Effect Of Extracellular Calcium Research Articles

MO092THE ROLE OF CALCIUM IN UROMODULIN EXPRESSION AND SECRETION FROM RENAL MEDULLARY EPITHELIAL CELLS OF HYPERTENSIVE AND NORMOTENSIVE RATS

Abstract Background and Aims Uromodulin (UMOD) is the most abundantly secreted protein found within the urine, primarily produced by medullary thick ascending limb (mTAL) epithelial cells of the kidneys. There is accruing genetic evidence implicating UMOD in blood pressure regulation and consequently hypertension. The molecular signaling induced by calcium in the kidney and its influence on blood pressure are not well understood. The aim of this study was to investigate the potential role of extracellular calcium and the calcium-sensing receptor (CaSR) in mTAL on UMOD production and secretion in TAL cells with the hope of defining novel clinical targets for the treatment of hypertension. Method Kidneys were harvested from normotensive Wistar-Kyoto (WKY) and stroke-prone spontaneously hypertensive (SHRSP) female rats. To determine the effect of extracellular calcium on UMOD secretion, mTAL tubules were incubated in media with and without 1mM calcium, nifedipine (10µM), NPS2143 (1 or 5 µM) and spermine (2mM). Extracellular and intracellular UMOD protein levels were detected by Western blot. Gene expression of Umod was determined by qRT-PCR. Results Calcium increased mTAL tubule UMOD secretion in WKY and SHRSP. Nifedipine slightly decreased UMOD secretion in WKY without calcium. In both strains, NPS2143 increased calcium-induced UMOD secretion, with an enhanced effect in SHRSP. Stimulation of CaSR with spermine decreased UMOD secretion in WKY. Analysis of intracellular UMOD levels in these conditions demonstrated increased accumulation when extracellular secretion was low, and vice versa. Incubation of primary mTAL cells with calcium confirmed increased localisation of UMOD at the membrane compared to the cytosol, without any major differences in cell morphology. The Umod mRNA level changes were not statistically significant among conditions. Conclusion Trafficking of UMOD in the mTAL is influenced by the type of CaSR ligand and the biased nature of G-protein coupled CaSR signalling. Unravelling the signalling events post-calcium will be necessary for identification of key regulators of UMOD secretion and provide new sites for therapeutic intervention in hypertension.

English

The effect of extracellular calcium on isolated uterine muscle contractions stimulated by acetone leaf extract of Anogeissus leiocarpa was investigated in a rat. About 12 m segment of the uterine muscle strip was mounted initially in a thermostatically regulated organ bath (37°C) containing normal Physiological Salt Solution (PSS; De Jalon) and later inphysiological salt solution devoid of CaCl2. The extract contracted the uterine muscle preparation in a concentration-related manner in normal PSS, with 0.53 mg/ml as the lowest active concentration. This contractile response was abolished by the calcium channel blocker, verapamil HCl (2 µg/ml). The extract however, did not evoke any contractile activity on the isolated tissue in a calcium-free media (PSS), even when caffeine (2 mmol) known to release calcium via the calcium induced-calcium release (CICR) mechanism was added to the perfusate. The results demonstrated the requirement for extracellular calcium for the extract mediated contractions and the inability of the extract to access calcium from intracellular storage sites such as the sarcoplasmic reticulum and the mitochondria. Therefore, the influx of Ca2+ into the cell cytosol is a pre-requisite for the extract-mediated uterine muscle contraction. Key word: Anogeissus leiocarpa, thermostatically organ bath, verapamil, caffeine, uterus.

Effects of Extracellular Calcium and Vitamin D on Cultured Human Sebocytes.

Effects of Extracellular Calcium and Vitamin D on Cultured Human Sebocytes.

