Beneficial effect of zinc supplementation on biomechanical properties of femoral distal end and femoral diaphysis of male rats chronically exposed to cadmium

The present study was aimed at estimate, based on the rat model of human moderate and relatively high chronic exposure to cadmium (Cd), whether zinc (Zn) supplementation may prevent Cd-induced weakening in the bone biomechanical properties. For this purpose, male Wistar rats were administered Cd (5 or 50 mg/l) or/and Zn (30 or 60 mg/l) in drinking water for 6 and 12 months. Bone mineral density (BMD) and biomechanical properties (yield load, ultimate load, post-yield load, displacement at yield and at ultimate, stiffness, work to fracture, yield stress, ultimate stress and Young modulus of elasticity) of the femoral distal end and femoral diaphysis were examined. Biomechanical properties of the distal femur were estimated in a compression test, whereas those of the femoral diaphysis -- in a three-point bending test. Exposure to Cd, in a dose and duration dependent manner, decreased the BMD and weakened the biomechanical properties of the femur at its distal end and diaphysis. Zn supplementation during Cd exposure partly, but importantly, prevented the weakening in the bone biomechanical properties. The favorable Zn influence seemed to result from an independent action of this bioelement and its interaction with Cd. However, Zn supply at the exposure to Cd had no statistically significant influence on the BMD at the distal end and diaphysis of the femur. The results of the present paper suggest that Zn supplementation during exposure to Cd may have a protective influence on the bone tissue biomechanical properties, and in this way it can, at least partly, decrease the risk of bone fractures. The findings seem to indicate that enhanced dietary Zn intake may be beneficial for the skeleton in subjects chronically exposed to Cd.     

Apparent Young's modulus of human radius using inverse finite-element method

The ability to assess the elastic and failure properties of cortical bone at the radial diaphysis has a clinical importance. A new generation of quantitative ultrasound (QUS) devices and peripheral quantitative computed tomography (p-QCT) has been developed to assess non-invasively bone material and structural properties at the distal radius. This anatomical site is characterized by a thin cortical thickness that complicates traditional mechanical testing methods on specimens. Until now, mechanical properties of cortical bone at distal radius (e.g., elastic modulus, yield stress and strain) remain rarely studied probably due to experimental difficulties. The present study introduces an inverse finite-element method strategy to measure the elastic modulus and yield properties of human cortical specimens of the radial diaphysis. Twenty millimeter-thick portions of diaphysis were cut from 40 human radii (ages 45-90) for biomechanical test. Subsequently the same portion was modeled in order to obtain a specimen-specific three dimensional finite-element model (3D-FEM). Longitudinal elastic constants at the apparent level and stress characterizations were performed by coupling mechanical parameters with isotropic linear-elastic simulations. The results indicated that the mean apparent Young's modulus for radial cortical bone was 16 GPa (SD 1.8) and the yield stress was 153 MPa (SD 33). Breaking load was 12,946 N (SD 3644), cortical thickness 2.9 mm (SD 0.6), structural effective strain at the yield (epsilon(y)=0.0097) and failure (epsilon(u)=0.0154) load were also calculated. The 3D-FEM strategy described here may help to investigate bone mechanical properties when some difficulties arise from machining mechanical sample.     

Hypercholesterolemia suppresses Kir channels in porcine bone marrow progenitor cells in vivo

OBJECTIVE: Inwardly-rectifying K(+) (Kir) channels are responsible for maintaining membrane potentials in a variety of cell types including endothelial cells where they modulate endothelium-dependent vasorelaxation. The goal of this study is to determine the functional expression of Kir channels in porcine bone marrow-derived side population (BM-SP) cells that demonstrate phenotypes of endothelial progenitor cells (EPCs). We further asses the hypercholesterolemia sensitivity of Kir channels in BM-SP cells, which may play a key role in hypercholesterolemia-mediated regulation of EPCs. METHODS: To assess the effect of hypercholesterolemia on Kir channels in BM-SP, Kir currents were recorded in SP cells sorted from the bone marrow of healthy or hypercholesterolemic animals. RESULTS: We found Kir channels constitute the major conductance in porcine bone marrow-derived side population (BM-SP) cells. These cells are defined by their efficiency of Hoechst dye efflux and have been reported to differentiate into multiple cell lineages including endothelium in vivo. We demonstrate here that porcine BM-SP cells differentiate to an endothelial lineage (CD31(+), vWF(+)) supporting the hypothesis that these cells are endothelial progenitor cells. Also, BM-SP cells express Kir with biophysical properties recapitulating those in mature endothelial cells, but with a much higher current density. Flow cytometric (FACS) analysis indicated that the number of SP cells was unaffected by hypercholesterolemia. However, hypercholesterolemia significantly inhibited Kir channels in BM-SP cells. CONCLUSIONS: We successfully demonstrate that BM side population cells represent an origin of endothelial progenitor cells. This study further shows, for the fist time, that the functional expression of Kir channels in bone marrow (BM)-derived SP. Moreover, we demonstrate that hypercholesterolemia condition significantly suppresses the Kir channels in BM-SP cells, suggesting that hypercholesterolemia-mediated regulation of Kir channels may be an important factor not only in dysfunction of mature endothelium but also in dysfunction of BM-SP cells.     

