Structural characterization and calcium absorption-promoting effect of sucrose-calcium chelate in Caco-2 monolayer cells and mice.

Sucrose emerges as a chelating agent to form a stable sucrose-metal-ion chelate that can potentially improve metal-ion absorption. This study aimed to analyze the structure of sucrose-calcium chelate and its potential to promote calcium absorption in both Caco-2 monolayer cells and mice. The characterization results showed that calcium ions mainly chelated with hydroxyl groups in sucrose to produce sucrose-calcium chelate, altering the crystal structure of sucrose (forming polymer particles) and improving its thermal stability. Sucrose-calcium chelate dose dependently increased the amount of calcium uptake, retention, and transport in the Caco-2 monolayer cell model. Compared to CaCl2 , there was a significant improvement in the proportion of absorbed calcium utilized for transport but not retention (93.13 ± 1.75% vs. 67.67 ± 7.55%). Further treatment of calcium channel inhibitors demonstrated the active transport of sucrose-calcium chelate through Cav1.3. Cellular thermal shift assay and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays indicated that the ability of sucrose-calcium chelate to promote calcium transport was attributed to its superior ability to bind with PMCA1b, a calcium transporter located on the basement membrane, and stimulate its gene expression compared to CaCl2 . Pharmacokinetic analysis of mice confirmed the calcium absorption-promoting effect of sucrose-calcium chelate, as evident by the higher serum calcium level (44.12 ± 1.90 mg/L vs. 37.42 ± 1.88 mmol/L) and intestinal PMCA1b gene expression than CaCl2 . These findings offer a new understanding of how sucrose-calcium chelate enhances intestinal calcium absorption and could be used as an ingredient in functional foods to treat calcium deficiency. PRACTICAL APPLICATION: The development of high-quality calcium supplements is crucial for addressing the various adverse symptoms associated with calcium deficiency. This study aimed to prepare a sucrose-calcium chelate and analyze its structure, as well as its potential to enhance calcium absorption in Caco-2 monolayer cells and mice. The results demonstrated that the sucrose-calcium chelate effectively promoted calcium absorption. Notably, its ability to enhance calcium transport was linked to its strong binding with PMCA1b, a calcium transporter located on the basement membrane, and its capacity to stimulate PMCA1b gene expression. These findings contribute to a deeper understanding of how the sucrose-calcium chelate enhances intestinal calcium absorption and suggest its potential use as an ingredient in functional foods for treating calcium deficiency.

Intestinal segment and vitamin D3 concentration affect gene expression levels of calcium and phosphorus transporters in broiler chickens.

Two experiments were conducted in this research. Experiment 1 investigated the spatial expression characteristics of calcium (Ca) and phosphorus (P) transporters in the duodenum, jejunum, and ileum of 21-day-old broilers provided with adequate nutrient feed. Experiment 2 evaluated the effects of dietary vitamin D3 (VD3) concentration (0, 125, 250, 500, 1,000, and 2,000 IU/kg) on growth performance, bone development, and gene expression levels of intestinal Ca and P transporters in 1-21-day-old broilers provided with the negative control diet without supplemental VD3. Results in experiment 1 showed that the mRNA levels of calcium-binding protein 28-kDa (CaBP-D28k), sodium-calcium exchanger 1 (NCX1), plasma membrane calcium ATPase 1b (PMCA1b), and IIb sodium-phosphate cotransporter (NaPi-IIb) were the highest in the broiler duodenum. By contrast, the mRNA levels of inorganic phosphate transporter 1 (PiT-1) and 2 (PiT-2) were the highest in the ileum. Results in experiment 2 showed that adding 125 IU/kg VD3 increased body weight gain (BWG), feed intake (FI), bone weight, and percentage and weight of Ca and P in the tibia and femur of 1-21-day-old broilers compared with the negative control diet (p < 0.05). The rise in dietary VD3 levels from 125 to 1,000 IU/kg further increased the BWG, FI, and weights of the bone, ash, Ca, and P (p < 0.05). No difference in growth rate and leg bone quality was noted in the broilers provided with 1,000 and 2,000 IU/kg VD3 (p > 0.05). Supplementation with 125-2,000 IU/kg VD3 increased the mRNA abundances of intestinal Ca and P transporters to varying degrees. The mRNA level of CaBP-D28k increased by 536, 1,161, and 28 folds in the duodenum, jejunum, and ileum, respectively, after adding 1,000 IU/kg VD3. The mRNA levels of other Ca and P transporters (PMCA1b, NCX1, NaPi-IIb, PiT-1, and PiT-2) increased by 0.57-1.74 folds by adding 1,000-2,000 IU/kg VD3. These data suggest that intestinal Ca and P transporters are mainly expressed in the duodenum of broilers. Moreover, the addition of VD3 stimulates the two mineral transporter transcription in broiler intestines.

