-epidioxyvitamin D (vitamin D endoperoxides, 2 and 2'), were chemically synthesized, and their biological activity in inducing differentiation of a human myeloid leukemia cell line (HL-60 cells) was examined. The potency of the endoperoxides derived from vitamin D derivatives possessing the 1 alpha-hydroxyl group such as 1 alpha, 25-dihydroxyvitamin D3 endoperoxides (2b and 2b') was markedly (10(-2)) diminished relative to the respective parent vitamin D compounds. In contrast, 25-hydroxyvitamin D3 endoperoxides [25-(OH)D3 endoperoxides, 2a and 2a'] and their analogues fluorinated at the 24- or 26- and 27-positions were 2.5-10 times more potent than 25-hydroxyvitamin D3 (1a) in spite of the absence of the conjugated triene structure typical of vitamin D compounds. The potency of these vitamin D endoperoxides (2 and 2'), especially those lacking the 1 alpha-hydroxyl group, in inducing differentiation of HL-60 cells was not correlated with their activity in binding to the cytosol receptor for 1 alpha, 25-dihydroxyvitamin D3 (1b). The binding efficiency to the receptor was relatively lower than the differentiating activity. To examine the action of vitamin D endoperoxides, carbon analogues of 25-(OH)D3 endoperoxides, two C-6 epimers of 25-hydroxy-6,19-dihydro-6,19-ethanovitamin D3 (6 and 6'), were synthesized. The carbon analogues (6 and 6') had no potential to induce differentiation of HL-60 cells. These results suggest that vitamin D endoperoxides (2 and 2') express their biological activity probably after being converted to some other compounds.     

In vitro biological activities of a series of 2 beta-substituted analogues of 1 alpha,25-dihydroxyvitamin D3

Biological activities of a series of 2beta-substituted analogues of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] were evaluated in vitro in terms of their binding affinity with regard to calf thymus cytosolic vitamin D receptor (VDR) and rat plasma vitamin D-binding protein (DBP). Additionally, reporter gene luciferase activities using either a rat 25-hydroxyvitamin D3-24-hydroxylase gene promoter, including two vitamin D-responsive elements (VDREs), in transfected rat osteoblast-like ROS17/2.8 cells, or a human VDR-GAL4 modified two-hybrid system in transfected human epitheloid carcinoma, cervix HeLa cells were examined. Binding affinity for VDR, transactivation potency on the target gene and VDR-mediated gene regulation of the hydroxyalkyl and hydroxyalkoxy 2beta-substituted analogues were almost comparable to those of 1alpha,25(OH)2D3, while the alkyl and alkenyl analogues were much less active than 1alpha,25(OH)2D3. This study investigated the biological evaluation of a series of 2beta-substituted analogues at the molecular level, with regard to the structural differences of alkyl, alkenyl, hydroxyalkyl, hydroxyalkoxy, alkoxy, hydroxy and chloro substituents at the 2beta-position of 1alpha,25(OH)2D3.     

Effects of synthetic vitamin D analogues on breast cancer cell proliferation in vivo and in vitro.

Calcipotriol (MC903) is a novel vitamin D analogue which effects cellular differentiation and proliferation in vitro and has reduced effects on calcium metabolism in vivo. In the present study its in vitro activity was evaluated using the MCF-7 breast cancer cell line, and its effects on calcium metabolism and mammary tumour growth were measured in vivo in adult female rats. Calcipotriol was compared to the natural metabolite of vitamin D3, 1 alpha,25-dihydroxycholecalciferol [1,25(OH)2D3] and its synthetic analogue 1 alpha hydroxycholecalciferol [1 alpha(OH)D3]. Both calcipotriol and 1,25(OH)2D3 produced significant inhibition of MCF-7 cell proliferation at a concentration of 5 x 10(-11) M. Intraperitoneal administration of calcipotriol to normal female rats showed that the analogue was 100-200 times less active than 1,25(OH)2D3 in raising serum calcium concentration and urinary calcium excretion. Anti-tumour activity of the vitamin D analogues was investigated in vivo using the nitrosomethylurea-induced rat mammary tumor model. Rats, maintained on a low calcium diet, were treated with 1 alpha(OH)D3 (0.25 and 1.25 micrograms/kg). Both doses produced a response rate of 25% but hypercalcaemia developed. Treatment with calcipotriol (50 micrograms/kg) of rats maintained on a normal laboratory diet caused inhibition of tumour progression (response rate 17%) without the development of severe hypercalcaemia. This study supports the concept that vitamin D derivatives may inhibit breast cancer cell proliferation in vivo.     