辽宁建平古人类肱骨形态结构分析

1957年,在辽宁省建平县发现了一根古人类肱骨化石,编号PA103。通过同一批龙骨中筛选的哺乳动物化石,吴汝康推断PA103应该为更新世晚期古人类,并对该化石进行了表面形态特征观察和描述。为了对PA103化石的内外结构进行更全面的了解,除了线性测量数据的对比,本文还通过计算机断层扫描技术,结合生物力学和形态示量图分析对建平古人类右侧肱骨化石PA103进行了分析。通过本研究发现,PA103骨干横断面的生物力学粗壮度和力学形状指数明显小于尼安德特人,而与同时期欧亚大陆古人类不利手侧最为接近,这说明建平人右侧肱骨可能不是惯用手,同时,建平人的行为活动应该与同时期同地区的古人类处于同一水平,而小于尼安德特人。整体来看,PA103骨干骨密质厚度和截面惯性矩与近现代人的分布模式较为接近,除局部数值增大外,其整体数值小于近现代人的平均水平,这可能与遗传或行为活动有关,由于缺少古人类化石对比数据,更详细的了解还需后期开展更多相关的研究。     

The bone behind a low areal bone mineral density: peripheral quantitative computed tomographic analysis in a woman with osteogenesis imperfecta

Areal bone mineral density (BMD) is the most widely used densitometric parameter. However, this approach makes it difficult to understand the structural basis of bone diseases, because a large number of bone properties are integrated into a single number. This is exemplified in the present case of a 27-year-old woman with osteogenesis imperfecta type I. Peripheral quantitative computed tomographic analysis at the radial metaphysis and at the radial diaphysis revealed a decreased areal BMD at both sites (z score -3.9 and -3.4, respectively). Yet, the structural basis for this decrease was different for the two locations: At the distal radius areal BMD was decreased because volumetric BMD was very low, whereas bone size was above the mean of the reference range. At the proximal radius areal BMD was decreased, because bone size was very low but volumetric BMD was above average. Bone mineral content of the radial diaphysis was very low for forearm muscle size, a finding which is compatible with Frost's hypothesis that the mechanostat setpoint is increased in osteogenesis imperfecta.     

A proteomic analysis of adult rat bone reveals the presence of cartilage/chondrocyte markers

The non-mineral component of bone matrix consists of 90% collagenous, 10% non-collagenous proteins. These proteins regulate mineralization, growth, cell signaling and differentiation, and provide bone with its tensile strength. Expression of bone matrix proteins have historically been studied individually or in small numbers owing to limitations in analytical technologies. Current mass-spectrometric and separations technologies allow a global view of protein expression patterns in complex samples. To our knowledge, no proteome profile of bone matrix has yet been reported. Therefore, we have used mass spectrometry as a tool to generate a profile of proteins present in the extracellular matrix of adult rat bone. Overall, 108 and 25 proteins were identified with high confidence in the metaphysis and diaphysis, respectively, using a bottom up proteomic technique. Twenty-one of these proteins were present in both the metaphysis and diaphysis including the bone specific proteins, osteocalcin, type I collagen, osteopontin, osteoregulin, and bone sialoprotein. Interestingly, type II collagen, a protein thought to be exclusively expressed in cartilage, was identified in both the metaphysis and diaphysis. This observation was validated by Western blot. Additionally, the presence of aggrecan, another protein expressed in cartilage was identified in the bone matrix extracts by Western blot. The proteome profile generated using this technology represents an initial survey of the acid soluble proteins of bone matrix which provides a reference for the analysis of deviations from the normal composition due to perturbations or disease states.     