Ca2+ Homeostasis by Plasma Membrane Ca2+ ATPase (PMCA) 1 Is Essential for the Development of DP Thymocytes.

The strength of Ca2+ signaling is a hallmark of T cell activation, yet the role of Ca2+ homeostasis in developing T cells before expressing a mature T cell receptor is poorly understood. We aimed to unveil specific functions of the two plasma membrane Ca2+ ATPases expressed in T cells, PMCA1 and PMCA4. On a transcriptional and protein level we found that PMCA4 was expressed at low levels in CD4-CD8- double negative (DN) thymocytes and was even downregulated in subsequent stages while PMCA1 was present throughout development and upregulated in CD4+CD8+ double positive (DP) thymocytes. Mice with a targeted deletion of Pmca1 in DN3 thymocytes had an almost complete block of DP thymocyte development with an accumulation of DN4 thymocytes but severely reduced numbers of CD8+ immature single positive (ISP) thymocytes. The DN4 thymocytes of these mice showed strongly elevated basal cytosolic Ca2+ levels and a pre-mature CD5 expression, but in contrast to the DP thymocytes they were only mildly prone to apoptosis. Surprisingly, mice with a germline deletion of Pmca4 did not show any signs of altered progression through the developmental thymocyte stages, nor altered Ca2+ homeostasis throughout this process. PMCA1 is, therefore, non-redundant in keeping cellular Ca2+ levels low in the early thymocyte development required for the DN to DP transition.

PMCA1 depletion in mouse eggs amplifies calcium signaling and impacts offspring growth†.

Egg activation in mammals is triggered by oscillations in egg intracellular calcium (Ca2+) level. Ca2+ oscillation patterns can be modified in vitro by changing the ionic composition of culture media or in vivo by conditions affecting mitochondrial function, such as obesity and inflammation. In mice, disruption of Ca2+ oscillations in vitro impacts embryo development and offspring growth. Here we tested the hypothesis that, even without in vitro manipulation, abnormal Ca2+ signaling following fertilization impacts offspring growth. Plasma membrane Ca2+ ATPases (PMCA) extrude cytosolic Ca2+ to restore Ca2+ homeostasis. To disrupt Ca2+ signaling in vivo, we conditionally deleted PMCA1 (cKO) in oocytes. As anticipated, in vitro fertilized cKO eggs had increased Ca2+ exposure relative to controls. To assess the impact on offspring growth, cKO females were mated to wild type males to generate pups that had high Ca2+ exposure at fertilization. Because these offspring would be heterozygous, we also tested the impact of global PMCA1 heterozygosity on offspring growth. Control heterozygous pups that had normal Ca2+ at fertilization were generated by mating wild type females to heterozygous males; these control offspring weighed significantly less than their wild type siblings. However, heterozygous offspring from cKO eggs (and high Ca2+ exposure) were larger than heterozygous controls at 12 week-of-age and males had altered body composition. Our results show that global PMCA1 haploinsufficiency impacts growth and support that abnormal Ca2+ signaling after fertilization in vivo has a long-term impact on offspring weight. These findings are relevant for environmental and medical conditions affecting Ca2+ handling and for design of culture conditions and procedures for domestic animal and human assisted reproduction.

Cardiomyocyte-specific loss of plasma membrane calcium ATPase 1 impacts cardiac rhythm and is associated with ventricular repolarisation dysfunction.