Effects of Extracellular Calcium and 1,25 dihydroxyvitamin D3 on Sebaceous Gland Cells In vitro and In vivo

Calcium and 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) are promoters of epithelial cell functions; however their effects on sebaceous glands are unknown. In this study, morphology, ultrastructure, cell numbers, lipid synthesis and apoptosis of SZ95 sebocytes were assessed in vitro under different concentrations of extracellular calcium with or without 1,25(OH)2D3. Moreover, serum calcium and 1,25(OH)2D3 levels were assessed in acne and non-acne patients (controls). Under conditions of low extracellular calcium, lipogenesis and cell detachment were observed. Increasing extracellular calcium enhanced sebocyte numbers, induced epithelial morphology and reduced lipogenesis. Moreover, a reduction in extracellular calcium reduced E-cadherin and enhanced caspase 3/7 activity (apoptosis), whereas calcium chelation by EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) resulted in enhanced lipogenesis. 1,25(OH)2D3 decreased sebaceous lipogenesis, but also induced signs of autophagy. In the clinical study, patients and controls exhibited normal serum calcium levels. Younger acne patients presented lower 1,25(OH)2D3 levels than did older ones. In conclusion, extracellular calcium and 1,25(OH)2D3 regulate sebocyte morphology, increase cell numbers, decrease sebaceous lipogenesis and induce cell autophagy in vitro. The increased ionized calcium and the reduced 1,25(OH)2D3 levels detected in the serum of younger patients with acne may contribute respectively to increased sebaceous gland volume and enhanced lipogenesis.

Osteogenic differentiation capacity of human mesenchymal stromal cells in response to extracellular calcium with special regard to connexin 43

The effects of extracellular calcium on osteogenic differentiation capacity of human bone-derived mesenchymal stromal cells with special regard to connexin 43 (cx43) have been investigated by means of cell culture experiments. Mesenchymal stromal cells isolated from human cancellous bone were cultured on tissue culture plates at different calcium ion (Ca2+) concentrations (1.8mmoll−1, 10mmoll−1, 20mmoll−1). Cell responses were evaluated by quantitative RT-PCR, immunofluorescence staining, and Lucifer Yellow fluorescence uptake experiments. It could be shown that increasing Ca2+ concentrations correlate with increasing cx43 and bone sialoprotein mRNA levels as well as with enhanced cx43 fluorescence signaling and matrix mineralization of the cultures as shown by von Kossa staining. Hemichannel gating – assessed by Lucifer Yellow uptake – increases with increasing extracellular Ca2+ concentrations suggesting that regulatory effects at the hemichannel level are calcium-dependent.

The Effect of Extracellular Calcium on Oxytocin-Induced Contractility in Naive and Oxytocin-Pretreated Human Myometrium In Vitro

Prolonged exposure to oxytocin during augmentation of labor is a significant risk factor for uterine atony, resulting in the desensitization phenomenon, a decrease in the responsiveness of myometrium to further oxytocin. The importance of extracellular calcium is well established in spontaneous myometrial contractility; however, its significance is unknown in the context of desensitized myometrium. We aimed to investigate the effect of low, normal, and high extracellular calcium concentration on oxytocin-induced contractility in oxytocin-pretreated human myometrium in vitro. We hypothesized that extracellular normocalcemia would provide superior oxytocin-induced contractility in both naive and oxytocin-pretreated myometrium compared with hypocalcemia and hypercalcemia. Myometrial tissue was obtained from women undergoing elective cesarean deliveries and was dissected into longitudinal strips. Each strip was mounted in a single organ bath with physiological salt solution (PSS) under homeostatic conditions and then pretreated for 2 hours with either oxytocin 10 M or PSS (control). The tissue was then washed with PSS, and calcium concentrations were altered to reflect low (1.25 mM), normal (2.5 mM), or high (3.75 mM) levels, thereby providing 6 study groups. After equilibration in the desired calcium concentration, a dose-response testing to oxytocin 10 M to 10 M was performed. Contractile parameters were measured and compared among groups after square root transformation. The primary outcome was motility index (frequency × amplitude), and secondary outcomes included frequency, amplitude, and area under the curve. One hundred seventy-four experiments were conducted from samples obtained from 36 women. In the control group, the mean motility index (√g·contractions/10 min) was significantly lower in the hypocalcemic group than in the normocalcemic group (estimated difference, -0.43; 95% confidence interval [CI], -0.82 to -0.04; P = 0.03). In addition, the mean frequency of contractions (√contractions/10 min) was significantly lower in the hypocalcemic (estimated difference, -0.27; 95% CI, -0.46 to -0.09; P = 0.002) and hypercalcemic groups (estimated difference, -0.18; 95% CI, -0.34 to -0.02; P = 0.03) compared with the normocalcemic group. In the oxytocin-pretreated group, there were no significant differences in the values of any of the contractility parameters of the hypocalcemic or hypercalcemic groups compared with the normocalcemic group (mean motility index [√g·contractions/10 min] estimated difference, 0.10; 95% CI, -0.23 to 0.43; P = 0.74 and -0.39; 95% CI, -1.10 to 0.32; P = 0.39, respectively). In oxytocin-naive myometrium, normocalcemia provides superior oxytocin-induced contractility compared with hypocalcemic and hypercalcemic conditions. We were unable to draw conclusions regarding oxytocin-pretreated myometrium because of the small sample size relative to the large variability of the data. These observations warrant further investigations in laboratory and clinical settings.