Comparative effects of alendronate and alfacalcidol on cancellous and cortical bone mass and bone mechanical properties in ovariectomized rats.

The purpose of the present study was to compare the effects of alendronate and alfacalcidol on cancellous and cortical bone mass and bone mechanical properties in ovariectomized rats. Twenty-six female Sprague-Dawley rats, 7 months of age, were randomized by the stratified weight method into four groups: the sham-operated control (Sham) group and the three ovariectomy (OVX) groups, namely, OVX + vehicle, OVX + alendronate (2.5 mg/kg, p.o., daily), and OVX + alfacalcidol (0.5 mug/kg, p.o., daily). At the end of the 8-week experimental period, bone histomorphometric analyses of cancellous bone at the proximal tibial metaphysis and cortical bone at the tibial diaphysis were performed, and the mechanical properties of the femoral distal metaphysis and femoral diaphysis were evaluated. OVX decreased cancellous bone volume per total tissue volume (BV/TV), and the maximum load of the femoral distal metaphysis, as a result of increases in serum osteocalcin (OC) levels, and also the number of osteoclasts (N.Oc), osteoclast surface (OcS) and bone formation rate (BFR) per bone surface (BS), and BFR/BV, without any effect on cortical area (Ct Ar), or maximum load of the femoral diaphysis. Alendronate prevented this decrease in cancellous BV/TV by suppressing increases in N.Oc/BS, OcS/BS, BFR/BS, and BFR/BV, without any apparent effect on Ct Ar, or maximum load of the femoral distal metaphysis and femoral diaphysis. On the other hand, alfacalcidol increased cancellous BV/TV, Ct Ar, and the maximum load of the femoral distal metaphysis and femoral diaphysis, by mildly decreasing trabecular BFR/BV, maintaining trabecular mineral apposition rate and osteoblast surface per BS, increasing periosteal and endocortical BFR/BS, and preventing an increase in endocortical eroded surface per BS. The present study clearly showed the differential skeletal effects of alendronate and alfacalcidol in ovariectomized rats. Alendronate prevented OVX-induced cancellous bone loss by suppressing bone turnover, while alfacalcidol improved cancellous and cortical bone mass and bone strength by suppressing bone resorption and maintaining or even increasing bone formation.     

Semiautomatic extraction of cortical thickness and diaphyseal curvature from CT scans

The understanding of locomotor patterns, activity schemes, and biological variations has been enhanced by the study of the geometrical properties and cortical bone thickness of the long bones measured using CT scan cross‐sections. With the development of scanning procedures, the internal architecture of the long bones can be explored along the entire diaphysis. Recently, several methods that map cortical thickness along the whole femoral diaphysis have been developed. Precise homology is vital for statistical examination of the data; however, the repeatability of these methods is unknown and some do not account for the curvature of the bones. We have designed a semiautomatic workflow that improves the morphometric analysis of cortical thickness, including robust data acquisition with minimal user interaction and considering the bone curvature. The proposed algorithm also performs automatic landmark refinement and rigid registration on the extracted morphometric maps of the cortical thickness. Because our algorithm automatically reslices the diaphysis into 100 cross‐sections along the medial axis and uses an adaptive thresholding method, it is usable on CT scans that contain soft tissues as well as on bones that have not been oriented specifically prior to scanning. Our approach exhibits considerable robustness to error in user‐supplied landmarks, suppresses distortion caused by the curvature of the bones, and calculates the curvature of the medial axis.     

Animal welfare in modern dog breeding

Background

While gross morphological changes in the skeleton between males and females are well know, differences between sexes in the histomorphology are less known. It is important to have knowledge on the bone structure of rabbits, as this is a widely used species in biomedical research. A study was performed to evaluate the association between sex and the compact bone morphology of the femoral diaphysis in juvenile rabbits.

