Rodent osteoblastic cells express voltage-sensitive calcium channels lacking a gamma subunit.

Voltage-sensitive calcium channels (VSCC) open in response to external stimuli, including calcitropic hormones, that alter plasma membrane calcium (Ca2+) permeability. Ca2+ that enters the cell through these channels serves a second messenger function, eliciting cellular responses that include secretion and changes in gene expression. In osteoblasts, VSCCs serve as key regulators of Ca2+ permeability and are a major class of calcitropic hormone-sensitive Ca2+ channels present in the plasma membrane. The members of the VSCC family exist as a complex of polypeptide subunits that are comprised of a pore-forming alpha1 subunit, an intracellular beta subunit, a dimer of disulfide-linked alpha2 and delta subunits, and in some tissues, a gamma subunit. Previous studies in our laboratory have shown that the major functional alpha1 subunit present in osteoblasts is the alpha1C (CaV1.2). To determine the complement of auxiliary subunits present in rodent osteoblastic cells, we employed RT-PCR using a battery of subunit specific primers and appropriate tissue controls. Immunohistochemistry also was performed, using available subunit specific antibodies, to measure protein expression and localization. Cell types examined included MC3T3-E1 at various stages of differentiation, ROS 17/2.8 osteosarcoma, and primary cultures of rat calvarial osteoblasts. The results indicate that all cells expressed multiple beta subunit classes and alpha2delta dimers, but no gamma subunits, regardless of differentiation state. We propose a structure for the functional osteoblast VSCC that consists of alpha1, beta, alpha2delta subunits and is devoid of a gamma subunit.

Expression of the heparan sulfate proteoglycan, perlecan, during mouse embryogenesis and perlecan chondrogenic activity in vitro.

Expression of the basement membrane heparan sulfate proteoglycan (HSPG), perlecan (Pln), mRNA, and protein has been examined during murine development. Both Pln mRNA and protein are highly expressed in cartilaginous regions of developing mouse embryos, but not in areas of membranous bone formation. Initially detected at low levels in precartilaginous areas of d 12.5 embryos, Pln protein accumulates in these regions through d 15.5 at which time high levels are detected in the cartilage primordia. Laminin and collagen type IV, other basal lamina proteins commonly found colocalized with Pln, are absent from the cartilage primordia. Accumulation of Pln mRNA, detected by in situ hybridization, was increased in d 14.5 embryos. Cartilage primordia expression decreased to levels similar to that of the surrounding tissue at d 15.5. Pln accumulation in developing cartilage is preceded by that of collagen type II. To gain insight into Pln function in chondrogenesis, an assay was developed to assess the potential inductive activity of Pln using multipotential 10T1/2 murine embryonic fibroblast cells. Culture on Pln, but not on a variety of other matrices, stimulated extensive formation of dense nodules reminiscent of embryonic cartilaginous condensations. These nodules stained intensely with Alcian blue and collagen type II antibodies. mRNA encoding chondrocyte markers including collagen type II, aggrecan, and Pln was elevated in 10T1/2 cells cultured on Pln. Human chondrocytes that otherwise rapidly dedifferentiate during in vitro culture also formed nodules and expressed high levels of chondrocytic marker proteins when cultured on Pln. Collectively, these studies demonstrate that Pln is not only a marker of chondrogenesis, but also strongly potentiates chondrogenic differentiation in vitro.

Calcium signals and calcium channels in osteoblastic cells.

Calcium (Ca2+) channels are present in non-excitable as well as in excitable cells. In bone cells of the osteoblast lineage, Ca2+ channels play fundamental roles in cellular responses to external stimuli including both mechanical forces and hormonal signals. They are also proposed to modulate paracrine signaling between bone-forming osteoblasts and bone-resorbing osteoclasts at local sites of bone remodeling. Calcium signals are characterized by transient increases in intracellular Ca2+ levels that are associated with activation of intracellular signaling pathways that control cell behavior and phenotype, including patterns of gene expression. Development of Ca2+ signals is a tightly regulated cellular process that involves the concerted actions of plasma membrane and intracellular Ca2+ channels, along with Ca2+ pumps and exchangers. This review summarizes the current state of knowledge concerning the structure, function, and role of Ca2+ channels and Ca2+ signals in bone cells, focusing on the osteoblast.