Distribution of alginate and cellulose and regulatory role of calcium in the cell wall of the brown alga Ectocarpus siliculosus (Ectocarpales, Phaeophyceae).

This work investigated a correlation between the three-dimensional architecture and compound-components of the brown algal cell wall. Calcium greatly contributes to the cell wall integrity. Brown algae have a unique cell wall consisting of alginate, cellulose, and sulfated polysaccharides. However, the relationship between the architecture and the composition of the cell wall is poorly understood. Here, we investigated the architecture of the cell wall and the effect of extracellular calcium in the sporophyte and gametophyte of the model brown alga, Ectocarpus siliculosus (Dillwyn) Lyngbye, using transmission electron microscopy, histochemical, and immunohistochemical studies. The lateral cell wall of vegetative cells of the sporophyte thalli had multilayered architecture containing electron-dense and negatively stained fibrils. Electron tomographic analysis showed that the amount of the electron-dense fibrils and the junctions was different between inner and outer layers, and between the perpendicular and tangential directions of the cell wall. By immersing the gametophyte thalli in the low-calcium (one-eighth of the normal concentration) artificial seawater medium, the fibrous layers of the lateral cell wall of vegetative cells became swollen. Destruction of cell wall integrity was also induced by the addition of sorbitol. The results demonstrated that electron-dense fibrils were composed of alginate-calcium fibrous gels, and electron negatively stained fibrils were crystalline cellulose microfibrils. It was concluded that the spatial arrangement of electron-dense fibrils was different between the layers and between the directions of the cell wall, and calcium was necessary for maintaining the fibrous layers in the cell wall. This study provides insights into the design principle of the brown algal cell wall.

Effects of extracellular calcium and surgical techniques on restoration of axonal continuity by polyethylene glycol fusion following complete cut or crush severance of rat sciatic nerves.

Complete crush or cut severance of sciatic nerve axons in rats and other mammals produces immediate loss of axonal continuity. Loss of locomotor functions subserved by those axons is restored only after months, if ever, by outgrowths regenerating at ∼1 mm/day from the proximal stumps of severed axonal segments. The distal stump of a severed axon typically begins to degenerate in 1-3 days. We recently developed a polyethylene glycol (PEG) fusion technology, consisting of sequential exposure of severed axonal ends to hypotonic Ca(2+) -free saline, methylene blue, PEG in distilled water, and finally Ca(2+) -containing isotonic saline. This study examines factors that affect the PEG fusion restoration of axonal continuity within minutes, as measured by conduction of action potentials and diffusion of an intracellular fluorescent dye across the lesion site of rat sciatic nerves completely cut or crush severed in the midthigh. Also examined are factors that affect the longer-term PEG fusion restoration of lost behavioral functions within days to weeks, as measured by the sciatic functional index. We report that exposure of cut-severed axonal ends to Ca(2+) -containing saline prior to PEG fusion and stretch/tension of proximal or distal axonal segments of cut-severed axons decrease PEG fusion success. Conversely, trimming cut-severed ends in Ca(2+) -free saline just prior to PEG fusion increases PEG fusion success. PEG fusion prevents or retards the Wallerian degeneration of cut-severed axons, as assessed by measures of axon diameter and G ratio. PEG fusion may produce a paradigm shift in the treatment of peripheral nerve injuries. © 2016 Wiley Periodicals, Inc.