Methods

Seventeen clinically healthy 2–3 month-old rabbits (9 females, 8 males) were included in the study. The rabbits were euthanized and the right femur was sampled for analysis. 70–80 microns thick bone sections of the femoral diaphysis were prepared using standard histological equipment. The qualitative histological characteristics were determined according to internationally accepted classification systems while the quantitative parameters were assessed using the software Scion Image. Areas, perimeters, minimum and maximum diameters of primary osteons' vascular canals, Haversian canals and secondary osteons were measured. Additionally, blood plasma concentrations of progesterone, corticosterone, IGF-I, testosterone and estradiol were analyzed.

Results

Qualitative histological characteristics were similar for both sexes. However, variations of certain quantitative histological characteristics were identified. Measured parameters of the primary osteons' vascular canals were higher in males than for females. On the other hand, females had significant higher values of secondary osteons parameters. Differences in Haversian canals parameters were only significant for minimum diameter.

Conclusion

The study demonstrated that quantitative histological characteristics of compact bone tissue of the femoral diaphysis in juvenile rabbits were sex dependent. The variations may be associated with different growth and modeling of the femur through influence by sex-specific steroids, mechanical loads, genetic factors and a multitude of other sources. The results can be applied in experimental studies focusing on comparison of the skeletal biology of the sexes.     

An assessment of ADAMs in bone cells: absence of TACE activity prevents osteoclast recruitment and the formation of the marrow cavity in developing long bones

ADAMs (A Disintegrin And Metalloprotease domain) are metalloprotease-disintegrin proteins that have been implicated in cell adhesion, protein ectodomain shedding, matrix protein degradation and cell fusion. Since such events are critical for bone resorption and osteoclast recruitment, we investigated whether they require ADAMs. We report here which ADAMs we have identified in bone cells, as well as our analysis of the generation, migration and resorptive activity of osteoclasts in developing metatarsals of mouse embryos lacking catalytically active ADAM 17 [TNFalpha converting enzyme (TACE)]. The absence of TACE activity still allowed the generation of cells showing an osteoclastic phenotype, but prevented their migration into the core of the diaphysis and the subsequent formation of marrow cavity. This suggests a role of TACE in the recruitment of osteoclasts to future resorption sites.     

Autologous mesenchymal stem cells loaded in Gelfoam<Superscript>®</Superscript> for structural bone allograft healing in rabbits

This study was designed to evaluate the effect of autologous bone marrow mesenchymal stem cells (MSCs) seeded into Gelfoam^® on structural bone allograft healing. Thirty New Zealand white rabbits were divided into two groups. Segmental bone defect was created on diaphysis of the femur, and the defect was reconstructed with structural bone allograft. In experimental group, structural allograft was wrapped around by Gelfoam^® containing autologous MSCs, whereas cells were not included in control group. At 4, 8, 12 weeks, the femur of rabbits underwent radiographic and histologic evaluation for bony union. Bone morphogenic protein-2 (BMP-2), BMP-4, BMP-7, vascular endothelial growth factor (VEGF), and receptor activator of nuclear factor-kappa B ligand (RANKL) were measured within the grafted periosteal tissue. Bony union was not achieved in both groups at 4 and 8 weeks. At 12 weeks, three out of five femurs in experimental group were united, but one out of five in control group was united. Mean Taira scores were significantly different between two groups. The expression of BMP-2 was significantly higher at 4, 8 weeks, the expressions of BMP-4 and BMP-7 were significantly higher at 8 and 12 weeks, and the expression of VEGF and RANKL were significantly higher at all time points in experimental group. Incorporation of the structural bone allograft could be enhanced if allograft is covered with Gelfoam^® containing autologous MSCs. MSCs have influence on not only bone formation, but neo-angiogenesis, and bone resorption.     

Inhibition of prostate cancer-meditated osteoblastic bone lesions by the low-calcemic analog 1alpha-hydroxymethyl-16-ene-26,27-bishomo-25-hydroxy vitamin D3

