Mechanisms involved in calcium oxalate endocytosis by Madin-Darby canine kidney cells

Calcium oxalate (CaOx) crystals adhere to and are internalized by tubular renal cells and it seems that this interaction is related (positively or negatively) to the appearance of urinary calculi. The present study analyzes a series of mechanisms possibly involved in CaOx uptake by Madin-Darby canine kidney (MDCK) cells. CaOx crystals were added to MDCK cell cultures and endocytosis was evaluated by polarized light microscopy. This process was inhibited by an increase in intracellular calcium by means of ionomycin (100 nM; N = 6; 43.9% inhibition; P<0.001) or thapsigargin (1 µM; N = 6; 33.3% inhibition; P<0.005) administration, and via blockade of cytoskeleton assembly by the addition of colchicine (10 µM; N = 8; 46.1% inhibition; P<0.001) or cytochalasin B (10 µM; N = 8; 34.2% inhibition; P<0.001). Furthermore, CaOx uptake was reduced when the activity of protein kinase C was inhibited by staurosporine (10 nM; N = 6; 44% inhibition; P<0.01), or that of cyclo-oxygenase by indomethacin (3 µM; N = 12; 17.2% inhibition; P<0.05); however, the uptake was unaffected by modulation of potassium channel activity with glibenclamide (3 µM; N = 6), tetraethylammonium (1 mM; N = 6) or cromakalim (1 µM; N = 6). Taken together, these data indicate that the process of CaOx internalization by renal tubular cells is similar to the endocytosis reported for other systems. These findings may be relevant to cellular phenomena involved in early stages of the formation of renal stones.

Study of urinary acidification in patients with idiopathic hypocitraturia

Hypocitraturia (HCit) is one of the most remarkable features of renal tubular acidosis, but an acidification defect is not seen in the majority of hypocitraturic patients, whose disease is denoted idiopathic hypocitraturia. In order to assess the integrity of urinary acidification mechanisms in hypocitraturic idiopathic calcium stone formers, we studied two groups of patients, hypocitraturic (HCit, N = 21, 39.5 ± 11.5 years, 11 females and 10 males) and normocitraturic (NCit, N = 23, 40.2 ± 11.7 years, 16 females and 7 males) subjects, during a short ammonium chloride loading test lasting 8 h. During the baseline period HCit patients showed significantly higher levels of titratable acid (TA). After the administration of ammonium chloride, mean urinary pH (3rd to 8th hour) and TA and ammonium excretion did not differ significantly between groups. Conversely, during the first hour mean urinary pH was lower and TA and ammonium excretion was higher in HCit. The enhanced TA excretion by HCit during the baseline period and during the first hour suggests that the phosphate buffer mechanism is activated. The earlier response in ammonium excretion by HCit further supports other evidence that acidification mechanisms react promptly. The present results suggest that in the course of lithiasic disease, hypocitraturia coexists with subtle changes in the excretion of hydrogen ions in basal situations.

Zinc accumulation in phosphate granules of Ucides cordatus hepatopancreas

Amorphous phosphate granules are present in vertebrate and invertebrate organisms. The functions attributed to these structures depend on their mineral contents and organic matrix composition. In the present study we have determined zinc concentrations in the hepatopancreas of the crab Ucides cordatus from regions contaminated with zinc, and the elemental composition of hepatopancreal phosphate granules. Organisms were collected from the contaminated areas of Sepetiba Bay (SB) and Guanabara Bay (GB), and from a non-contaminated area, Ribeira Bay (RB). The first two sites are located near the metropolitan region of Rio de Janeiro city, Brazil. Atomic absorption spectroscopy (AAS) showed a significant difference (P<0.05) for zinc concentration in the hepatopancreas from organisms collected at the contaminated sites GB (210 ± 20 µg/g dry weight) and SB (181 ± 16 µg/g dry weight) compared to the non-contaminated site RB (76 ± 14 µg/g dry weight). Phosphate granules isolated from hepatopancreatic tissue were studied by electron diffraction (ED), energy dispersive X-ray analysis (EDX) and electron spectroscopic imaging (ESI). ED of granules presented no diffraction spots, indicating that these structures are in an amorphous state, while EDX of granules isolated from a contaminated area contained P, Ca and Zn. Mg, Cl and Fe were also found in some of the spectra. ESI showed that O, P and Ca were colocalized in the mineralized layers of most granules observed. The correlation between the results obtained by AAS and those obtained by microanalytical techniques suggests that the hepatopancreatic granules of U. cordatus may be related to the phenomenon of heavy metal retention.