Plasma membrane calcium ATPase 1 (PMCA1, Atp2b1) is emerging as a key contributor to cardiac physiology, involved in calcium handling and myocardial signalling. In addition, genome wide association studies have associated PMCA1 in several areas of cardiovascular disease including hypertension and myocardial infarction. Here, we investigated the role of PMCA1 in basal cardiac function and heart rhythm stability. Cardiac structure, heart rhythm and arrhythmia susceptibility were assessed in a cardiomyocyte-specific PMCA1 deletion (PMCA1CKO) mouse model. PMCA1CKO mice developed abnormal heart rhythms related to ventricular repolarisation dysfunction and displayed an increased susceptibility to ventricular arrhythmias. We further assessed the levels of cardiac ion channels using qPCR and found a downregulation of the voltage-dependent potassium channels, Kv4.2, with a corresponding reduction in the transient outward potassium current which underlies ventricular repolarisation in the murine heart. The changes in heart rhythm were found to occur in the absence of any structural cardiomyopathy. To further assess the molecular changes occurring in PMCA1CKO hearts, we performed proteomic analysis. Functional characterisation of differentially expressed proteins suggested changes in pathways related to metabolism, protein-binding, and pathways associated cardiac function including ß-adrenergic signalling. Together, these data suggest an important role for PMCA1 in basal cardiac function in relation to heart rhythm control, with reduced cardiac PMCA1 expression resulting in an increased risk of arrhythmia development.

Age quadratically affects intestinal calcium and phosphorus transporter gene expression in broiler chickens.

OBJECTIVE: This research aimed to evaluate the effects of age on growth, tibia development, and intestinal calcium (Ca) and phosphorus (P) transporter gene expressions in broiler chickens. METHODS: A total of 224 male Arbor Acres broilers were fed with nutrient-adequate diets and reared in eight cages (28 broilers per cage). Eight broilers (one broiler per cage) were selected and killed at 5, 10, 15, 20, 25, 30, 35, and 40 days of age, respectively. RESULTS: Body weight continuously increased with age of broiler chickens from 5 to 40 days. The bone weight, ash weight, diameter, and length of the tibia also increased with broiler age. By contrast, the tibia ash, Ca, and P percentages quadratically changed with age (p<0.001), and the highest values of mineral contents were observed at 20, 25, and 25 days of age, respectively. The mRNA abundances of calcium-binding protein 28-kDa (CaBP-D28k), sodium-calcium exchanger 1 (NCX1), and plasma membrane ATPase 1b (PMCA1b) increased from 5 to 25 days and then decreased up to 40 days. Similar results were noted in the mRNA abundances of IIb sodium-phosphate cotransporter (NaPi-IIb), inorganic phosphate transporter 1 (PiT-1), inorganic phosphate transporter 2 (PiT-2), nuclear vitamin D receptor (nVDR), and membrane vitamin D receptor (mVDR). The mRNA abundances of Ca and P transporters and VDRs were the highest at 25 days of age. CONCLUSION: These data indicate that age quadratically affects intestinal Ca and P transporter gene expression and mineral absorption capacity in broiler chickens.

The Influence of Thyroid Hormone on Ca2+ Signaling Pathways During Embryonal Development.

Thyroid hormones influence brain development through the regulation of gene expression. Ca2+-dependent gene expression is a major pathway controlled by the Ca2+/calmodulin-dependent protein kinase IV (CaMKIV), which in turn is induced by the thyroid hormone T3, as also demonstrated in a mouse embryonic stem cell line. In addition, T3 controls the expression of neurexin, synaptotagmin2 (SYT2), synaptotagmin-related gene1 (SRG1), and a number of other genes involved in neurotransmitter release in a Ca2+-dependent manner. It has been noticed that the development of dopaminergic neurons by evoking significant calcium entry occurs through TRPC calcium channels. It was also demonstrated that the T3-mediated development of an early neuronal network is characteristic for depolarizing GABAergic neurons concomitant with intracellular calcium transients. An important aspect of T3-dependent regulation of gene expression in the developing brain is its modulation by the transcription activator COUP-TF1. Regulation of alternative splicing by CaMKIV is another important aspect for embryonal neural development since it can lead to the expression of PMCA1a, the neuronal-specific isoform of the plasma membrane calcium pump. Maternal hypothyroidism or CaMKIV deficiency can have a severe influence on fetal brain development.

Plasma membrane Ca2+ ATPase 1 (PMCA1) but not PMCA4 is critical for B-cell development and Ca2+ homeostasis in mice.