Prostate cancer metastasizes almost exclusively into the bone whereby it induces primarily an osteoblastic response. Non-calcemic vitamin D analogs have been shown to inhibit proliferation of prostate cancer cells in culture and inhibit their growth as subcutaneous xenografts in mice. However, their effect on prostate cancer cell growth in the bone has not been examined. In the present study, we inoculated the osteoblastic prostate cancer cell line MDA-PCa 2b into the bone of male SCID mice and examined the effect of the low-calcemic hybrid analog 1alpha-hydroxymethyl-16-ene-26,27-bishomo-25-hydroxy vitamin D(3) (JK-1626-2) on their ability to induce bone lesions. We found that 7 weeks after inoculation of MDA-PCa 2b cells, 90% of the mice in the vehicle-treated group had significant bone lesions that were detectable by micro-computed tomography and characterized by thickening of the cortical bone and ossification of the epiphysis. Only 30% of the mice in the analog-treated group (daily injections of 4microg/kg, 5 days/week for up to 7 weeks) had detectable bone lesions. Histological examination of the decalcified tumor-bearing bones has shown that tumor cells completely replaced the bone marrow in the diaphysis, and destroyed the trabecular bone in the metaphysis in 90% of the vehicle-treated mice. In contrast, the metaphysis of 60% of analog-treated mice appeared normal, although tumor cells were still found in the diaphysis of 70% of the bones in the analog-treated group. There was no evidence of hypercalcemia in any of the analog-treated mice. In a co-culture, MDA-PCa 2b cells induced a profound mitogenic response in osteoblasts followed by enhanced differentiation. However, in the presence of the analog the mitogenic response of the osteoblasts to the malignant cells was significantly attenuated. These experiments led to the hypothesis that, in vivo, JK-1626-2 prevented the metastatic bone lesions by inhibiting the mitogenic response of osteoblasts to growth factors produced by MDA-PCa 2b cells.     

Molecular basis of ATP-sensitive K+ channels in rat vascular smooth muscles

ATP-sensitive K+ (K(ATP)) channels couple metabolic changes to membrane excitability in vascular smooth muscle cells (SMCs). While the electrophysiological properties of K(ATP) channels have been examined, little is known about the molecular basis of K(ATP) complex in vascular SMCs. We identified and cloned four K(ATP) subunit genes from rat mesenteric artery, namely rvKir6.1, rvKir6.2, rvKirSUR1, and rvSUR2B. These clones showed over 99.6% amino acid sequence identity with other previously reported isoforms. The mRNA expression patterns of the K(ATP) subunits varied among rat aorta, mesenteric artery, pulmonary artery, tail artery, hepatic artery, and portal vein. Heterologous co-expression of rvKir6.1 and rvSUR2B yielded functional K(ATP) channels that were inhibited by glibenclamide, and opened by pinacidil. Our results for the first time reported the expression of four K(ATP) subunits in same vascular tissues, unmasking the diversity of native K(ATP) channels in vascular SMCs.     

Cell population dynamics of periodontal ligament stimulated with parathyroid extract.

Young adult rats were injected with parathyroid extract (PTE). Periodontal ligament (PDL) adjacent to a previously resorbing alveolar bone surface was divided into four zones number I to IV, from bone to cementum. Zones I and IV were within 25 mmu of the bone and cementum surfaces, respectively, while a line bisecting the center of the PDL separated Zones II and III. A net increase of about 16 total nuclei in all zones was observed during the first five hours after PTE administration. Since local mitosis accounted for no more than two nuclei, approximately 14 cells apparently migrated into the area. Over the first five hours Zones I and II combined showed a 21-cell increase, being apparently the sole recipients of cells migrating into the field (14) plus approximately seven more from Zone III, which lost cells during the time period. The concurrent histological observation in Zone II, of increased intravascular monocytes and perivascular macrophages during the first five hours, suggests cells are migrating into Zone II via vascular channels. The data suggest two sources for increased PDL cellularity associated with stimulated osteoclast histogenesis: (1) local PDL cellular proliferation and (2) influx of migrating cells (probably through vascular channels) during first five hours after PTE.     

Functional plasticity of the human humerus: Shape,rigidity, and muscular entheses

The relationship between the mechanical loading undergone by a bone and its form has been widely assumed as a premise in studies aiming to reconstruct behavioral patterns from skeletal remains. Nevertheless, this relationship is complex due to the existence of many factors affecting bone structure and form, and further research combining structural and shape characteristics is needed. Using two‐block PLS, which is a test to analyze the covariance between two sets of variables, we aim to investigate the relationship between upper‐limb entheseal changes, cross‐sectional properties, and contour shape of the humeral diaphysis. Our results show that individuals with strongly marked entheseal changes have increased diaphyseal rigidities. Bending rigidities are mainly related to entheseal changes of muscles that cross the shoulder. Moreover, the entheseal changes of muscles that participate in the rotation of the arm are related to mediolaterally flatter and ventrodorsally broader humeral shapes in the mid‐proximal diaphysis. In turn, this diaphyseal shape is related to diaphyseal rigidity, especially to bending loadings. The shape of the diaphysis of the rest of the humerus does not covary either with rigidity or with entheseal changes. The results indicate that large muscular scars, such as those found in the mid‐proximal diaphyses, seem to be related to diaphyseal shape, whereas this relationship is not seen for areas with less direct influences of powerful muscles. Am J Phys Anthropol 150:609–617, 2013. © 2013 Wiley Periodicals, Inc.     

