The progression of coronary artery calcification in predialysis patients on calcium carbonate or sevelamer.

Coronary artery calcification is more prevalent in dialysis patients than in patients without kidney disease and this is associated with high serum phosphorus. In this study, we evaluate the effect of calcium carbonate or sevelamer treatments on the progression of calcification in 90 predialysis patients. Inclusion criteria were stable serum calcium, phosphorus, parathyroid hormone, and a similar baseline total calcium score (TCS). These patients were not treated by phosphate binder, vitamin D, or statin. They were given low-phosphorus diets without or with daily calcium carbonate or sevelamer throughout the study that averaged 2 years. Baseline demographic or clinical characteristics along with biochemical parameters were not different among the three groups. The TCS significantly increased in patients on the low-phosphorus diet alone, to a lesser extent in calcium carbonate-treated patients, and not at all in sevelamer-treated patients. The progression of coronary calcification paralleled that of the calcium score. Our study shows that sevelamer treatment should not be restricted to dialysis patients; however, a larger study should be undertaken to confirm these results.

Guanosine 3': 5'-cyclic monophosphate-dependent pathway alterations in ventricular cardiomyocytes of spontaneously hypertensive rats.

1. We investigated the effect of the NO-donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) on cardiomyocytes isolated from control normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. 2. Ventricular cardiomyocytes were isolated from SHR and WKY hearts and imaging analysis of fura-2-loaded cells was performed in order to evaluate calcium transient in electrical field paced (0.5 Hz) cells. 3. In WKY cardiomyocytes, 1 - 200 microM SNAP dose-dependently increased cyclic GMP content. In basal conditions, cyclic GMP content of SHR cardiomyocytes was significantly higher than in WKY, but SNAP failed to further increase cyclic GMP over the basal level. 4. In control conditions, the Delta F/F and decay time of the calcium transient were similar in both strains. In WKY cardiomyocytes, SNAP (1 - 100 microM) reduced the decay time. In SHR cardiomyocytes, SNAP was ineffective. Dibutyryl cyclic GMP (10(-6) - 10(-8) M), a membrane permeable cyclic GMP analogue, behaved similarly to SNAP. 5. In WKY and SHR cardiomyocytes, 10(-8) M isoprenaline similarly increased Delta F/F and decreased the decay time. SNAP and dibutyryl cyclic GMP prevented the effect of isoprenaline in WKY, whereas both molecules were ineffective in SHR cardiomyocytes. In WKY, SNAP effects were blocked by pretreating cells with the cGK inhibitor KT-5823. 6. Western blotting analysis of cGK type I showed that the enzyme was expressed in WKY isolated cardiomyocytes, but absent in four out of five SHR preparations. 7. We concluded that the low expression of cGKI may determine the lack of NO/cyclic GMP-dependent regulation on calcium transient in SHR cardiomyocytes. This alteration may contribute to the development of heart hypertrophy in hypertensive status.

Lack of nitric oxide- and guanosine 3':5'-cyclic monophosphate-dependent regulation of alpha-thrombin-induced calcium transient in endothelial cells of spontaneously hypertensive rat hearts.

