Linkage between O6-methylguanine-DNA methyltransferase (O6-MT) activity and cellular resistance to antitumour nitrosoureas in cultured rat brain tumour cell strains

Summary We have examined O⁶-methylguanine-DNA methyltransferase (O⁶-MT) activity of rat brain tumour cell strains with reference to cellular resistance to antitumour nitrosoureas, 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (nimustine, ACNU) and methyl-6-[3-(2-chloroethyl)-3-nitrosoureido]-6-deoxy--D-glucopyranoside (ramustine, MCNU). The values of O⁶-MT activity were 52 and 160 fmol/mg protein extract in 9 L and C 6 rat brain tumour cells, respectively; while HeLa S 3 cells, as a methyl excision repair positive (Mer⁺) cell strain, revealed a rather high value of 488 fmol/mg. 9 L cells indicative of a low O⁶-MT activity showed 13 M for ACNU and 18 M for MCNU at a 10% survival dose (SD₁₀), determined by a clonogenic cell assay as an index of cellular resistance. In contrast to this, C 6 cells revealed a SD₁₀ value of 67 M and 36 M for ACNU and MCNU, respectively, indicating higher resistance than 9 L cells. HeLa S 3 cells showed the highest SD₁₀ value as follows: 84 M for ACNU and 73 M for MCNU. The relationship between the O⁶-MT activity and the cellular resistance was almost linear, with relatively resistant cell lines exhibiting the higher levels of the O⁶-MT activity. This correlation between the O⁶-MT activity and the cellular resistance to nitrosoureas as ACNU and MCNU was not observed among other antitumour drugs, which included bleomycin (BUM), neocarzinostatin (NCS),cis-diamminedichloroplatinum (II) (CDDP), and etoposide (VP-16) in clinical use for brain tumour chemotherapy. This indicates that O⁶-MT activity can be an indicator of cellular resistance to antitumour nitrosoureas in the chemotherapy of brain tumours.     

Parathyroid hormone stimulates phosphoinositide metabolism and intracellular calcium mobilization in osteoblast-like clone MC3T3-E1 cells

The effects of parathyroid hormone (rat 1–34 fragment, rPTH) on phosphoinositide metabolism and intracellular Ca²⁺ mobilization were studied in a osteoblast-like clone MC3T3-E1 cells. rPTH caused a transient rise in intracellular Ca²⁺ in a dose-dependent manner. Significant Ca²⁺ increase was still observed under the extracellular Ca²⁺-free condition. In addition, 100nM rPTH stimulated rapid formation of both 1,2-diacylglycerol (1,2-DAG) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃), indicating agonist-triggered hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂). The current observations suggested that the rPTH-induced increase of intracellular Ca²⁺ was in part due to internal Ca²⁺ release mediated by Ins(1,4,5)P₃. These results indicate possible involvement of phosphoiniositide metabolism in signal transduction of PTH-stimulated osteoblastic cells.     

Comparison of parathyroid hormone and calcium ionophore A23187 effects on bone resorption and nucleic acid synthesis in cultured fetal rat bone

Summary It has recently been demonstrated that calcium ionophore A23187 mimics certain of the effects of parathyroid hormone (PTH) on bone in vitro, including stimulation of⁴⁵Ca release and cAMP formation. To further examine the relative effects of these two agents on bone cell metabolism, we compared the effects of synthetic PTH 1–34 (50 ng/ml) and calcium ionophore A23187 (0.5μg/ml) on⁴⁵Ca release, DNA concentration, and nucleic acid synthesis in fetal rat forelimb rudiments cultured for periods up to 120 h. Both agents stimulated⁴⁵Ca release; however, the effects of PTH were apparent after a shorter period of exposure. Bone DNA concentration (expressed asμg DNA/mg bone) was not affected by PTH but was significantly increased relative to control values by exposure to A23187 for 8–120 h of incubation. PTH increased the incorporation of³H-thymidine into DNA at 30 and 48 h, and increased the incorporation of¹⁴C-uridine into RNA at 48 h, time points which corresponded to a period of accelerated PTH stimulation of⁴⁵Ca release. In contrast,³H-thymidine and¹⁴C-uridine incorporation were both uniformly suppressed by A23187 at all time points examined. Thus the increased DNA concentration observed in A23187-treated rudiments appeared to be the result of a decreased rate of bone maturation and mineralization. The markedly different patterns of nucleic acid synthesis in response to PTH and A23187 suggest that these agents differ significantly in their mechanisms of action on bone cell metabolism.     