The amplitude and duration of Ca2+ signaling is crucial for B-cell development and self-tolerance; however, the mechanisms for terminating Ca2+ signals in B cells have not been determined. In lymphocytes, plasma membrane Ca2+ ATPase (PMCA) isoforms 1 and 4 (PMCA1 and PMCA4, aka ATP2B1 and ATP2B4) are the main candidates for expelling Ca2+ from the cell through the plasma membrane. We report here that Pmca4 (Atp2b4) KO mice had normal B-cell development, while mice with a conditional KO of Pmca1 (Atp2b1) had greatly reduced numbers of B cells, particularly splenic follicular B cells, marginal zone B cells, and peritoneal B-1a cells. Mouse and naïve human B cells showed only PMCA1 expression and no PMCA4 by western blot, in contrast to T cells, which did express PMCA4. Calcium handling was normal in Pmca4-/- B cells, but Pmca1 KO B cells had elevated basal levels of Ca2+ , elevated levels in ER stores, and reduced Ca2+ clearance. These findings show that the PMCA1 isoform alone is required to ensure normal B-cell Ca2+ signaling and development, which may have implications for therapeutic targeting of PMCAs and Ca2+ in B cells.

The effect of steroid hormone on the expression of the calcium-processing proteins in the immature female rat brain.

The cytosolic calcium concentration is regulated by calcium-processing proteins such as transient receptor potential cation channel subfamily V member 5 (TRPV5), TRPV6, sodium-calcium exchanger 1 (NCX1), and plasma membrane Ca2+ ATPase 1 (PMCA1). Those calcium-processing proteins are important for physiological functions in the brain. The effects of steroid hormones on calcium-processing protein expressions in the brains are unknown. Thus, the effects of steroid hormones on the distribution, localization, and expressions of calcium-processing proteins in the brain were analyzed. Immature female rats were injected with estrogen (E2), progesterone (P4), dexamethasone (DEX), and their antagonists (ICI 182,780 and RU486). We found that TRPV5 and TRPV6 proteins were highly expressed in the cerebral cortex (CT), hypothalamus (HY), and brain stem (BS) compared to that in the olfactory bulb (OB) and cerebellum (CB). Also, the NCX1 protein was highly expressed in CT and BS compared to that in OB, HY, and CB, and PMCA1 protein was highly expressed in CT compared to that in other brain regions. Furthermore, expression levels of TRPV5, TRPV6, NCX1, and PMCA1 proteins were regulated by E2, P4, and/or DEX in the CT and HY. In summary, calcium-processing proteins are widely expressed in the immature rat brain, and expressions of calcium-processing proteins in CT and HY indicated that they may regulate by E2, P4, and/or DEX and can be attenuated by antagonist treatment. These results indicate that steroid hormone regulation of TRPV5, TRPV6, NCX1, and PMCA1 proteins may serve as a critical regulator of cytosolic calcium absorption and release in the brain.

Vitamin D and the intestine: Review and update.

The central role of vitamin D in calcium homeostasis is to increase calcium absorption from the intestine. This article describes the early work that served as the foundation for the initial model of vitamin D mediated calcium absorption. In addition, other research related to the role of vitamin D in the intestine, including those which have challenged the traditional model and the crucial role of specific calcium transport proteins, are reviewed. More recent work identifying novel targets of 1,25(OH)2D3 action in the intestine and highlighting the importance of 1,25(OH)2D3 action across the proximal/distal and crypt/villus axes in the intestine is summarized.

Immunolocalization of calcium sensing and transport proteins in the murine endolymphatic sac indicates calciostatic functions within the inner ear.

An exceptionally low calcium (Ca2+) concentration in the inner ear endolymph ([Ca2+]endolymph) is crucial for proper auditory and vestibular function. The endolymphatic sac (ES) is believed to critically contribute to the maintenance of this low [Ca2+]endolymph. Here, we investigated the immunohistochemical localization of proteins that are presumably involved in the sensing and transport of extracellular Ca2+ in the murine ES epithelium. Light microscopic and fluorescence immunolabeling in paraffin-embedded murine ES tissue sections (male C57BL/6 mice, 6-8 weeks old) demonstrated the presence of the calcium-sensing receptor CaSR, transient receptor potential cation channel subtypes TRPV5 and TRPV6, sarco/endoplasmic reticulum Ca2+-ATPases SERCA1 and SERCA2, Na+/Ca2+ exchanger NCX2, and plasma membrane Ca2+ ATPases PMCA1 and PMCA4 in ES epithelial cells. These proteins exhibited (i) membranous (apical or basolateral) or cytoplasmic localization patterns, (ii) a proximal-to-distal labeling gradient within the ES, and (iii) different distribution patterns among ES epithelial cell types (mitochondria-rich cells (MRCs) and ribosome-rich cells (RRCs)). Notably, in the inner ear membranous labyrinth, CaSR was exclusively localized in MRCs, suggesting a unique role of the ES epithelium in CaSR-mediated sensing and control of [Ca2+]endolymph. Structural loss of the distal ES, which is consistently observed in Meniere's disease, may therefore critically disturb [Ca2+]endolymph and contribute to the pathogenesis of Meniere's disease.

