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Voltage operated calcium channels (VOCCs) are important in stimulus-response coupling in osteoblasts. We have investigated the expression of VOCCs in the mouse osteocyte cell line, MLO-Y4. Using the whole-cell patch clamp technique we were unable to detect any VOCC currents (n = 436) even in the presence of the L-type VOCC agonist Bay K 8644 (n = 350). Reverse transcription polymerase chain reaction (RT-PCR), using primers to detect alpha(1C), alpha(1D), and alpha(1G) VOCC subunits (all of which are expressed in primary osteoblasts), did not generate detectable products with mRNA from MLO-Y4 cells. However, after treatment with physiological levels of hormones, VOCC alpha(1) subunit mRNAs were detected in MLO-Y4 cells. PTH, 17beta-estradiol, and dexamethasone-treatment induced expression of L-type (alpha(1C), alpha(1D)) subunit transcripts. ATP-treatment induced expression of T-type (alpha(1G)) transcripts. Using whole-cell patch clamp we detected VOCC currents in 5-10% of cells after treatment. Current characteristics (L- or T-type) were consistent with the transcript expressed., (Copyright 2001 Academic Press.)

Femur-derived osteoblasts cultured from rat femora were loaded with Fluo-3 using the AM ester. A quantifiable stretch was applied and [Ca(2+)]i levels monitored by analysis of fluorescent images obtained using an inverted microscope and laser scanning confocal imaging system. Application of a single pulse of tensile strain via an expandable membrane resulted in immediate increase in [Ca(2+)]i in a proportion of the cells, followed by a slow and steady decrease to prestimulation levels. Application of parathyroid hormone (10(-6) M) prior to mechanical stimulation potentiated the load-induced elevation of [Ca(2+)]i. Mechanically stimulating osteoblasts in Ca(2+)-free media or in the presence of either nifedipine (10 microM; L-type Ca(2+)-channel blocker) or thapsigargin (1 microM; depletes intracellular Ca(2+) stores) reduced strain-induced increases in [Ca(2+) ]i. Furthermore, strain-induced increases in [Ca(2+)]i were enhanced in the presence of Bayer K 8644 (500 nm), an agonist of L-type calcium channels. The effects of mechanical strain with and without inhibitors and agonists are described on the total cell population and on single cell responses. Application of strain and strain in the presence of the calcium-channel agonist Bay K 8644 to periosteal-derived osteoblasts increased levels of the extracellular matrix proteins osteopontin and osteocalcin within 24 h postload. This mechanically induced increase in osteopontin and osteocalcin was inhibited by the addition of the calcium-channel antagonist, nifedipine. Our results suggest an important role for L-type calcium channels and a thapsigargin-sensitive component in early mechanical strain transduction pathways in osteoblasts., (Copyright 2000 Wiley-Liss, Inc.)

Changes in strain distribution across the vertebrate skeleton induce modelling and remodelling of bone structure. This relationship, like many in biomedical science, has been recognised since the 1800s, but it is only the recent development of in vivo and in vitro models that is allowing detailed investigation of the cellular mechanisms involved. A number of secondary messenger pathways have been implicated in load transduction by bone cells, and many of these pathways are similar to those proposed for other load-responsive cell types. It appears that load transduction involves interaction between several messenger pathways, rather than one specific switch. Interaction between these pathways may result in a cascade of responses that promote and maintain bone cell activity in remodelling of bone. The paper outlines research on the early rapid signals for load transduction and, in particular, activation of membrane channels in osteoblasts. The involvement of calcium channels in the immediate load response and the modulation of intracellular calcium as an early signal are discussed. These membrane channels present a possible target for manipulation in the engineering of bone tissue repair.

The expression of voltage-operated Ca2+ currents (VOCCs) in bone marrow stromal cells cultured for 3-30 days has been studied by the use of the whole-cell patch-clamp technique. Both low-voltage-activated (LVA) and high-voltage-activated (HVA) VOCCs were recorded. LVA currents were first detectable after 6-7 days in culture and reached a peak of expression at 8 days, after which both the amplitude and frequency of expression of the current fell rapidly. The current was virtually undetectable in cells cultured for more than 15 days. The HVA current was detectable after 3 days in culture and reached a peak of both amplitude and frequency of expression after 1-2 weeks. This current was expressed consistently throughout the remaining culture period. In cultures treated with dexamethasone (10(-8) mol/L) peak expression of LVA currents still occurred at 7-8 days, but currents were enhanced approximately threefold. Expression of LVA currents was maintained to the end of the culture period. Expression of HVA currents was not significantly modified by treatment of cultures with dexamethasone. Examination of the biophysical and pharmacological (blockade by Ni2+ and diphenylhydantoin) properties of the LVA current in these cells suggests that they may have similarities with the LVA T currents of neuronal cells.

1. The whole-cell patch-clamp technique was used to study Ca2+ channel currents in stromal cells of 7-10 day dexamethasone-treated and control rat bone marrow cultures. In saline containing either 108 mM Ba2+ or a 2.5 mM Ca(2+)-1 mM Mg2+ mixture, most cells expressed both fast-inactivating, low-voltage-activated (LVA) and slow-inactivating, high-voltage-activated (HVA) currents. 2. Repeated application of 400 ms voltage steps to 60 mV above the holding potential (Vh, -90 mV in Ca(2+)-Mg2+ mixture and -60 mV in Ba2+) at a frequency > or = 0.1 Hz resulted in a potentiation of the LVA component of the 2nd and subsequent currents. 3. LVA current potentiation was examined using a two-pulse (prepulse-test pulse) method. Prepulses to Vh + 150 mV induced an 80-100% increase in the amplitude of the LVA component of Ca2+ channel currents in saline containing either Ba2+ or Ca(2+)-Mg2+. This effect was also seen in non-dexamethasone-treated cultures. 4. Potentiation was not modified by omission of ATP and GTP from the pipette saline, and was not inhibited by extracellular application of the broad spectrum kinase inhibitors H-7 or RK252-a. 5. Potentiation was dependent on the amplitude and duration of the prepulse. Using the standard protocol, the threshold for potentiation was approximately Vh + 45 mV and saturation occurred at Vh + 150-180 mV. Further increases in prepulse amplitude did not modify potentiation. With a prepulse to +10 mV (Ba2+ saline) potentiation was half-maximal with a prepulse duration of 250-300 ms duration and saturated at 750-1000 ms. 6. Peak potentiation occurred 1-2 s after the prepulse. The time for total decay of potentiation varied from 10 to 90 s. 7. Voltage dependency of prepulse-induced potentiation did not resemble that of inactivation induced by similar prepulses. 8. Current kinetics, I-V relationship and sensitivity to blockade by Ni2+ and diphenylhydantoin of prepulse-recruited current resembled those of control LVA current. 9. The amplitude of prepulse-recruited current was positively correlated with control LVA current amplitude. 10. LVA currents supported regenerative potentials under current clamp. Repeated activation reduced spike latency. 11. It is suggested that current potentiation may be recruited physiologically, possibly in association with activation of stretch-sensitive channels, causing enhanced activation of HVA Ca2+ currents.