Selective Permeability of [3H]-D-Mannitol and [l4C]-Carboxyl-lnulin Across the Blood-Brain Barrier and Blood-Spinal Cord Barrier in the Rabbit

Abstract

Selective permeability across the blood-brain and blood-spinal cord barriers was studied in a rabbit experimental model. To maintain steady state levels of the tracers in the circulation, 50μCi each of [³H]-D-mannitol and [¹⁴C]-cartoxyl-inulin were administered as a bolus and by slow intravenous infusion for 60, 90 and 120 minutes; 90 min proved to be the optimal equilibration time for uptake of radioactive tracers into central nervous system (CNS) regions. Kinetic transfer of [³H]-D-mannitol and [¹⁴C]-carboxyl-inulin from blood into CNS was computed as apparent transfer constant (K_D). The K_D for [³H]-D-mannitol in regions of brain (cerebrum, cerebellum, mid-brain, pons and brain stem), and spinal cord (cervical, thoracic and lumbar) were 0.033 to 0.054 μl/min/g^-1 and 0.053 to 0.065 μ/min/g^-1, respectively. The K_D for [¹⁴C]-carboxyl-inulin into brain and spinal cord regions were 0.016 to 0.033 and 0.037 to 0.054 μl/min/g^-1, respectively. Of the regions of spinal cord and brain examined, lumbar cord appears to be the most permeable. The K_D values pooled for samples of the spinal cord show a significant increase in uptake of [³H]-D-mannitol (p=0.0043) and [¹⁴C]-carboxyl-inulin (p=0.0001) compared to samples of brain. The blood-spinal cord barrier was more permeable to these substances than the blood-brain barrier. (J Spinal Cord Med, 18:221–226)     

Caribbean maitotoxin elevates [Ca2+]i and activates non-selective cation channels in HIT-T15 cells

AIM: To investigate the cytotoxic mechanism of caribbean maitotoxin (MTX-C) in mammalian cells.METHODS: We used whole-cell patch-clamp techniques and fluorescence calcium imaging to determine the cellular toxic mechanisms of MTX-C in insulin secreting HIT-T15 cells, which is a system where the effects of MTX have been observed. HIT-T15 cells stably express L-type calcium current, making it a suitable model for this study. Using the fluorescence calcium indicator Indo-1 AM, we found that there is a profound increase in HIT-T15 intracellular free calcium 3 min after application of 200 nmol/L MTX-C.RESULTS: About 3 min after perfusion of MTX-C, a gradual increase in free calcium concentration was observed. This elevation was sustained throughout the entire recording period. Application of MTX-C did not elicit the L-type calcium current, but large cationic currents appeared after applying MTX-C to the extracellular solution. The current-voltage relationship of the cation current is approximately linear within the voltage range from -60 to 50 mV, but flattened at voltages at -80 and -100 mV. These results indicate that MTX-C induces a non-voltage activated, inward current under normal physiological conditions, which by itself or through a secondary mechanism results in a large amount of cationic influx. The biophysical mechanism of MTX-C is different to its isoform, pacific maitotoxin (MTX-P), when the extracellular calcium is removed.CONCLUSION: We conclude that MTX-C causes the opening of non-selective, non-voltage-activated ion channels, which elevates level of intracellular calcium concentration and leads to cellular toxicities.     

Demonstration of 1,25(OH)2 vitamin D3 receptors and actions in vascular smooth muscle cellsIn vitro

Summary Binding of [³H] 1,25 (OH)₂D₃ and effects of 1,25 (OH)₂D₃ on cell ultrastructure were evaluated in vascular smooth muscle cells (VSMC) primary cultures (aortic media). Specific reversible binding of [³H] 1,25 (OH)₂D₃ by a 3.5 S macromolecule with DNA binding, K_D 6.2×10⁻¹⁰M and N_max 16 fmol/mg protein was demonstrated. Incubation of VSMC with 10⁻⁸ M 1,25 (OH)₂D₃, but not 25 (OH)D₃, in the presence of 10% FCS for up to three weeks caused rapid reversible appearance in the cytoplasm of membrane-bounded electron-dense lysosomal particles which on electronspectroscopic imaging contained Ca and Pi. VSMC are targets for vitamin D.     