A moderate decrease in temperature inhibits the calcium signaling mechanism(s) of the regulatory volume decrease in chick embryo cardiomyocytes

Chick cardiomyocytes, when submitted to hyposmotic swelling, exhibit a partial regulatory volume decrease (RVD). A Ca2+ influx by stretch-activated channels signals a taurine efflux and the RVD at 37°C. We evaluated the cell's performance at room temperature. Cardiomyocytes isolated and cultured from 11-day-old chick embryos were submitted to a hyposmotic solution (180 mOsm/kg H2O) at 37°C and at room temperature (26°C). Under these conditions we measured the changes in cell volume as well as the intracellular free Ca2+ (using fura-2). During hyposmotic swelling, cells at 37°C displayed a peak relative volume of 1.61 ± 0.03 and recovery to 1.22 ± 0.04 (N = 14), while cells at 26°C presented a peak swell relative volume of 1.74 ± 0.06 and did not recover (1.59 ± 0.09, N = 9). Transient increases in intracellular Ca2+, which are characteristic of the normal RVD, were observed at both temperatures (29.1 ± 4.5% (N = 8) and 115.2 ± 42.8% (N = 5) increase at 37° and 26°C (P<0.05), respectively). A delay in the Ca2+ transient increase was also observed when the cells were at 26°C (109 ± 34 s compared to 38 ± 9 s at 37°C, P<0.05). At room temperature the RVD does not occur because the calcium transient increase, which is an early event in the signaling of the RVD, is delayed. Also, free calcium is not cleared as in the 37°C RVD. In the normal RVD the free calcium returns to baseline levels. The very high and persistent free calcium levels seen at room temperature can lead to unregulated enzyme activities and may promote irreversible injury and cell death.

Involvement of calcium in pain and antinociception

Calcium ions are widely recognized to play a fundamental role in the regulation of several biological processes. Transient changes in cytoplasmic calcium ion concentration represent a key step for neurotransmitter release and the modulation of cell membrane excitability. Evidence has accumulated for the involvement of calcium ions also in nociception and antinociception, including the analgesic effects produced by opioids. The combination of opioids with drugs able to interfere with calcium ion functions in neurons has been pointed out as a useful alternative for safer clinical pain management. Alternatively, drugs that reduce the flux of calcium ions into neurons have been indicated as analgesic alternatives to opioids. This article reviews the manners by which calcium ions penetrate cell membranes and the changes in these mechanisms caused by opioids and calcium antagonists regarding nociceptive and antinociceptive events.

Extracellular calcium-sensing receptor: structural and functional features and association with diseases

The recently cloned extracellular calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays an essential role in the regulation of extracellular calcium homeostasis. This receptor is expressed in all tissues related to this control (parathyroid glands, thyroid C-cells, kidneys, intestine and bones) and also in tissues with apparently no role in the maintenance of extracellular calcium levels, such as brain, skin and pancreas. The CaR amino acid sequence is compatible with three major domains: a long and hydrophilic aminoterminal extracellular domain, where most of the activating and inactivating mutations described to date are located and where the dimerization process occurs, and the agonist-binding site is located, a hydrophobic transmembrane domain involved in the signal transduction mechanism from the extracellular domain to its respective G protein, and a carboxyterminal intracellular tail, with a well-established role for cell surface CaR expression and for signal transduction. CaR cloning was immediately followed by the association of genetic human diseases with inactivating and activating CaR mutations: familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism are caused by CaR-inactivating mutations, whereas autosomal dominant hypoparathyroidism is secondary to CaR-activating mutations. Finally, we will comment on the development of drugs that modulate CaR function by either activating (calcimimetic drugs) or antagonizing it (calcilytic drugs), and on their potential therapeutic implications, such as medical control of specific cases of primary and uremic hyperparathyroidism with calcimimetic drugs and a potential treatment for osteoporosis with a calcilytic drug.

