A. J. El Haj

Bone marrow stromal cells are load responsive in vitro

Mechanical load-related effects on bone marrow stromal cellsin vitro have been investigated. A dose response of a cyclical load of 1 Hz between 350 ustrain and 2500 ustrain applied to 10-day-old cultures resulted in elevated alkaline phosphatase levels and the number of cells expressing this protein after 2 days. No significant changes in the number of cells expressing or the production of collagen type I was observed. A critical stage of development of the cultures must be reached before load-related elevation in alkaline phosphatase expression could be measured independent of the stage at which loading was applied. Using a prostaglandin inhibitor at concentrations previously usedin vivo, the load response was abolished. We have demonstrated that bone marrow stromal cells are load responsive in culture and have made preliminary studies into determining the involvement of prostaglandins in this process.

Load-induced proteoglycan orientation in bone tissuein vivo andin vitro

SummaryPrevious studies of Alcian blue-induced birefringence in adult avian cortical bone showed that a short period of intermittent loading rapidly produces an increased level of orientation of proteoglycans within the bone tissue. In the absence of further loading, this persists for over 24 hours. We have proposed that this phenomenon could provide a means for “capturing” the effects of transient strains, and so provide a persistent, constantly updated strain-related influence on osteocyte populations related to the bones averaged recent strain history, in effect, a “strain memory” in bone tissue. In our present study, we use the Alcian blue-induced birefringence technique to demonstrate that proteoglycan orientation also occurs after intermittent loading of both cortical and cancellous mammalian bonein vivo andin vitro. We also show that the change in birefringence is proportional to the magnitude of the applied strain, and that the reorientation occurs rapidly, reaching a maximal value after only 50 loading cycles. Examination of electron micrographs of bone tissue after staining with cupromeronic blue allows direct visualization and quantification of the change in proteoglycan orientation produced by loading. This shows that intermittent loading is associated with a realignment of the proteoglycan protein cores, bringing them some 5 degrees closer to the direction of collagen fibrils in the bone matrix.

Seasonal and latitudinal adaptation to temperature in crustaceans

Abstract Eurythermal crustaceans survive seasonal, diurnal or tidal changes in environmental temperature by developing several capacity and resistance adaptations to change their behaviour, physiology, growth and metabolism. In temperature climates, marked physiological differences have been observed between summer and winter crayfish with seasonal changes in haemocyanin oxygen affinity and changes in the relationship between pH and temperature. Rates of protein synthesis in the leg and abdominal muscles of the native British crayfish, Austropotamobius pallipes , decreased with temperature at an exaggerated Q 10 value of 5–10 suggesting metabolic depression at the low winter temperatures. A similar response was noted in the stenothermal Antarctic isopod, Glyptonotus antarcticus , as whole animal rates of protein synthesis were extremely low in this species when compared to data collected from the temperate marine isopod, Idotea rescata . This was due to the relatively high energetic cost of protein synthesis in G. antarcticus in association with low rates of oxygen uptake. Temperature also has an effect on rates of transcription of several proteins in the muscle, including actin and myosin heavy chain (MHC), with an increase in levels of expression as temperature increases in temperate and Antarctic species. The potential role of heat shock proteins as an additional strategy in the response of temperate crustaceans to thermal stress is discussed.

Expression of voltage-operated Ca2+ channels in rat bone marrow stromal cells in vitro

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.

An ecdysteroid-responsive gene in a lobster - a potential crustacean member of the steroid hormone receptor superfamily.

The role of ecdysteroids in modulating exoskeletal growth during the moult cycle of Crustacea has been well described. However, little is known about the action of ecdysteroids at the level of gene transcription and regulation in Crustacea. This paper reports the cloning of an ecdysteroid responsive gene, HHR3, a potential Manduca sexta MHR3 homologue in the American lobster, Homarus americanus. Levels of HHR3 expression are up-regulated in response to in vivo injections of premoult concentrations (10(-6) M) of 20-hydroxyecdysone in the epidermal and muscle tissue of the lobster after 6 h. Maximal mRNA levels are observed after 21 h before returning to basal levels. In muscle tissue, elevated levels of HHR3 mRNA follow a time course similar to elevated actin mRNA expression in response to hormonal injection. In contrast, in eyestalk tissue, the HHR3 levels decline up to 21 h post-injection before rising to basal levels after 48 h. Eyestalk, epidermal and leg muscle tissue was extracted over the moult cycle to determine the levels of expression. In muscle, HHR3 is high during the premoult period that corresponds to the period of the moult cycle when the ecdysteroid titre is high. In the epidermis, HHR3 levels are also high during the premoult with elevated levels maintained into the postmoult period. In the eyestalk, mRNA levels of HHR3 show an opposite pattern of expression with low levels during premoult and postmoult and high levels found during the intermoult period. Our results provide novel evidence for an ecdysteroid responsive gene in a crustacean that has many similarities to MHR3 in Manduca and DHR3 in Drosophila melanogaster. This raises the question of whether a similar cascade of ecdysteroid responsive genes exist in other members of Arthropoda such as the Crustacea, as has been demonstrated in Drosophila. In addition, we provide further evidence for negative feedback regulation of ecdysteroids at the site of moult-inhibiting hormone (MIH) production in the lobster eyestalk.