Plasma membrane Ca2+ -ATPase 1 is required for maintaining atrial Ca2+ homeostasis and electrophysiological stability in the mouse.

KEY POINTS: The role of plasma membrane Ca2+ -ATPase 1 (PMCA1) in Ca2+ homeostasis and electrical stability in atrial tissue has been investigated at both organ and cellular levels in mice with cardiomyocyte-specific deletion of PMCA1 (PMCA1cko ) The PMCA1cko hearts became more susceptible to atrial arrhythmic stress conditions than PMCA1loxP/loxP hearts. PMCA1 deficiency alters cellular Ca2+ homeostasis under both baseline and stress conditions. PMCA1 is required for maintaining cellular Ca2+ homeostasis and electrical stability in murine atria under stress conditions. ABSTRACT: To determine the role of plasma membrane Ca2+ -ATPase 1 (PMCA1) in maintaining Ca2+ homeostasis and electrical stability in the atrium under physiological and stress conditions, mice with a cardiomyocyte-specific deletion of PMCA1 (PMCA1cko ) and their control littermates (PMCA1loxP/loxP ) were studied at the organ and cellular levels. At the organ level, the PMCA1cko hearts became more susceptible to atrial arrhythmias under rapid programmed electrical stimulation compared with the PMCA1loxP/loxP hearts, and such arrhythmic events became more severe under Ca2+ overload conditions. At the cellular level, the occurrence of irregular-type action potentials of PMCA1cko atrial myocytes increased significantly under Ca2+ overload conditions and/or at higher frequency of stimulation. The decay of Na+ /Ca2+ exchanger current that followed a stimulation protocol was significantly prolonged in PMCA1cko atrial myocytes under basal conditions, with Ca2+ overload leading to even greater prolongation. In conclusion, PMCA1 is required for maintaining Ca2+ homeostasis and electrical stability in the atrium. This is particularly critical during fast removal of Ca2+ from the cytosol, which is required under stress conditions.

Histone Deacetylase Inhibitor Treatment Increases the Expression of the Plasma Membrane Ca2+ Pump PMCA4b and Inhibits the Migration of Melanoma Cells Independent of ERK.

Several new therapeutic options emerged recently to treat metastatic melanoma; however, the high frequency of intrinsic and acquired resistance among patients shows a need for new therapeutic options. Previously, we identified the plasma membrane Ca2+ ATPase 4b (PMCA4b) as a metastasis suppressor in BRAF-mutant melanomas and found that mutant BRAF inhibition increased the expression of the pump, which then inhibited the migratory and metastatic capability of the cells. Earlier it was also demonstrated that histone deacetylase inhibitors (HDACis) upregulated PMCA4b expression in gastric, colon, and breast cancer cells. In this study, we treated one BRAF wild-type and two BRAF-mutant melanoma cell lines with the HDACis, SAHA and valproic acid, either alone, or in combination with the BRAF inhibitor, vemurafenib. We found that HDACi treatment strongly increased the expression of PMCA4b in all cell lines irrespective of their BRAF mutational status, and this effect was independent of ERK activity. Furthermore, HDAC inhibition also enhanced the abundance of the housekeeping isoform PMCA1. Combination of HDACis with vemurafenib, however, did not have any additive effects on either PMCA isoform. We demonstrated that the HDACi-induced increase in PMCA abundance was coupled to an enhanced [Ca2+]i clearance rate and also strongly inhibited both the random and directional movements of A375 cells. The primary role of PMCA4b in these characteristic changes was demonstrated by treatment with the PMCA4-specific inhibitor, caloxin 1c2, which was able to restore the slower Ca2+ clearance rate and higher motility of the cells. While HDAC treatment inhibited cell motility, it decreased only modestly the ratio of proliferative cells and cell viability. Our results show that in melanoma cells the expression of both PMCA4b and PMCA1 is under epigenetic control and the elevation of PMCA4b expression either by HDACi treatment or by the decreased activation of the BRAF-MEK-ERK pathway can inhibit the migratory capacity of the highly motile A375 cells.