Disposition and metabolism of F6-1alpha,25(OH)2 vitamin D3 and 1alpha,25(OH)2 vitamin D3 in the parathyroid glands of rats dosed with tritium-labeled compounds.

26,26,26,27,27,27-Hexafluoro-1alpha,25(OH)2 vitamin D3, a hexafluorinated analog of 1alpha,25(OH)2 vitamin D3, has been reported to be several times more potent than the parent compound with respect to some vitamin D actions. The reason for enhanced biological activity in the bones, kidneys, and small intestine appears to be related to F6-1alpha,25(OH)2 vitamin D3 metabolism to ST-232 (26,26,26,27,27,27-hexafluoro-1alpha,23S,25-trihydroxyvitamin D3), a bioactive 23S-hydroxylated form that is resistant to further metabolism. We compared the disposition and metabolism of [1beta-3H]F6-1alpha,25(OH)2 vitamin D3 and [1beta-3H]1alpha,25(OH)2 vitamin D3 in parathyroid glands of rats intravenously administered with labeled compounds at a dose of 10 microg/kg. In the [1beta-3H]F6-1alpha,25(OH)2 vitamin D3-dosed group, radioactivity was highly detected in the kidneys, parathyroid glands, and the small intestine. The radioactivity in the parathyroid glands remained high until 48 h postdosing, with values of 2.5, 8.4, and 14.6 times higher at 6, 24, and 48 h postdosing than after dosing with [1beta-3H] 1alpha,25(OH)2 vitamin D3. In the group given [1beta-3H]F6-1alpha,25(OH)2 vitamin D3, the unchanged compound was mainly detected with a small amount of ST-232 at 6 h postdosing. At the 24- and 48-h time points, over half of the radioactivity was observed as ST-232, and additionally, ST-233, the 23-oxo form, accounted for a small amount at the 48-h time point. The present study demonstrated local retention of [1beta-3H]F6-1alpha,25(OH)2 vitamin D3 and the bioactive metabolite ST-232 in parathyroid glands after intravenous administration. The findings may indicate one of the reasons for the higher potency of F6-1alpha,25(OH)2 vitamin D3 than 1alpha,25(OH)2 vitamin D3 in parathyroid.     

Vasodilatation induced by pinacidil in dogs. Comparison with hydralazine and nifedipine

In helical strips of dog arteries precontracted with prostaglandin (PG) F2 alpha, pinacidil and nifedipine produced a dose-related relaxation. The potencies of pinacidil were in the order of coronary and renal greater than mesenteric greater than basilar and middle cerebral arteries, whereas those of nifedipine were in the order of basilar and renal greater than mesenteric and coronary arteries. Pinacidil caused a greater relaxation in mesenteric veins than in the arteries. Hydralazine consistently relaxed the arteries only at 10(-3) M. In mesenteric artery strips exposed to Ca2+-free, high K+ media, contractions induced by Ca2+ were reduced by 10(-8) M nifedipine, but they were not influenced by 10(-5) M pinacidil or by 10(-4) M hydralazine. In the arteries exposed to Ca2+-free media and stimulated by PGF2 alpha or norepinephrine, tonic contractions induced by Ca2+ were reduced moderately by 10(-5) M pinacidil but only slightly by 10(-8) M nifedipine. In Ca2+-free media, PGF2 alpha-induced contractions were inhibited only by pinacidil. In isolated mesenteric vasculature, perfusion pressure was lowered by pinacidil and hydralazine. It may be concluded that pinacidil produces vasodilatation due to interference with the transmembrane influx of Ca2+ into smooth muscle evoked by receptor stimulation but not that due to inhibition in the Ca2+ influx associated with K+-induced membrane depolarization. Decreased release of Ca2+ from intracellularly stored sites or increased sequestration to the sites may also be involved. Pinacidil appears to dilate arteries and veins as well as resistance vessels, whereas hydralazine appears to act exclusively on resistance vessels.     

Inhibition of angiogenesis by vitamin D3 analogues.