Voltage‐dependent potentiation of low‐voltage‐activated Ca2+ channel currents in cultured rat bone marrow 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.

Regulation of Muscle Gene Expression Over the Moult in Crustacea

Abstract Muscle growth in crustacea occurs over the moult and involves a corresponding increase in fractional rates of sarcomeric protein synthesis. Heterologous and homologous cDNA probes for the myofibrillar protein, actin and myosin, have been used to investigate the factors responsible for controlling muscle growth in crustaceans at the molecular level. Factors such as passive stretch, caused by ecdysial expansion; moulting hormones such as ecdysteroids, which regulate the cycle of moult related events; and temperature, an environmental factor known to influence the rates of many biochemical reactions, all play a role in modulating muscle growth in crustacea. In isopod crustaceans, the regulation of muscle growth produces new problems as these crustaceans are characterised by a biphasic moult cycle in which the posterior half of the old exoskeleton is shed hours to days before the anterior half. These animals therefore provide an ideal model for the study of differential muscle growth. In both decapod and isopod crustaceans, transcriptional regulation alone may not be sufficient to account for the the upregulation of sarcomeric protein synthesis during intermittent growth. Both passive stretch and elevated ecdysteroid titre increase protein synthesis rates for actin in lobster muscles, in vivo. These factors may be involved in the promotion of ribosomal activity and translational processing during ecdysial muscle growth. Temperature has a direct effect on protein synthesis rates and levels of actin mRNA expression and may be a principal factor in the control of seasonal moult cycles.

Haemolymph oxygen transport and acid-base status in Glyptonotus antarcticus eights

Haemolymph samples were withdrawn from routinely active male intermoult Glyptonotus held at 0u2009±u20090.5°C, and analysed for blood-gas and acid-base variables. In both the arterialised (a) and venous (v) haemolymph, over 50% of the oxygen was transported as dissolved oxygen at PaO2 and PvO2 levels of 12.0u2009±u20091.15 and 7.70u2009±u20091.89 kPa, respectively. The maximum oxygen-carrying capacity of the haemocyanin (CmaxHcO2) was relatively low at 0.19u2009±u20090.05u2009mmol l−1, accompanied by relatively low protein and [Cu2+] levels indicating low circulating haemocyanin concentrations. Arterialised haemolymph had a mean pHu2009of 7.88u2009±u20090.02(6) at a PCO2 of 0.12u2009±u20090.01(6) kPa and a bicarbonate level of 12.95u2009±u20090.80(6) mequiv l−1 with small differences in PCO2 and pHu2009between arterial and venous haemolymph. The non-bicarbonate buffering capacity of Glyptonotus haemolymph was low at −2.0 mequiv l−1 HCO3− pHu2009unit−1. Haemolymph [l-lactate] and [d-glucose] levels were similar at <u20091u2009mmol l−1 in animals held in the laboratory and those sampled in Antarctica. The blood-gas and acid-base status of Glyptonotus haemolymph may be a reflection of the low and stable temperatures experienced by this Antarctic crustacean.

Novel culture procedure permitting the synthesis of proteins by rat calvarial cells cultured on hydroxyapatite particles to be quantified

A simple culture procedure and assay conditions are described which have permitted us to quantify the synthesis of proteins which are associated with an osteoblastic phenotype, by rat calvarial periosteal cells grown on particulate materials. The main feature of the method is the use of an adhesive which does not permit cells to attach to itself but allows attachment and growth of cells on material particles embedded in it on glass coverslips. Cells were cultured for 27 d on hydroxyapatite particle-coated coverslips. Alkaline phosphatase, osteopontin and collagen type I were monitored in cell lysates from d 10 to d 20. After Western blotting, osteopontin and collagen type I were quantified using specific antisera and enhanced chemiluminescence. Maximum levels coincided with peak alkaline phosphatase activity, after 10 and 17 d. The procedures described will be generally applicable to the comparison of cell behaviour on particulate substrata.

Hormonal regulation of [Ca2+]i in periosteal-derived osteoblasts: effects of parathyroid hormone, 1,25(OH)2D3 and prostaglandin E2

The effects of hormonal modulators of osteoblast function, parathyroid hormone, 1,25(OH)2D3 and prostaglandins on [Ca2+]i in periosteal‐derived osteoblasts from rat femurs have been investigated. Our results show that application of parathyroid hormone PTH (10−5 M) and prostaglandin E2 (PGE2) (4 μM) result in a rapid heterogeneous elevation in [Ca2+]i that, in the case of PTH, is dependent on both extracellular and intracellular sources of calcium. Variable responses to treatments have been found within populations of cells. The PGE2 response is dose dependent. Treatment with 1,25(OH)2D3 (10−8 M) induces a brief (60–90 sec) elevation in [Ca2+]i that is almost totally abolished in EGTA‐buffered Ca2+‐free medium. Interactive effects of multiple hormone treatments have been observed. Pretreatment with 1,25(OH)2D3 results in near‐total inhibition of the PTH and PGE2 responses. In conclusion, modulation of [Ca2+]i appears to play a role not only in the direct effects of osteotropic hormones on osteoblasts but also in the synergistic and antagonistic effects between circulating hormones. J. Cell. Physiol. 183:163–171, 2000.