Down-regulation of L-type Ca2+ channel transcript levels by 1,25-dihyroxyvitamin D3. Osteoblastic cells express L-type alpha1C Ca2+ channel isoforms.

Osteoblast Ca2+ channels play a fundamental role in controlling intracellular and systemic Ca2+ homeostasis. A reverse transcription-polymerase chain reaction strategy was used to determine the molecular identity of voltage-sensitive calcium channels present in ROS 17/2.8 osteosarcoma cells. The amino acid sequences encoded by the two resultant PCR products matched the alpha1C-a and the alpha1C-d isoforms. The ability of 1, 25-dihydroxyvitamin D3 (1,25(OH)2D3) and structural analogs to modulate expression of voltage-sensitive calcium channel mRNA transcripts was then investigated. ROS 17/2.8 cells were cultured for 48 h in the presence of either 1,25(OH)2D3,1,24-dihydroxy-22-ene-24-cyclopropyl D3 (analog BT) or 25-hydroxy-16-ene-23-yne-D3 (analog AT), and the levels of mRNA encoding alpha1C were quantitated using a competitive reverse transcription-polymerase chain reaction assay. We found that 1, 25(OH)2D3 and analog BT reduced steady state levels of alpha1C mRNA. Conversely, the Ca2+-mobilizing analog AT did not alter steady state levels of voltage-sensitive calcium channel mRNA. Since analog BT, but not analog AT, binds and transcriptionally activates the nuclear receptor for 1,25(OH)2D3, these findings suggest that the down-regulation of voltage-sensitive calcium channel mRNA levels may involve the nuclear receptor.

Beta-adrenergic receptors in porcine adipocyte membranes: modification by animal age, depot site, and dietary protein deficiency.

Adipocyte lipid metabolism is primarily regulated by insulin and the catecholamines norepinephrine and epinephrine. Stimulation of the beta-adrenergic receptors (beta-AR) by catecholamines causes an increase in the rates of adipocyte lipid degradation and a decrease in the rates of lipid synthesis. These catabolic effects are in opposition to insulin, which causes net anabolic effects. Because most of the postnatal development of adipose tissue mass in pigs results from hypertrophy of adipocytes (rapid in first few weeks of life) caused by increased net synthesis of triacylglycerol, there is interest in the modulation of beta-AR in adipocytes of growing pigs. The beta-AR are characterized by measuring ligand binding to the receptor to ascertain the affinity of the ligand for the receptor and the receptor number. We found the affinity of the receptor did not vary with animal age (10, 28, and 75 d), with adipose tissue depot site, or in adipocytes of protein-deficient pigs. The beta-AR in obese pigs tended to have greater affinity than those in crossbred pigs of the same age and weight. The beta-AR number was not different when expressed per milligram of adipocyte membrane protein in pigs of different age, in obesity, in different adipose tissue depots, or during protein deficiency. The number expressed per cell or per unit adipocyte surface area did not differ between depots or during protein deficiency. The number per cell tended to be greater in the larger cells from 75-d-old pigs than in the smaller cells from 10- and 28-d pigs. It was greatest in obese pigs with the largest adipocytes. Under the various experimental conditions (age, obesity, depot, protein deficiency), the membrane fatty acid composition was greatly different, but in most cases there was no modulation of beta-AR affinity.

Effect of sera from lean and obese pigs on the differentiation of porcine adipose stromal-vascular cells in culture.

Primary cultures of stromal-vascular (S-V) cells from adipose tissue of new-born pigs were used to evaluate the characteristics of four sera obtained from male and female genetically lean or obese pigs weighing 50-60 kg. (2) Lean pig sera (LPS) stimulated greater levels of sn-glycerol-3-phosphate dehydrogenase (GPDH) activity in cultured cells than obese sera (OPS). Male LPS tended to promote higher GPDH specific activity than female LPS or male and female OPS. When GPDH was expressed on a per DNA basis, male LPS significantly (P < 0.05) increased GPDH per unit DNA compared with female LPS or male and female OPS.