Enhancement of intrarenal plasma membrane calcium pump isoform 1 expression in chronic angiotensin II-infused mice.

Plasma membrane calcium pump isoform 1 (PMCA1) is encoded by ATPase plasma membrane Ca2+transporting 1 (ATP2B1), the most likely candidate gene responsible for hypertension. Although PMCA1 is highly expressed in the kidney, little is known about regulation of its renal expression in various pathological conditions in vivo. Our study was designed to elucidate regulation of renal PMCA1 expression in mice. We employed three mouse models for kidney disease. These were the unilateral ureteral obstruction (UUO), the remnant kidney using 5/6 nephrectomy, and chronic angiotensin II administration models. Mice were assessed for systolic blood pressure and renal injury in accordance with the damage induced in the specific model. Kidney PMCA1 mRNA levels were measured in all mice. The UUO model showed renal fibrosis but no changes in blood pressure or renal PMCA1 mRNA expression. Similarly, the 5/6 nephrectomy model exhibited declined renal function without changes in blood pressure or renal PMCA1 mRNA expression. In contrast, chronic angiotensin II administration increased albuminuria and blood pressure as well as significantly increasing renal PMCA1 mRNA and protein expression. These results suggest that renal PMCA1 has a role as one of the molecules involved in angiotensin II-induced hypertension and kidney injury.

Effects of estrogen on esophageal function through regulation of Ca2+-related proteins.

BACKGROUND: The calcium ion is important for physiological functions in all tissues and organs and essential to many vital functions, including hormone secretion and muscle contraction. The intracellular concentration of calcium is regulated by calcium related proteins such as CaBP-9k, PMCA1, and NCX1. In this study, we investigated the relationship between calcium regulation and esophageal functions such as mucin secretion and smooth muscle contraction. METHODS: To evaluate the influence of sex steroid hormones, immature rats were treated for 3 days with estradiol (E2), progesterone (P4), and their antagonists (ICI 182,780, and RU486). Esophageal function, transcription level, and localization of CaBP-9k, PMCA1, NCX1, ERα, and MUC2 were examined in the esophagus. RESULTS: Transcriptional level of Cabp-9k and Muc2 was increased by E2, but not by P4. CaBP-9k, PMCA1, and MUC2 were mainly localized in the mucosal layer. Acidic mucosubstances in the esophagus were increased by E2 and recovered by ICI treatment. Unlike the expression of Cabp-9k, mRNA levels of Pmca1, Ncx1, and Erα were only decreased in response to E2, and recovered by ICI co-treatment group. The contraction of the esophagus and mRNA level of Mylk were reduced by E2. Overall, E2 upregulated mucus secretion, but downregulated muscle contraction in the esophagus through regulation of the expression of calcium related genes and the resultant intracellular calcium level. CONCLUSIONS: The regulation of E2 in the function of esophagus may be applied to treat esophageal diseases such as reflux esophagitis, achalasia, and esophageal cancer.

Implications of the thyroid hormone on neuronal development with special emphasis on the calmodulin-kinase IV pathway.

Thyroid hormones influence brain development through regulation of gene expression. This is especially true for Ca2+-dependent regulation since a major pathway is controlled by the Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) which in turn is induced by the thyroid hormone T3. In addition, CaMKIV is involved in regulation of alternative splicing of a number of protein isoforms, among them PMCA1a, the neuronal specific isoform of the plasma membrane calcium pump. On the other hand, hypothyroidism or CaMKIV deficiency can have a severe influence on brain development. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Trans- and paracellular calcium transport along the small and large intestine in horses.