The effects of vitamin D3 and two analogues on embryonic angiogenesis were studied in 4.5-day-old chick embryo chorioallantoic membranes. The active metabolite of vitamin D3, 1 alpha,25-dihydroxyvitamin D3, and a synthetic vitamin D3 analogue, 22-oxa-1 alpha,25-dihydroxyvitamin D3, inhibited angiogenesis in a dose-dependent manner, the inhibition occurring in the picomolar range. In contrast, vitamin D3 was not effective. The results suggest that these two vitamin D3 analogues might be promising anti-angiogenic agents for controlling the angiogenesis which occurs in several pathological conditions, including tumor development.     

Manipulation of thromboxane synthesis by novel 26,27-dialkyl analogues of 1 alpha,25-dihydroxyvitamin D3 in human promyelocytic leukemia (HL-60) cells.

Using new steroidal side-chain-lengthened 26,27-dialkyl analogues of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3], we manipulated the synthesis of thromboxane and thromboxane-producing enzymes, cyclo-oxygenase and thromboxane synthase, in human promyelocytic leukemia (HL-60) cells in serum-free culture. The order of potency of the analogues for stimulating thromboxane B2 synthetic activity from arachidonic acid (reflecting combined cyclo-oxygenase activity and thromboxane synthase activity) and from prostaglandin H2 (thromboxane synthase activity only) as well as for cyclo-oxygenase induction was 1 alpha,25-(OH)2D3 > or = 1 alpha,25-(OH)2-26,27-CH3)2D3 > 1 alpha,25-(OH)2-26,27-(C2H5)2D3 > 1 alpha,25-(OH)2-26,27-(C3H7)2D3. These results suggest that there are functional and structural limits to the chain length of C-26 and C-27 dialkyl groups flanking the C-25-OH group in the 1 alpha,25-(OH)2D3 molecule for expressing thromboxane synthetic activity in HL-60 cells. Removal of the C-1 alpha-OH group from 1 alpha,25-(OH)2D3 led to markedly decreased thromboxane synthetic activity in HL-60 cells. These structure-activity relationships indicate that both the C-25-OH and C-1 alpha-OH groups in the 1 alpha,25-(OH)2D3 molecule are essential for expressing thromboxane synthesis in HL-60 cells. Also, the rank order for stimulating thromboxane synthesis correlated well with the binding affinity of these dialkyl analogues for the 1 alpha,25-(OH)2D3 receptor of HL-60 cells, suggesting a 1 alpha,25-(OH)2D3 receptor-mediated induction mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detailed molecular understanding of agonistic and antagonistic vitamin D receptor ligands

The vitamin D receptor (VDR) is an endocrine member of the nuclear receptor superfamily and binds the biologically most active vitamin D metabolite, 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). The VDR ligand-binding domain is a molecular switch, since its ligand-triggered interactions with corepressor and coactivator proteins are the central molecular events of nuclear 1alpha,25(OH)2D3 signaling. 1alpha,25(OH)2D3 analogues have been developed with the goal to improve the biological profile of the natural hormone for a therapeutic application either in hyperproliferative diseases, such as psoriasis and different types of cancer, or in bone disorders, such as osteoporosis. Most of the analogues described to date are agonists, with a few having been identified as antagonists. Only the two side chain analogue Gemini and some of its derivatives act under restricted conditions as inverse agonists. In this review we discuss the molecular mechanisms of these different type of analogues based on crystal structure data, molecular dynamics simulations and biochemical assays.     

Seocalcitol (EB 1089): a vitamin D analogue of anti-cancer potential. Background, design, synthesis, pre-clinical and clinical evaluation

It is well established that the metabolically active form of vitamin D, 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) plays a key role in the establishment and maintenance of the calcium metabolism in the body. In addition to this classic effect of 1alpha,25(OH)2D3, substantial evidence has emerged demonstrating that 1alpha,25(OH)2D3 is able to regulate cell growth and differentiation in a number of different cell types, including cancer cells. However, the clinical usefulness of 1alpha,25(OH)2D3 is limited by its tendency to cause hypercalcaemia. Much effort has therefore been directed to identifying new vitamin D analogues with potent cell regulatory effects, but with weaker effects on the calcium metabolism than those of 1alpha,25(OH)2D3. One of these new synthetic analogues is Seocalcitol (EB 1089). Despite being 50-200 times more potent than 1alpha,25(OH)2D3 with respect to regulation of cell growth and differentiation in vitro as well as in vivo, EB 1089 displays a reduced calcaemic activity in vivo compared to that of 1alpha, 25(OH)2D3. These characteristics make EB 1089 a potentially useful compound for the treatment of cancer. Recent clinical evaluation of EB 1089 has focused mainly on establishing a maximum tolerated dose in cancer patients. Early results confirm that the low calcaemic activity observed in animals can be reproduced in the clinic. Furthermore, EB 1089 has been shown to induce regression of tumours, especially in hepatocellular carcinoma where complete remission has been obtained. In conclusion, the development of EB 1089 as an anti-cancer drug holds promise. However, its final evaluation must await the completion of ongoing controlled clinical trials.     