Cosmos 1887: morphology, histochemistry, and vasculature of the growing rat tibia

Light microscopy, electron microscopy, and enzyme histochemistry were used to study the effects of spaceflight on metaphyseal and cortical bone of the rat tibia. Cortical cross-sectional area and perimeter were not altered by a 12.5-day spaceflight in 3-month-old male rats. The endosteal osteoblast population and the vasculature near the periosteal surface in flight rats compared with ground controls showed more pronounced changes in cortical bone than in metaphyseal bone. The osteoblasts demonstrated greater numbers of transitional Golgi vesicles, possibly caused by a decreased cellular metabolic energy source, but no difference in the large Golgi saccules or the cell membrane-associated alkaline phosphatase activity. The periosteal vasculature in the diaphysis of flight rats often showed lipid accumulations within the lumen of the vessels, occasional degeneration of the vascular wall, and degeneration of osteocytes adjacent to vessels containing intraluminal deposits. These changes were not found in the metaphyseal region of flight animals. The focal vascular changes may be due to ischemia of bone or a developing fragility of the vessel walls as a result of spaceflight.     

微动脉平滑肌细胞K^＋通道的研究进展

K^＋通道维持着血管平滑肌细胞的静息膜电位。目前发现血管微动脉平滑肌细胞上主要表达内向整流型K^＋通道、ATP敏感型K^＋通道、电压依赖型K^＋通道和大电导钙激活型K^＋通道等四种K^＋通道。本文对微动脉平滑肌细胞K^＋通道最新进展做一综述。     

The dependence of transversely isotropic elasticity of human femoral cortical bone on porosity

The objective of this study was to examine the dependence of the elastic properties of cortical bone as a transversely isotropic material on its porosity. The longitudinal Young's modulus, transverse Young's modulus, longitudinal shear modulus, transverse shear modulus, and longitudinal Poisson's ratio of cortical bone were determined from eighteen groups of longitudinal and transverse specimens using tensile and torsional tests on a servo-hydraulic material testing system. These cylindrical waisted specimens of cortical bone were harvested from the middle diaphysis of three pairs of human femora. The porosity of these specimens was assessed by means of histology. Our study demonstrated that the longitudinal Young's and shear moduli of human femoral cortical bone were significantly (p<0.01) negatively correlated with the porosity of cortical bone. Conversely, the elastic properties in the transverse direction did not have statistically significant correlations with the porosity of cortical bone. As a result, the transverse elastic properties of cortical bone were less sensitive to changes in porosity than those in the longitudinal direction. Additionally, the anisotropic ratios of cortical bone elasticity were found to be significantly (p<0.01) negatively correlated with its porosity, indicating that cortical bone tended to become more isotropic when its porosity increased. These results may help a number of researchers develop more accurate micromechanics models of cortical bone.     

Regulation of calcium channels in smooth muscle: New insights into the role of myosin light chain kinase

Smooth muscle myosin light chain kinase (MLCK) plays a crucial role in artery contraction, which regulates blood pressure and blood flow distribution. In addition to this role, MLCK contributes to Ca²⁺ flux regulation in vascular smooth muscle (VSM) and in non-muscle cells, where cytoskeleton has been suggested to help Ca²⁺ channels trafficking. This conclusion is based on the use of pharmacological inhibitors of MLCK and molecular and cellular techniques developed to down-regulate the enzyme. Dissimilarities have been observed between cells and whole tissues, as well as between large conductance and small resistance arteries. A differential expression in MLCK and ion channels (either voltage-dependent Ca²⁺ channels or non-selective cationic channels) could account for these observations, and is in line with the functional properties of the arteries. A potential involvement of MLCK in the pathways modulating Ca²⁺ entry in VSM is described in the present review.