Effect of extracellular calcium on regucalcin expression and cell viability in neoplastic and non-neoplastic human prostate cells

Extracellular calcium (Ca2+o) and its receptor, the Ca2+-sensing receptor (CaSR), play an important role in prostate physiology, and it has been shown that the deregulation of Ca2+ homeostasis and the overexpression of CaSR are involved in prostate cancer (PCa). Regucalcin (RGN), a Ca2+-binding protein that plays a relevant role in intracellular Ca2+ homeostasis, was identified as an under-expressed protein in human PCa. Moreover, RGN was associated with suppression of cell proliferation, suggesting that the loss of RGN may favor development and progression of PCa. This work aims to unveil the role of Ca2+o on RGN expression and viability of non-neoplastic (PNT1A) and neoplastic (LNCaP) prostate cell lines. It was demonstrated that Ca2+o up-regulates RGN expression in both cell lines, but important differences were found between cells for dose- and time-responses to Ca2+o treatment. It was also shown that high [Ca2+]o triggers different effects on cell proliferation of neoplastic and non-neoplastic PCa cells, which seems to be related with RGN expression levels. This suggests the involvement of RGN in the regulation of cell proliferation in response to Ca2+o treatment. Also, the effect of Ca2+o on CaSR expression seems to be dependent of RGN expression, which is strengthened by the fact that RGN-knockdown in PNT1A cells increases the CaSR expression, whereas transgenic rats overexpressing RGN exhibit low levels of CaSR. Overall, our results highlighted the importance of RGN as a regulatory protein in Ca2+-dependent signaling pathways and its deregulation of RGN expression by Ca2+o may contribute for onset and progression of PCa.

Effects of extracellular calcium on viability and osteogenic differentiation of bone marrow stromal cells in vitro

Bone marrow stromal cells (BMSCs) have been extensively used for tissue engineering. However, the effect of Ca(2+) on the viability and osteogenic differentiation of BMSCs has yet to be evaluated. To determine the dose-dependent effect of Ca(2+) on viability and osteogenesis of BMSCs in vitro, BMSCs were cultured in calcium-free DMEM medium supplemented with various concentrations of Ca(2+) (0, 1, 2, 3, 4, and 5mM) from calcium citrate. Cell viability was analyzed by MTT assay and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) assay, Von Kossa staining, and real-time PCR. Ca(2+) stimulated BMSCs viability in a dose-dependent manner. At slightly higher concentrations (4 and 5mM) in the culture, Ca(2+) significantly inhibited the activity of ALP on days 7 and 14 (P<0.01 or P<0.05), significantly suppressed collagen synthesis (P<0.01 or P<0.05), and significantly elevated calcium deposition (P<0.01) and mRNA levels of osteocalcin (P<0.01 or P<0.05) and osteopontin (P<0.01 or P<0.05). Therefore, elevated concentrations of extracellular calcium may promote cell viability and late-stage osteogenic differentiation, but may suppress early-stage osteogenic differentiation in BMSCs.

Differential Activation of Intracellular Calcium Oscillations by Multiple Calcium and L-Phenylalanine Binding Sites Located at the Extracellular Domain of Calcium-Sensing-Receptor

Calcium sensing receptor (CaSR), along other members of the family C G protein-coupled receptors (GPCRs), play very important roles in responding to changes in the extracellular calcium concentrations and in circulating levels of amino acids and integrating these extracellular signals into alterations in intracellular signaling pathways. We have reported several potential calcium-binding sites located within the CaSR's extracellular domain using our developed computational algorithms based on geometric factors and surface electrostatic potentials. In the present study, we first report the differential effects of several disease-related mutations located at the predicted calcium binding sites on the inhibition and activation of intracellular calcium responses using both a cell population assay and single cell imaging. We then identify a potential amino acid binding site using computational methods and site-directed mutagenesis. The effect of amino acid binding in altering intracellular calcium responses, especially calcium oscillations, and its synergistic interaction with the effects of extracellular calcium on these parameters are also investigated. A common activation mechanism for CaSR and other family C GPCRs, such as mGluR1 by extracellular calcium and amino acid is been proposed. These results could have important implications for our understanding of how the CaSR integrates information about these two completely different classes of agonists--one an inorganic divalent cation, the other a nutrient--and how the receptor senses these agonists in health and in disease states.

