R- and L-type Ca2+ channels are insensitive to eliprodil in rat cultured cerebellar granule neurons.

We have investigated, by using the whole-cell patch-clamp technique, the Ca2+ channel antagonist properties of eliprodil in cultured cerebellar granule cells which are known to express L-, N-, P- as well as Q- and R-type Ca2+ channels. Eliprodil maximally antagonized 50% of the voltage-dependent Ba2+ current with an IC50 of 4 microM. omega-Conotoxin-GVIA (3.2 microM) and omega-agatoxin-IVA (0.5 microM) blocked 28 and 43% of the current, respectively. When eliprodil (30 microM) was added to omega-conotoxin-GVIA or omega-agatoxin-IVA the magnitude of the maximal inhibition was identical to that obtained with eliprodil alone confirming a full blockade by eliprodil of N-, P- and Q-type Ca2+ channels. The L-type channel antagonist nimodipine (10 microM) blocked 24% of the current; this blockade was fully additive to that of eliprodil, indicating that the nimodipine-sensitive component of the current is eliprodil-insensitive. In the presence of eliprodil and nimodipine a residual Cd2+ sensitive current (25%), identified as the R-type current, remained unblocked. We conclude that in cerebellar granule neurons R- and L-type Ca2+ channels are insensitive to eliprodil. The nimodipine-sensitive channels present in cerebellar granule neurons may represent a neuronal subtype of L channels distinct from that (eliprodil-sensitive/nimodipine-sensitive) present in cortical or hippocampal neurons.

Effects of spaceflight and recovery on rat humeri and vertebrae: histological and cell culture studies.

Skeletal changes associated with spaceflight in the rat have been well documented, but few data are available on bone tissue and bone cell metabolism after subsequent on-Earth recovery. We therefore investigated the effects of microgravity and subsequent recovery on trabecular bone morphology and cellular activities in rat humeri and thoracic vertebrae and compared histomorphometric parameters in caudal vertebrae with the behavior of vertebral osteoblastic cells in culture. We report here that humeral weight showed normal growth during the experiment but was unaffected by spaceflight or recovery from spaceflight. However, the 14-day spaceflight resulted in inhibition of static indexes of bone formation in humeral proximal metaphyses and thoracic vertebral bodies. This was associated with a decrease in bone volume in humeral metaphyses. After 14 days of on-Earth recovery, osteoblastic and osteoid surfaces returned toward normal and bone volume was normalized in humeri, whereas the static bone formation parameters were not restored in thoracic vertebrae. In addition, histological indexes of bone formation and osteoblastic cell growth in vitro were not affected by spaceflight in caudal vertebrae. This study shows that rat humeri and thoracic and caudal vertebrae exhibit different patterns of response to spaceflight and subsequent on-Earth recovery, which could be due, at least in part, to the different loading pattern of these bones, and also to differences in bone turnover rate.

Effects of the tripeptide glycyl-L-histidyl-L-lysine copper complex on osteoblastic cell spreading, attachment and phenotype.

We have studied the effects of the complex Glycyl-L-Histidyl-L-Lysine:Cu (GHK:Cu), the GHK sequence present in the alpha 2 (I) chain of human collagen (Coll I), and bone matrix glycoproteins containing either RGD (fibronectin, FN), or RGD and GHK (Coll I), on the spreading, attachment and markers of the osteoblast phenotype in rat calvaria cells (RC), human trabecular osteoblastic cells (HT) and human marrow stromal cells (HM). Coll I (20 micrograms/ml) and FN (20 micrograms/ml) coating enhanced osteoblastic cell spreading, whereas free GHK:Cu and GHK coating (10(-10)-10(-8) M) had no effect. FN and Coll I, as well as GHK:Cu and GHK, increased the attachment of RC and HT cells. The attachment of both total number of cells and alkaline phosphatase (ALP)-positive osteoblastic cells was increased, showing no preferential effect on cells expressing this early marker of the osteoblast phenotype. In addition, immunocytochemical analysis showed that FN, Coll I and GHK:Cu coating increased both the total number of HM cells and the number of HM cells expressing Coll I or osteocalcin, indicating that GHK:Cu and RGD-containing proteins acted similarly on cells expressing different maturational stages. In contrast to its effect on cell attachment, GHK:Cu coating slightly inhibited the basal and 1,25(OH)2D-induced stimulation of ALP activity or osteocalcin production in rat and human osteoblastic cells. The finding that GHK promotes cell attachment and decreases the phenotype of normal rat and human osteoblastic cells suggests that osteoblasts may interact with free GHK or GHK-containing proteins in the bone matrix.