GPRC6A mediates responses to osteocalcin in β‐cells in vitro and pancreas in vivo

A bone‐pancreas endocrine loop has been identified recently that involves insulin secreted from β‐cells in the pancreas stimulating insulin receptors in osteoblasts, leading to osteoblastic differentiation and increased secretion of osteocalcin (Ocn), a bone‐derived hormone that regulates insulin secretion in β‐cells. The identity of the Ocn‐sensing receptor in β‐cells is a missing component of this endocrine loop. The abnormalities in glucose homeostasis in Gprc6a null mice suggests that this pertussis toxin–sensitive G protein–coupled receptor is a candidate for mediating the effects of Ocn on insulin secretion in the pancreas. In support of this possibility, we found that transfection of non‐Gprc6a‐expressing HEK‐293 cells with a full‐length Gprc6a cDNA imparted a dose‐dependent response to Ocn (5 to 60 ng/mL), as measured by PKD1 and ERK phosphorylation. In addition, Gprc6a is highly expressed in mouse pancreatic tissue and in the mouse TC‐6 pancreatic β‐cell line. Ocn also stimulated ERK activity in TC‐6 pancreatic β‐cells. Finally, intraperitoneal injection of Ocn stimulated ERK activity in the pancreas and increased serum insulin levels in wild‐type mice, but these responses were markedly attenuated in Gprc6a^?/? mice. These findings suggest that GPRC6A is a candidate for mediating the response to Ocn in the bone‐pancreas endocrine loop regulating insulin signaling. © 2011 American Society for Bone and Mineral Research.     

Intracellular free calcium and phosphatidyl inositol — 1,4,5 — triphosphate in bone cells cultured in a low calcium environment

The movement of intracellular free calcium([Ca²⁺]i) and phosphatidyl inositol-1, 4, 5-triphosphate (IP₃) was studied in bone cells cultured in a low-calcium environment. The [Ca²⁺]i was 98.0±10.2(n=6)nM/10⁶ cells for the control group (bone cells cultured in control medium) and 21.3±2.8(n=6)nM/10⁶ cells for the low Ca group (bone cells cultured in low Ca medium). After the addition of exogenous CaCl₂ to the calibration solution, [Ca²⁺]i increased significantly more in the low Ca group than in the control group(p<0.01). The IP₃ content/2×10⁶ cells was 12.40 pmoles in the control group and less than 0.19 pmoles in the low Ca group. After the stimulation with phospholipase C (PLC), the IP₃ content in the bone cells increased markedly more in the low Ca group than in the control group. These findings suggest that a low-calcium environment around cells and organsin vivo may inhibit the intracellular signal tranduction system.     

Actions of parathyroid hormone and 1,25-dihydroxycholecalciferol on citrate decarboxylation in osteoblast-like bone cells differ in calcium requirement and in sensitivity to trifluoperazine

Summary The actions of PTH in OB bone cells appear to involve both calcium and cAMP. At present little information exists regarding the relationship, if any, between these two putative second messengers of hormone action in bone cells. In this report the molecular role of calcium in the actions of PTH and 1,25(OH)₂D₃ has been compared, since like PTH, the steroid 1,25(OH)₂D₃ is a potent bone resorbing hormone that exerts inhibition of citrate decarboxylation in OB cells, but unlike PTH does not activate adenylate cyclase. It was found that 1,25(OH)₂D₃ could initiate near maximum inhibition of citrate decarboxylation at extracellular calcium levels as low as 0.05 mM, whereas PTH effects began to be apparent only at 0.1 mM calcium, and maximum inhibition of citrate decarboxylation by PTH required 0.5 mM Ca. In addition, PTH-induced decrease in citrate decarboxylation was inhibited by low doses of TFP, an inhibitor of calmodulin and calcium-dependent, phospholipid-sensitive protein kinases, in contrast to 1,25(OH)₂D₃, whose effects were not reduced by this agent. These results suggest that: (a) the actions of 1,25(OH)₂D₃ may not be directly dependent on calcium influx; (b) in OB cell response to PTH a relationship probably exists between cAMP and calcium; and (c) this relationship may involve calmodulin, or calcium-dependent protein kinases that can be inhibited by TFP.