CaSR Mediated Gastric Acid Secretion via the Vacuolar H+-ATPase - With Special Emphasis on the Effect of Extracellular Calcium and Changes in Systemic pH

Purpose: One of the most important functions of the stomach is gastric acid secretion. Since the H+, K+ATPase is the main proton pump in the production of gastric acid, little attention has been paid to the vacuolar H+-ATPase. Therefore, our goal was to evaluate whether this ion channel can be activated by calcium via the CaSR like the H+, K+ATPase. We also hypothesized that the activity of the H+-ATPase is influenced by extracellular pH disturbances such as alkalosis and acidosis, therefore leading to a change in acid secretion. Methods: Isolated gastric glands from male Sprague-Dawley rats were used to measure intracellular pH recovery rates, which were monitored using the pH sensitive dye BCECF. Recovery rates were measured after maximally alkalizing the cells using the NH4Cl prepulse technique. To make sure that the recovery rate was due to the vacuolar H+-ATPase we used sodium and potassium free flushing solutions. Calcimimetic effects were induced by adding R641 (400 nM), an allosteric modifier of the CaSR, to the Na+ and K+ free solution. For imitating pH disturbances, all solutions were titrated to pH 7.2 (for acidosis) and 7.6 (for alkalosis). Finally, we compared the two different influences (calcium and pH-change) in order to determine which factor had a greater influence on gastric acid secretion. Results: Allosteric modification of the CaSR via R641 significantly increases vacuolar H+-ATPase activity and thus gastric acid secretion (0.09815± 0.00780 versus 0.03001±0.00305, p-value <0.01). Metabolic acidosis decreases (0.02467±0.00203 versus 0.03160±0.00187, p-value=0.0179) and metabolic alkalosis increases hydrochloric acid secretion (0.04833 ±0.00256 versus 0.03160±0.00187, p-value <0.01). Combining the two influences (Ca2+ and pH change) we discovered that acid-induced decreased acid secretion can be stimulated again through addition of R641. In contrast, metabolic alkalosis-induced acid secretion cannot be further increased by adding a calcimimetic. Conclusion: The stomach has a regulatory function on the acid-base homeostasis of the body through its secretion of hydrochloric acid. Additionally, extracellular-induced pH changes influence gastric acid secretion via the H+-ATPase. Hypercalcemic conditions, such as in patients with hyperparathyroidism or end stage renal disease, significantly increase gastric acid secretion through stimulation of the H+-ATPase via the CaSR. This fact might be able to explain the comparatively high numbers of coexisting gastric ulcers in those patients.

The effects of extracellular calcium on motility, pseudopod and uropod formation, chemotaxis, and the cortical localization of myosin II in Dictyostelium discoideum

Extracellular Ca(++), a ubiquitous cation in the soluble environment of cells both free living and within the human body, regulates most aspects of amoeboid cell motility, including shape, uropod formation, pseudopod formation, velocity, and turning in Dictyostelium discoideum. Hence it affects the efficiency of both basic motile behavior and chemotaxis. Extracellular Ca(++) is optimal at 10 mM. A gradient of the chemoattractant cAMP generated in the absence of added Ca(++) only affects turning, but in combination with extracellular Ca(++), enhances the effects of extracellular Ca(++). Potassium, at 40 mM, can partially substitute for Ca(++). Mg(++), Mn(++), Zn(++), and Na(+) cannot. Extracellular Ca(++), or K(+), also induce the cortical localization of myosin II in a polar fashion. The effects of Ca(++), K(+) or a cAMP gradient do not appear to be similarly mediated by an increase in the general pool of free cytosolic Ca(++). These results suggest a model, in which each agent functioning through different signaling systems, converge to affect the cortical localization of myosin II, which in turn effects the behavioral changes leading to efficient cell motility and chemotaxis. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.

Effects of extracellular calcium on the growth‐differentiation switch in immortalized keratinocyte HaCaT cells compared with normal human keratinocytes

The keratinocyte growth and differentiation switch, tightly regulated by several mechanisms, is generally associated with decreased proliferation, cell cycle arrest in G0/G1 phase and expression of epidermal differentiation markers, such as keratin 1 (K1), keratin 10 (K10) and involucrin. In vitro, the spontaneously immortalized human keratinocyte cell line HaCaT is often used as a model to study keratinocyte functions. Comparative differentiation studies between HaCaT cells and normal human keratinocytes (NHK) over an extended time-period have rarely been reported. Therefore, we studied their switch from a proliferating to a differentiated state over 13 days. As culture conditions involved changes in cellular responses, cells were cultured in a specific medium for keratinocyte growth and differentiation was induced by increasing extracellular calcium concentration from 0.09 to 1.2 mm. In NHK, addition of calcium-induced morphological changes and concomitant decreased proliferation. For HaCaT cells, calcium addition resulted in morphological changes, but in an unexpected manner, cells were more proliferative than when cultured at low calcium levels. HaCaT cell hyperproliferation correlated with cell cycle analysis, showing an accumulation in S/G2-M phases. Furthermore, RT-PCR and western blot analysis revealed a delay in the expression of the differentiation markers K1, K10 and involucrin in HaCaT cells compared with NHK. In conclusion, even though calcium-induced differentiation was not associated with a decreased cell proliferation, HaCaT cells conserved properties characteristic of differentiation.