Low pH and calcium effects on net Na+ and K+ fluxes in two catfish species from the Amazon River (Corydoras: Callichthyidae)

The present study analyzes Na+ and K+ disturbances caused by low pH in two catfish species from the Amazon River. Corydoras adolfoi inhabits ion-poor, black-stained, low pH (3.5-4.0) waters, while C. schwartzi is native to ion-rich waters at circumneutral pH. Fish were exposed to pH 3.5 Ca2+-free, and Ca2+-enriched (~500 µmol/l) water to determine the protective effects of calcium. Net Na+ and K+ fluxes were measured in the water collected from the fish experimental chambers. C. adolfoi was unable to control the Na+ efflux at low pH, exhibiting Na+ loss up to -594 ± 84 nmol g-1 h-1 during the first hour. After 3 and 6 h, net Na+ flux increased by 7- and 23-fold, respectively. In C. schwartzi, at pH 3.5, the initial high Na+ loss (-1,063 ± 73 nmol g-1 h-1) was gradually attenuated. A K+ loss occurred in both species, but remained relatively constant throughout exposure. High [Ca2+] affected ion losses in both species. C. adolfoi had 70% loss attenuation, indicating incapacity to control Na+ efflux. In C. schwartzi, elevated [Ca2+] completely prevented the Na+ losses caused by exposure to low pH. Rather different patterns were seen for K+ fluxes, with C. adolfoi showing no K+ disruption when exposed to low pH/high [Ca2+]. Thus, C. adolfoi loses Na+ during acid exposure, but has the ability to control K+ loss, while C. schwartzi controls diffusive Na+ loss but exhibits a slightly higher K+ loss. Ion balance was influenced by [Ca2+] at low pH in C. schwartzi but not in C. adolfoi.

Effects of elevated calcium on motor and exploratory activities of rats

The effects of serum and brain calcium concentration on rat behavior were tested by maintaining animals on either distilled water (N = 60) or water containing 1% calcium gluconate (N = 60) for 3 days. Animals that were maintained on high calcium drinking water presented increased serum calcium levels (control = 10.12 ± 0.46 vs calcium treated = 11.62 ± 0.51 µg/dl). Increase of brain calcium levels was not statistically significant. In the behavioral experiments each rat was used for only one test. Rats that were maintained on high calcium drinking water showed increased open-field behavior of ambulation (20.68%) and rearing (64.57%). On the hole-board, calcium-supplemented animals showed increased head-dip (67%) and head-dipping (126%), suggesting increased ambulatory and exploratory behavior. The time of social interaction was normal in animals maintained on drinking water containing added calcium. Rats supplemented with calcium and submitted to elevated plus-maze tests showed a normal status of anxiety and elevated locomotor activity. We conclude that elevated levels of calcium enhance motor and exploratory behavior of rats without inducing other behavioral alterations. These data suggest the need for a more detailed analysis of several current proposals for the use of calcium therapy in humans, for example in altered blood pressure states, bone mineral metabolism disorders in the elderly, hypocalcemic states, and athletic activities.

Interactions between intracellular Ca2+ stores: Ca2+ released from the NAADP pool potentiates cADPR-induced Ca2+ release

Cells possess multiple intracellular Ca2+-releasing systems. Sea urchin egg homogenates are a well-established model to study intracellular Ca2+ release. In the present study the mechanism of interaction between three intracellular Ca2+ pools, namely the nicotinic acid adenine dinucleotide phosphate (NAADP), the cyclic ADP-ribose (cADPR) and the inositol 1',4',5'-trisphosphate (IP3)-regulated Ca2+ stores, is explored. The data indicate that the NAADP Ca2+ pool could be used to sensitize the cADPR system. In contrast, the IP3 pool was not affected by the Ca2+ released by NAADP. The mechanism of potentiation of the cADPR-induced Ca2+ release, promoted by Ca2+ released from the NAADP pool, is mediated by the mechanism of Ca2+-induced Ca2+ release. These data raise the possibility that the NAADP Ca2+ store may have a role as a regulator of the cellular sensitivity to cADPR.

Effect of calcium intake on urinary oxalate excretion in calcium stone-forming patients

Dietary calcium lowers the risk of nephrolithiasis due to a decreased absorption of dietary oxalate that is bound by intestinal calcium. The aim of the present study was to evaluate oxaluria in normocalciuric and hypercalciuric lithiasic patients under different calcium intake. Fifty patients (26 females and 24 males, 41 ± 10 years old), whose 4-day dietary records revealed a regular low calcium intake (<=500 mg/day), received an oral calcium load (1 g/day) for 7 days. A 24-h urine was obtained before and after load and according to the calciuria under both diets, patients were considered as normocalciuric (NC, N = 15), diet-dependent hypercalciuric (DDHC, N = 9) or diet-independent hypercalciuric (DIHC, N = 26). On regular diet, mean oxaluria was 30 ± 14 mg/24 h for all patients. The 7-day calcium load induced a significant decrease in mean oxaluria compared to the regular diet in NC and DIHC (20 ± 12 vs 26 ± 7 and 27 ± 18 vs 32 ± 15 mg/24 h, respectively, P<0.05) but not in DDHC patients (22 ± 10 vs 23 ± 5 mg/24 h). The lack of an oxalate decrease among DDHC patients after the calcium load might have been due to higher calcium absorption under higher calcium supply, with a consequent lower amount of calcium left in the intestine to bind with oxalate. These data suggest that a long-lasting regular calcium consumption <500 mg was not associated with high oxaluria and that a subpopulation of hypercalciuric patients who presented a higher intestinal calcium absorption (DDHC) tended to hyperabsorb oxalate as well, so that oxaluria did not change under different calcium intake.