Involvement of interleukin 1 and tumour necrosis factor alpha as endogenous growth factors in human osteoblastic cells.

Normal human osteoblastic (OB) cells produce several haematopoietic factors, including IL-1, IL-6, PGE2 and TNF-alpha. However, it is unknown whether these factors play a role as autocrine mitogenic factors. We show here that some of these cytokines released by OB cells are endogenous growth factors for OB cells. Conditioned medium (CM) obtained from quiescent OB cells, dose-dependently stimulated OB cell proliferation, suggesting the production of autocrine growth factors by OB cells. Treatment with exogenous rhIL-1 and rhTNF-alpha increased OB cell growth. We found that neutralizing antibodies against IL-1 and TNF-alpha at concentrations that specifically inhibited the mitogenic activity of these cytokines, suppressed part of the mitogenic effect of CM on quiescent OB cells cultured at low or high density. In contrast, treatment of OB cells with indomethacin at a dose (10(-6) M) that inhibits endogenous prostaglandin production, increased OB cell proliferation in the presence or absence of CM, indicating that the mitogenic effect of CM on OB cells was not due to PGE2. In addition, exogenous recombinant human (rh)IL-6, or a specific neutralizing anti-IL-6 antibody, did not affect the OB cell proliferation. The results indicate that, in contrast to PGE2 and IL-6, IL-1 and TNF-alpha released by OB cells act as endogenous mitogenic factors for human osteoblasts.

Systemic administration of transforming growth factor-beta 2 prevents the impaired bone formation and osteopenia induced by unloading in rats.

We investigated the effect of recombinant human transforming growth factor beta 2 (rhTGF-beta 2) administration on trabecular bone loss induced by unloading in rats. Hind limb suspension for 14 d inhibited bone formation and induced osteopenia as shown by decreased bone volume, calcium and protein contents in long bone metaphysis. Systemic infusion of rhTFG-beta 2 (2 micrograms/kg per day) maintained normal bone formation rate, and prevented the decrease in bone volume, bone mineral content, trabecular thickness and number induced by unloading. In vitro analysis of tibial marrow stromal cells showed that rhTGF-beta 2 infusion in unloaded rats increased the proliferation of osteoblast precursor cells, but did not affect alkaline phosphatase activity or osteocalcin production. Northern blot analysis of RNA extracted from the femoral metaphysis showed that rhTGF-beta 2 infusion in unloaded rats increased steady-state levels of type I collagen mRNA but not alkaline phosphatase mRNA levels. rhTGF-beta 2 infusion at the dose used had no effect on metaphyseal bone volume and formation, osteoblast proliferation or collagen expression in control rats. The results show that systemic administration of rhTGF-beta 2 enhances osteoblast precursor cell proliferation and type I collagen expression by osteoblasts, and prevents the impaired bone formation and osteopenia induced by unloading.

Size- and density-dependent elution of normal and pathological red blood cells by gravitational field-flow fractionation.

Elution of normal and pathological human red blood cells (RBCs) was performed by gravitational field-flow fractionation (GFFF). The reproducibility of the retention factor was lower than 10% and elution at high and low flow-rates confirmed the existence of "lifting forces". No direct correlation between size and retention was observed for normal RBCs in the absence of density information. Elution of pathological human RBCs, known to be modified in shape, density and rigidity, was performed. The elution parameters confirmed that the retention mechanism of RBCs is at least density dependent but that other factors can be involved, such as shape or deformity. Moreover, peak profile description parameters (standard deviation and asymmetry) can be qualitatively related to some biophysical parameters. Numerous elution characteristics can be linked to cell properties described in the literature and although GFFF appeared to have limited capabilities in terms of size analysis it appeared to be a versatile tool for studying cell biophysical characteristics.