Effect of extracellular calcium on the additive effect of theophylline on the cardiac response to catecholamine

At diff erent extracellular calcium concentrations, the positive inotropic eff ect of isoproterenol and isoproterenol in combination with theophylline, a phosphodiesterase inhibitor have been evaluated in the isolated frog heart and the isolated guinea pig left atria to investigate whether extracellular calcium produces any eff ect on the additive eff ect of the theophylline on the cardiac response to catecholamine. Cumulative dose response study of isoproterenol and isoproterenol in presence of theophylline at diff erent extracellular calcium concentration was performed. Th e study revealed an increase in additive eff ect of theophylline on the cardiac response to catecholamine with increase in extracellular calcium concentration, but increase in extracellular calcium concentration decreased the myocardial responsiveness to additive eff ect of theophylline and isoproterenol combination. Th e mechanism of positive inotropic eff ect of isoproterenol and in combination with theophylline involves increase in intracellular CAMP by diff erent pathways and extracellular calcium produces positive inotropic eff ect by initiating the interaction between the contractile proteins actin and myosin.The study revealed that an increase in the concentration of extracellular calcium increased the additive eff ect of theophylline and isoproterenol combination, but a decrease in the myocardial responsiveness was observed. Key words: Additive eff ect, cAMP, isoproterenol, positive inotropic eff ect, theophylline and calcium

Effect of extracellular calcium on the additive effect of theophylline on the cardiac response to catecholamine

Effect of extracellular calcium on the additive effect of theophylline on the cardiac response to catecholamine

Effects of extracellular calcium on cell membrane resealing in sonoporation

Sonoporation has been exploited as a promising strategy for intracellular drug and gene delivery. The technique uses ultrasound to generate pores on the cell membrane to allow entry of extracellular agents into the cell. Resealing of these non-specific pores is a key factor determining both the uptake and post-ultrasound cell survival. This study examined the effects of extracellular Ca 2+ on membrane resealing in sonoporation, using Xenopus oocytes as a model system. The cells were exposed to tone burst ultrasound (1.06 MHz, duration 0.2 s, acoustic pressure 0.3 MPa) in the presence of 0.1% Definity® at various extracellular [Ca 2+] (0–3 mM). Sonoporation inception and resealing in a single cell were monitored in real time via the transmembrane current of the cell under voltage clamp. The time-resolved measurements of transmembrane current revealed the involvement of two or more Ca 2+ related processes with different rate constants and characteristics. Rapid resealing occurred immediately after ultrasound application followed by a much slower resealing process. Complete resealing required [Ca 2+] above 0.54 mM. The cells resealed in 6–26 s at 1.8 mM Ca 2+, but took longer at lower concentrations, up to 58–170 s at 0.54 mM Ca 2+.

Calcium-sensing Receptor Attenuates AVP-induced Aquaporin-2 Expression via a Calmodulin-dependent Mechanism

Recent evidence suggests that arginine vasopressin (AVP)-dependent aquaporin-2 expression is modulated by the extracellular calcium-sensing receptor (CaSR) in principal cells of the collecting duct, but the signaling pathways mediating this effect are unknown. Using a mouse cortical collecting duct cell line (mpkCCD(cl4)), we found that increasing the concentration of apical extracellular calcium or treating with the CaSR agonists neomycin or Gd(3+) attenuated AVP-dependent accumulation of aquaporin-2 mRNA and protein; CaSR gene-silencing prevented this effect. Calcium reduced the AVP-induced accumulation of cAMP, but this did not occur by increased degradation of cAMP by phosphodiesterases or by direct inhibition of adenylate cyclase. Notably, the effect of extracellular calcium on AVP-dependent aquaporin-2 expression was prevented by inhibition of calmodulin. In summary, our results show that high concentrations of extracellular calcium attenuate AVP-induced aquaporin-2 expression by activating the CaSR and reducing coupling efficiency between V(2) receptor and adenylate cyclase via a calmodulin-dependent mechanism in cultured cortical collecting duct cells.