Effect of age on the differentiation of porcine adipose stromal-vascular cells in culture.

Stromal-vascular (S-V) cells isolated from adipose tissue of newborn pigs (NBPC) and mature pigs (MPC) by collagenase digestion were used to evaluate differences in preadipocyte culture and development. Cells were seeded at a density of 3 x 10(4) cells/cm2 on six-well (35-mm) tissue culture plates in 3 mL of DMEM/HAM's F12 medium plus 10% fetal calf serum and cultured at 37 degrees C under a humidified atmosphere of 95% air:5% CO2 for 24 h. Cells were then washed thoroughly in DMEM/HAM's F12 medium without fetal calf serum and maintained in serum free (SF) medium or SF medium supplemented with 2.5% newborn pig serum (NBPS) or mature pig serum (MPS) for 12 d. After 1 d, more NBPC adhered to the culture plates, as indicated by DNA values. After 12 d, protein per culture well was not significantly different, but DNA concentration per well remained higher (P < .05) in cultures of NBPC than in the MPC cultured in the same medium, indicating fewer MPC. Protein:DNA ratios were higher (P < .05) in cultures of MPC regardless of the medium, reflecting larger cell size. More cells containing fat deposits were seen with NBPC in all conditions in comparison with MPC, and more fat was deposited in NBPC in SF than in SF plus NBPS or MPS. The NBPC had higher (P < .05) sn-glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) per protein than MPC regardless of the medium. For both cell types, GPDH activity in either serum was less than activity of cells grown in SF.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of agonists and antagonists to the porcine adipose tissue beta-adrenergic receptor(s).

1. Affinities of agonists for porcine adipose tissue beta-adrenergic receptors, determined by competitive ligand binding with 3H-dihydroalprenolol to crude adipose tissue membranes in vitro, varied from 50 times > to 25 times < than isoproterenol. Affinities for antagonists varied from 8 times > to 1000 times < propranolol. 2. Receptor affinity was not related to the ability to stimulate or inhibit lipolysis, or to the agonist or antagonist purported receptor subtype specificity. 3. Modeling of ligand-binding data indicated more than one binding site for several ligands. The assignment of beta-adrenergic subtypes to the individual binding sites was unclear because this would depend on the individual ligands used to establish binding sites.

Effect of dietary cimaterol on Na(+)-K+ ATPase activity in rabbit tissues.

Cimaterol, a catecholamine analog that acts as a beta-adrenergic agonist, has been shown to have a growth promoting effect in rabbits. Catecholamines are also known to stimulate ion transport in many tissues. The objective of this study was to determine the effect of dietary cimaterol on rabbit tissue Na(+)-K+ ATPase activity. Twelve New Zealand White rabbits, fed either control diet or cimaterol-supplemented diet, were killed at the end of a 35-d feeding trial. Heart, kidney, and liver were weighed, and total ATPase and Na(+)-K+ ATPase activities of these tissues were determined. Cimaterol did not affect liver and kidney weights, but heart weight was significantly increased. Cimaterol increased both total and Na(+)-K+ ATPase activities in heart and kidney but had no effect in liver. These data indicate that increased dietary energy intake and body energy expenditure in cimaterol-fed animals can be attributed, in part, to elevated Na(+)-K+ ATPase activities in heart and kidney.

Effect of insulin and adrenergic agonists on glucose transport of porcine adipocytes.

1. Insulin increased basal 2-deoxyglucose uptake in isolated swine adipocytes by 75%. In the absence of insulin, isoproterenol did not inhibit basal 2-deoxyglucose transport. 2. Adenosine deaminase plus isoproterenol or theophylline alone reduced insulin effect by 10 and 40%, respectively. Isoproterenol alone or with 2-chloroadenosine did not inhibit insulin effect on glucose transport activity. 3. Insulin effect was inhibited by isoproterenol in the presence of theophylline but not in the presence of adenosine deaminase. 4. These results suggest that catecholamines do not counter-regulate basal and insulin-stimulated glucose transport in swine adipocytes.