Gender differences in vascular expression of endothelin and ET A/ET B receptors, but not in calcium handling mechanisms, in deoxycorticosterone acetate-salt hypertension

We determined if the increased vascular responsiveness to endothelin-1 (ET-1) observed in male, but not in female, DOCA-salt rats is associated with differential vascular mRNA expression of ET-1 and/or ET A/ET B receptors or with functional differences in Ca2+ handling mechanisms by vascular myocytes. Uninephrectomized male and female Wistar rats received DOCA and drinking water containing NaCl/KCl. Control rats received vehicle and tap water. Blood pressure and contractile responses of endothelium-denuded aortic rings to agents which induce Ca2+ influx and/or its release from internal stores were measured using standard procedures. Expression of mRNA for ET-1 and ET A/ET B receptors was evaluated by RT-PCR after isolation of total cell RNA from both aorta and mesenteric arteries. Systolic blood pressure was higher in male than in female DOCA rats. Contractions induced by Bay K8644 (which activates Ca2+ influx through voltage-operated L-type channels), and by caffeine, serotonin or ET-1 in Ca2+-free buffer (which reflect Ca2+ release from internal stores) were significantly increased in aortas from male and female DOCA-salt compared to control aortas. DOCA-salt treatment of male, but not female, rats statistically increased vascular mRNA expression of ET-1 and ET B receptors, but decreased the expression of ET A receptors. Molecular up-regulation of vascular ET B receptors, rather than differential changes in smooth muscle Ca2+ handling mechanisms, seems to account for the increased vascular reactivity to ET-1/ET B receptor agonists and higher blood pressure levels observed in male DOCA-salt rats.

Role of Tamm-Horsfall protein and uromodulin in calcium oxalate crystallization

One of the defenses against nephrolithiasis is provided by macromolecules that modulate the nucleation, growth, aggregation and retention of crystals in the kidneys. The aim of the present study was to determine the behavior of two of these proteins, Tamm-Horsfall and uromodulin, in calcium oxalate crystallization in vitro. We studied a group of 10 male stone formers who had formed at least one kidney stone composed of calcium oxalate. They were classified as having idiopathic nephrolithiasis and had no well-known metabolic risk factors involved in kidney stone pathogenesis. Ten normal men were used as controls, as was a group consisting of five normal women and another consisting of five pregnant women. Crystallization was induced by a fixed supersaturation of calcium oxalate and measured with a Coulter Counter. All findings were confirmed by light and scanning electron microscopy. The number of particulate material deposited from patients with Tamm-Horsfall protein was higher than that of the controls (P<0.001). However, Tamm-Horsfall protein decreased the particle diameter of the stone formers when analyzed by the mode of the volume distribution curve (P<0.002) (5.64 ± 0.55 µm compared to 11.41 ± 0.48 µm of uromodulin; 15.94 ± 3.93 µm and 12.45 ± 0.97 µm of normal men Tamm-Horsfall protein and uromodulin, respectively; 8.17 ± 1.57 µm and 9.82 ± 0.95 µm of normal women Tamm-Horsfall protein and uromodulin, respectively; 12.17 ± 1.41 µm and 12.99 ± 0.51 µm of pregnant Tamm-Horsfall protein and uromodulin, respectively). Uromodulin produced fewer particles than Tamm-Horsfall protein in all groups. Nonetheless, the total volume of the crystals produced by uromodulin was higher than that produced by Tamm-Horsfall protein. Our results indicate a different effect of Tamm-Horsfall protein and uromodulin. This dual behavior suggests different functions. Tamm-Horsfall protein may act on nucleation and inhibit crystal aggregation, while uromodulin may promote aggregation of calcium oxalate crystals.