Influence of temperature on calcium sensitivity in the ventricular myocardium of the South American lungfish: Effects of extracellular calcium and adrenaline

The mechanical properties of ventricular myocardium of the South American lungfish, Lepidosiren paradoxa, acclimated to 25 °C, were evaluated in vitro at 15, 25 and 35 °C. The inotropism (Fc—% of initial values) of ventricle strips was examined in response to adrenaline (from 10 −8 to 10 −5 M) and extracellular calcium (from 2.5 to 14.5 mM) in all experimental temperatures. At 15 and 25 °C, Fc rose when extracellular Ca 2+ or adrenaline were increased, while Fc remained unchanged at 35 °C. These results suggest that at lower temperatures Ca 2+ availability is a limiting step to cardiac performance and can be ameliorated by adrenergic stimulation. In contrast, since inotropic agents failed to increase cardiac inotropism at 35 °C, lungfish myocytes seem to show a high temperature sensitivity, which increases Ca 2+-buffering capacity and/or Ca 2+ transportation from and to cytosol as well as myofilaments Ca 2+ sensitivity.

93 CALCIUM REMOVAL INCREASES THE PROTECTIVE EFFECTS OF β-CYCLODEXTRIN PLUS CHOLESTEROL ON PORCINE SPERM DURING COLD SHOCK

Porcine sperm are extremely sensitive to the damaging effects of cold shock and cryopreservation. Cholesterol-binding molecules, such as 2-hydroxypropyl-�-cyclodextrin (HBCD), improve post-thaw and post-cooling porcine sperm viability when added to an egg yolk-based extender, but also enhance sperm capacitation in other species. Depending upon the environmental cholesterol content, HBCD can act either as a cholesterol shuttle or sink to increase or decrease, respectively, sperm plasma membrane cholesterol content. Increasing the sperm cholesterol to phospholipid ratio reduces cold shock sensitivity whereas decreasing the ratio initiates the process of sperm capacitation. An increase in protein tyrosine phosphorylation correlates with sperm capacitation and has been shown to be dependent upon the presence of extracellular calcium. Sperm intracellular calcium also increases during cold shock. The objective of this study was to determine the combined effects of extracellular calcium and membrane cholesterol manipulation on porcine sperm viability and protein tyrosine phosphorylation following cold shock (10�C for 10 min). Viability was assessed using CFDA/propidium iodide staining. Protein tyrosine phosphorylation, previously shown to correlate with porcine sperm capacitation, was evaluated via antiphosphotyrosine (clone 4G10) immunoblots. We report here that following cold shock, porcine sperm incubated in defined medium containing both 0.8 mM HBCD and 0.5 mM cholesterol 3-sulfate (ChS) incubated in the absence of added extracellular calcium and the presence of 6 mM EGTA have significantly improved viability (90.5 � 6.3%, n = 3) when compared with cold-shocked sperm incubated in either the same medium with calcium (46.1 � 3.8%), without HBCD or ChS (26.5 � 7.4% with calcium; 46.5 � 13.1% without calcium), or with HBCD alone (17.0 � 7.4% with calcium, 36.8 � 7.5% without calcium). As we have found previously, treatment with 0.8 mM HBCD plus 0.5 mM ChS completely inhibited the increase in protein tyrosine phosphorylation induced by the cold shock treatment. Although protein tyrosine phosphorylation correlates with porcine sperm capacitation, the ability of cold shock treatment to induce the same phosphorylation pattern indicates that other processes or pathways may contribute to its appearance. Removing extracellular calcium consistently decreased, but did not completely eliminate, the protein tyrosine phosphorylation induced by cold shock. These results indicate that cold shock-induced protein tyrosine phosphorylation is not dependent upon, but can be modulated by, extracellular calcium. The combined effects of calcium, HBCD and ChS on viability suggest that porcine sperm viability following cold shock is best maintained by removing extracellular calcium and increasing membrane cholesterol content via the cholesterol shuttle activity of HBCD. This work was supported by grants from PA Dept. Ag. (ME 443291) and the NIH (5-K08-HD041430).