Rapid effects of 1alpha,25(OH)2D3 in resting human peripheral blood mononuclear cells

The steroid hormone 1alpha,25(OH)2D3 produces biological responses via both genomic and nongenomic mechanisms. Stimulation of rapid, nongenomic responses by 1alpha,25(OH)2D3 has been postulated to result from interaction of the ligand with cell membrane 1alpha,25(OH)2D3 receptors and to involve membrane receptors. We examined the rapid effects of 1alpha,25(OH)2D3 on calcium mobilization and calcium entry into resting human peripheral blood mononuclear cells isolated from healthy volunteers. We also investigated the possible involvement of purinergic receptors in this action. 1alpha,25(OH)2D3 induced a time-dependent increase in intracellular calcium concentration ([Ca2+]i). The initial 1alpha,25(OH)2D3-stimulated calcium increment was sensitive to thapsigargin (Tg), indicating its origins in calcium release from intracellular stores. 2-Aminoethyldiphenyl borate (2APB), an inhibitor of capacitative calcium entry, caused a significant [Ca2+]i decrease in human cells treated with 1alpha,25(OH)2D3. Furthermore, in contrast to observations in osteoblasts and skeletal muscle cells, nifedipine had no effect on 1alpha,25(OH)2D3-induced calcium entry, suggesting that L-type calcium channels were not implicated in this action. Besides, 1alpha,25(OH)(2)D3 prevented the calcium entry induced by 3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate (BzATP), a specific agonist of purinergic P2X7 receptors. This finding was further confirmed by 1alpha,25(OH)2D3-induced reduction of BzATP- and 4-aminopyridine (4AP)-stimulated ethidium bromide fluorescence. The presented results demonstrate, for the first time in healthy, resting human peripheral blood mononuclear cells that 1alpha,25(OH)2D3 is capable of exerting a rapid, nongenomic effect on [Ca2+]i, while inhibiting of the P2X7 channel permeability.     

Lead inhibits the basal and stimulated responses of a rat osteoblast-like cell line ROS 17/2.8 to 1 alpha,25-dihydroxyvitamin D3 and IGF-I

Low-level exposure to lead impairs longitudinal growth in children and in experimental animals. The proposed mechanisms include decreased osteocalcin secretion in response to 1 alpha,25-(OH)2 vitamin D3 and decreased response to insulin-like growth factor-I. The interaction of lead, 1 alpha,25-(OH)2 vitamin D3, and insulin-like growth factor-I was investigated in an osteoblast-like cell line from rat osteosarcoma, ROS 17/2.8. Cells were cultured 24 hr in a serum-free medium with lead, 1 alpha,25-(OH)2 vitamin D3, and insulin-like growth factor-I. 1 alpha,25-(OH)2 vitamin D3 (10 nM) evoked a 4-5 X increase in osteocalcin secretion and a 100% increase in cellular alkaline phosphatase activity but no increase in DNA/cell layer. Insulin-like growth factor-I (92.5 ng/ml) evoked a 100% increase of osteocalcin secretion and a 20% increase in cellular DNA contents but no change in cellular alkaline phosphatase activity. Basal and stimulated cellular osteocalcin secretion, cellular alkaline phosphatase activity, and DNA contents were significantly inhibited by addition of 1-10 microM lead. The data are consistent with a toxic effect of lead on osteoblastic function and the cellular responses to 1 alpha,25-(OH)2 vitamin D3 and insulin-like growth factor-I. This interaction may be relevant to impaired childhood growth at low levels of lead exposure.     