Mitochondria, calcium and pro-apoptotic proteins as mediators in cell death signaling

Cellular Ca2+ signals are crucial in the control of most physiological processes, cell injury and programmed cell death through the regulation of a number of Ca2+-dependent enzymes such as phospholipases, proteases, and nucleases. Mitochondria along with the endoplasmic reticulum play pivotal roles in regulating intracellular Ca2+ content. Mitochondria are endowed with multiple Ca2+ transport mechanisms by which they take up and release Ca2+ across their inner membrane. During cellular Ca2+ overload, mitochondria take up cytosolic Ca2+, which in turn induces opening of permeability transition pores and disrupts the mitochondrial membrane potential (Dym). The collapse of Dym along with the release of cytochrome c from mitochondria is followed by the activation of caspases, nuclear fragmentation and cell death. Members of the Bcl-2 family are a group of proteins that play important roles in apoptosis regulation. Members of this family appear to differentially regulate intracellular Ca2+ level. Translocation of Bax, an apoptotic signaling protein, from the cytosol to the mitochondrial membrane is another step in this apoptosis signaling pathway.

Impaired beta-adrenergic response and decreased L-type calcium current of hypertrophied left ventricular myocytes in postinfarction heart failure

Infarct-induced heart failure is usually associated with cardiac hypertrophy and decreased ß-adrenergic responsiveness. However, conflicting results have been reported concerning the density of L-type calcium current (I Ca(L)), and the mechanisms underlying the decreased ß-adrenergic inotropic response. We determined I Ca(L) density, cytoplasmic calcium ([Ca2+]i) transients, and the effects of ß-adrenergic stimulation (isoproterenol) in a model of postinfarction heart failure in rats. Left ventricular myocytes were obtained by enzymatic digestion 8-10 weeks after infarction. Electrophysiological recordings were obtained using the patch-clamp technique. [Ca2+]i transients were investigated via fura-2 fluorescence. ß-Adrenergic receptor density was determined by [³H]-dihydroalprenolol binding to left ventricle homogenates. Postinfarction myocytes showed a significant 25% reduction in mean I Ca(L) density (5.7 ± 0.28 vs 7.6 ± 0.32 pA/pF) and a 19% reduction in mean peak [Ca2+]i transients (0.13 ± 0.007 vs 0.16 ± 0.009) compared to sham myocytes. The isoproterenol-stimulated increase in I Ca(L) was significantly smaller in postinfarction myocytes (Emax: 63.6 ± 4.3 vs 123.3 ± 0.9% in sham myocytes), but EC50 was not altered. The isoproterenol-stimulated peak amplitude of [Ca2+]i transients was also blunted in postinfarction myocytes. Adenylate cyclase activation through forskolin produced similar I Ca(L) increases in both groups. ß-Adrenergic receptor density was significantly reduced in homogenates from infarcted hearts (Bmax: 93.89 ± 20.22 vs 271.5 ± 31.43 fmol/mg protein in sham myocytes), while Kd values were similar. We conclude that postinfarction myocytes from large infarcts display reduced I Ca(L) density and peak [Ca2+]i transients. The response to ß-adrenergic stimulation was also reduced and was probably related to ß-adrenergic receptor down-regulation and not to changes in adenylate cyclase activity.

Melatonin and N-acetyl-serotonin cross the red blood cell membrane and evoke calcium mobilization in malarial parasites

The duration of the intraerythrocytic cycle of Plasmodium is a key factor in the pathogenicity of this parasite. The simultaneous attack of the host red blood cells by the parasites depends on the synchronicity of their development. Unraveling the signals at the basis of this synchronicity represents a challenging biological question and may be very important to develop alternative strategies for therapeutic approaches. Recently, we reported that the synchrony of Plasmodium is modulated by melatonin, a host hormone that is synthesized only during the dark phases. Here we report that N-acetyl-serotonin, a melatonin precursor, also releases Ca2+ from isolated P. chabaudi parasites at micro- and nanomolar concentrations and that the release is blocked by 250 mM luzindole, an antagonist of melatonin receptors, and 20 mM U73122, a phospholipase C inhibitor. On the basis of confocal microscopy, we also report the ability of 0.1 µM melatonin and 0.1 µM N-acetyl-serotonin to cross the red blood cell membrane and to mobilize intracellular calcium in parasites previously loaded with the fluorescent calcium indicator Fluo-3 AM. The present data represent a step forward into the understanding of the signal transduction process in the host-parasite relationship by supporting the idea that the host hormone melatonin and N-acetyl-serotonin generate IP3 and therefore mobilize intracellular Ca2+ in Plasmodium inside red blood cells.