Intestinal calcium absorption plays a key role in the maintenance of calcium homeostasis and may either occur by paracellular or transcellular mechanisms. The horse has some unique peculiarities in calcium homeostasis compared to other species including a high absorptive capacity for calcium in the intestine, high plasma calcium concentrations, high renal excretion, and low plasma concentrations of vitamin D metabolites. So far, knowledge about the underlying mechanisms and the regulation of intestinal calcium absorption is still limited concerning this species. Several studies have documented that intestinal calcium transport in horses is not as dependent on vitamin D as in other species. However, published data on other potential regulatory mechanisms are still lacking. In the present study, paracellular and transcellular transport mechanisms for intestinal calcium transport along the intestinal axis were identified in horses using the Ussing chamber technique. Furthermore, the expression of respective transport proteins including transient receptor potential vanilloid member 6, calbindin-D9k and calcium ATPase type 1 in line with the determined calcium flux rates was documented. In respect to regulation of transepithelial calcium transport, novel regulatory proteins for maintaining calcium homeostasis such as B-box and SPRY-domain containing protein and calmodulin were investigated for the first time in equine intestinal tissues in this study. This provides the basis for a new approach for a better understanding of equine calcium homeostasis regulation.

Deletion of the intestinal plasma membrane calcium pump, isoform 1, Atp2b1, in mice is associated with decreased bone mineral density and impaired responsiveness to 1, 25-dihydroxyvitamin D3.

The physiological importance of the intestinal plasma membrane calcium pump, isoform 1, (Pmca1, Atp2b1), in calcium absorption and homeostasis has not been previously demonstrated in vivo. Since global germ-line deletion of the Pmca1 in mice is associated with embryonic lethality, we selectively deleted the Pmca1 in intestinal absorptive cells. Mice with loxP sites flanking exon 2 of the Pmca1 gene (Pmca1(fl/fl)) were crossed with mice expressing Cre recombinase in the intestine under control of the villin promoter to give mice in which the Pmca1 had been deleted in the intestine (Pmca1(EKO) mice). Pmca1(EKO) mice were born at a reduced frequency and were small at the time of birth when compared to wild-type (Wt) littermates. At two months of age, Pmca1(EKO) mice fed a 0.81% calcium, 0.34% phosphorus, normal vitamin D diet had reduced whole body bone mineral density (P < 0.037), and reduced femoral bone mineral density (P < 0.015). There was a trend towards lower serum calcium and higher serum parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) concentrations in Pmca1(EKO) mice compared to Wt mice but the changes were not statistically significant. The urinary phosphorus/creatinine ratio was increased in Pmca1(EKO) mice (P < 0.004). Following the administration of 200 ng of 1α,25(OH)2D3 intraperitoneally to Wt mice, active intestinal calcium transport increased ∼2-fold, whereas Pmca1(EKO) mice administered an equal amount of 1α,25(OH)2D3 failed to show an increase in active calcium transport. Deletion of the Pmca1 in the intestine is associated with reduced growth and bone mineralization, and a failure to up-regulate calcium absorption in response to 1α,25(OH)2D3.

Expression of calcium-buffering proteins in rat intrinsic laryngeal muscles.

Intrinsic laryngeal muscles (ILM) are highly specialized muscles involved in phonation and airway protection, with unique properties that allow them to perform extremely rapid contractions and to escape from damage in muscle dystrophy. Due to that, they may differ from limb muscles in several physiological aspects. Because a better ability to handle intracellular calcium has been suggested to explain ILM unique properties, we hypothesized that the profile of the proteins that regulate calcium levels in ILM is different from that in a limb muscle. Calcium-related proteins were analyzed in the ILM, cricothyroid (CT), and tibialis anterior (TA) muscles from male Sprague-Dawley rats (8 weeks of age) using quantitative PCR and western blotting. Higher expression of key Ca(2+) regulatory proteins was detected in ILM compared to TA, such as the sarcoplasmic reticulum (SR) Ca(2+)-reuptake proteins (Sercas 1 and 2), the Na(+)/Ca(2+) exchanger, phospholamban, and the Ca(2+)-binding protein calsequestrin. Parvalbumin, calmodulin and the ATPase, Ca(2+)-transporting, and plasma membrane 1 were also expressed at higher levels in ILM compared to TA. The store-operated calcium entry channel molecule was decreased in ILM compared to the limb muscle and the voltage-dependent L-type and ryanodine receptor were expressed at similar levels in ILM and TA. These results show that ILM have a calcium regulation system profile suggestive of a better ability to handle calcium changes in comparison to limb muscles, and this may provide a mechanistic insight for their unique pathophysiological properties.