The biological activities of 1alpha,25-dihydroxyvitamin D3 and its synthetic analog 1alpha,25-dihydroxy-16-ene-vitamin D3 in normal human osteoblastic cells and human osteosarcoma SaOS-2 cells are modulated by 17-beta estradiol and dependent on stage of differentiation

We compared the effects of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and its analog, 1alpha,25-dihydroxy-16-ene-vitamin D3 [1alpha,25(OH)2-16-ene-D3], as well as their interactions with 17-beta estradiol (E2) on osteoblastic function in our human normal (HOB) and osteosarcoma SaOS-2 cell models representing two different stages of differentiation, the more differentiated HOB+DEX cells and SaOS+DEX cells, and the corresponding less differentiated HOB-DEX and SaOS-DEX cells. The differential effects of 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-D3 and the modulation by E2 on ALP activity in HOB-DEX and HOB+DEX cells were small but significant. The most significant effects were seen in SaOS+DEX cells, in which 1alpha,25(OH)2-16-ene-D3 was 100-fold more potent than 1alpha,25(OH)2D3, the maximal enhancement being exerted at 0.1 nM and 10 nM, respectively. E2 enhanced the stimulatory effects of both compounds, with ALP being increased 2-fold at 0.1 nM (p<0.001). Osteocalcin (OC) production in HOB-DEX cells was stimulated 1.3 to 1.4-fold by 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-D3 at a concentration of 0.01 nM, with E2 inhibiting the effect of 1alpha,25(OH)2-16-ene-D3. In SaOS-DEX and SaOS+DEX cells, 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-D3 stimulated OC production 1.6-fold at 0.1 nM with E2 slightly enhancing the effect of 1alpha,25(OH)2D3. Western blot analysis of 1alpha,25(OH)2D3 receptor (VDR) levels showed that in SaOS+DEX cells, the effect of 1alpha,25(OH)2D3 was larger than that of 1alpha,25(OH)2-16-ene-D3. These results show that 1alpha,25(OH)2-16-ene-D3 is biologically active in human osteoblasts.     

Metabolism of 26,26,26,27,27,27-F6-1 alpha,23S,25-trihydroxyvitamin D3 by human UDP-glucuronosyltransferase 1A3.

26,26,26,27,27,27-Hexafluoro-1alpha,25-dihydroxyvitamin D(3) [F(6)-1alpha, 25(OH)(2)D(3)], which is now clinically used as a drug for secondary hyperparathyroidism, is a hexafluorinated analog of the active form of vitamin D(3). Our previous studies demonstrated that CYP24A1 is responsible for the metabolism of F(6)-1alpha,25(OH)(2)D(3) in the target tissues and that F(6)-1alpha,25(OH)(2)D(3) was successively converted to F(6)-1alpha,23S,25(OH)(3)D(3) and F(6)-23-oxo-1alpha,25(OH)(2)D(3). In this study, we examined the metabolism of F(6)-1alpha,25(OH)(2)D(3),F(6)-1alpha,23S,25(OH)(3)D(3), and F(6)-23-oxo-1alpha,25(OH)(2)D(3) by human UDP-glucuronosyltransferases (UGTs). Of these compounds, F(6)-1alpha,23S,25(OH)(3)D(3) was remarkably glucuronidated both in human liver microsomes and in the recombinant system expressing human UGT. No significant interindividual differences were observed among 10 human liver samples. The recombinant system for 12 species of human UGTs revealed that F(6)-1alpha,23S,25(OH)(3)D(3) glucuronidation was specifically catalyzed by UGT1A3. The information obtained in this study seems very useful to predict the metabolism and efficacy of vitamin D analogs in human bodies before clinical trials. In addition, note that for the first time a possible probe substrate for UGT1A3 has been found.     

Characterization of the effects of a new Ca2+ channel activator, FPL 64176, in GH3 cells.

We examined the effects of the benzolpyrrole-type Ca2+ channel activator FPL 64176 on voltage-dependent L-type Ca2+ channels in rat anterior pituitary (GH3) cells. FPL 64176 increased K(+)-dependent Ca2+ influx into GH3 cells with an EC50 value of 1.2 x 10(-7) M but had no effect on the binding of [3H]PN200-110 to GH3 cell membranes at concentrations up to 10(-6) M. Whole-cell patch-clamp electrophysiology revealed that FPL 64176 (1 microM) increased L-type Ca2+ channel current amplitude and shifted the current-voltage relationship in the hyperpolarizing direction. Furthermore, Ca2+ channel current activation and deactivation were prolonged. Single-channel analysis showed that FPL 64176 increased both the probability of channel opening and the mean channel open time. Interestingly, the effect of FPL 64176 on channel open time was highly voltage dependent, with much longer openings being observed at more hyperpolarized potentials. We conclude that FPL 64176 represents a new class of L-type Ca2+ channel activator with a novel site and mechanism of action.