While the expression and/or activity of endothelial nitric oxide synthase (eNOS) has been characterized in spontaneously hypertensive (SHR) and normotensive Wistar Kyoto rat (WKY) hearts, in coronary endothelial cells (ECs) from both strains, the effect of NO on intracellular calcium concentration ([Ca(2+)](i)) is still unknown. Coronary microvascular ECs were isolated from SHR and WKY and characterized. Immunocytochemistry and Western blot analysis showed that eNOS was similarly expressed in ECs from both strains. Measuring [Ca(2+)](i) by imaging analysis of fura-2-loaded cells, we demonstrated that alpha-thrombin (3-180 U l(-1)) induced a superimposable dose-dependent calcium transient in ECs from both strains. In WKY ECs, S-nitroso-N-acetyl-DL-penicillamine (SNAP) dose-dependently (10 - 100 microM) and 0.1 microM atrial natriuretic factor (ANF) reduced the maximum and the decay time of alpha-thrombin-induced calcium transient. The inhibitory effects of SNAP and ANF were prevented by blocking cyclic GMP-dependent protein kinase. Non selective eNOS inhibitors prolonged the decay time of alpha-thrombin-induced calcium transient, while the selective inducible NOS inhibitor 1400 W was ineffective. SNAP (100 microM) and 0.1 microM ANF increased cyclic GMP content up to 22.9 and 42.3 fold respectively. In SHR ECs, alpha-thrombin-induced calcium transient was not modified by SNAP, ANF or eNOS inhibition. SNAP (100 microM) and 0.1 microM ANF increased cyclic GMP content up to 9. 3 and 51 fold respectively. In WKY ECs, SNAP dose-dependently (10 - 100 microM) reduced also bradykinin-induced calcium transient, while in SHR ECs was ineffective. We concluded that in SHR ECs, the cyclic GMP-dependent regulation of calcium transient is lost.

Calcium waves in unstimulated left ventricular cardiomyocytes isolated from aged spontaneously hypertensive and normotensive rats.

In this work, we described the incidence and the characteristics of calcium waves in cardiomyocytes isolated from aged normotensive rats (Wistar Kyoto, WKY) and age-matched spontaneously hypertensive rats (SHR) using imaging analysis of fura-2-loaded left ventricular cardiomyocytes. Left ventricular cardiomyocytes were isolated by enzymatic digestion from hearts of 18-20 month old WKY and aged-matched SHR. Intracellular calcium concentration did not differ in either strain, whereas the incidence of cells presenting calcium waves was greater in cardiomyocytes isolated from SHR. Moreover, cardiomyocytes isolated from SHR were significantly longer than those isolated from WKY. The calcium wave frequency was lower in SHR cardiomyocytes, while the velocity of the calcium waves was similar in both strains. Our results suggest that alterations in the calcium handling of SHR may contribute to the increased incidence of arrhythmias described in SHR hearts.

Calcium-dependent electrophysiological alterations in hypertrophied rat cardiomyocytes.

We compared the occurrence of calcium-dependent electrophysiological alterations in left ventricular myocytes isolated from the heart of old spontaneously hypertensive (SHR) and age-matched normotensive Wistar Kyoto rats (WKY). Electrical and mechanical activity were measured in patch-clamped myocytes in which intracellular Ca2+ was allowed to change freely. In parallel experiments, intracellular Ca2+ concentration was monitored in fura-2-AM preloaded cells using an image analysis system. Left ventricular myocytes from SHR showed a significantly greater membrane capacitance (an index of cell size), a prolonged duration of the action potential and a higher incidence of delayed afterdepolarization (DADs) and associated aftercontractions, and of spontaneous activity. DADs were likely generated by intracellular Ca2+ waves due to spontaneous and local Ca2+ release from intracellular stores. These results demonstrate that DADs, possibly related to spontaneous intracellular Ca2+ waves, occur frequently in hypertrophied rat cardiomyocytes and may be a relevant arrhythmogenic mechanism.

The effect of tetraethylammonium on intracellular calcium concentration in Alzheimer's disease fibroblasts with APP, S182 and E5-1 missense mutations.

It has been proposed that the lack of intracellular calcium concentration ([Ca2+]i) increase induced by the potassium channel blocker tetraethylammonium (TEA) in skin fibroblast cell lines identifies patients with both sporadic and familial Alzheimer's disease (AD). In order to verify this hypothesis, the effect of TEA on [Ca2+]i was studied in single fura-2-loaded skin fibroblast cell lines available in the Tissue Bank of the Italian Research Council. Four out of eight familial AD patients (one patient with S182 mutation, one patient with E5-1 mutation and two patients with 717 Val-->Ile APP mutation) and two out of five sporadic AD patients showed a positive response to TEA, whereas five out of 11 control lines were unresponsive. Our data suggest that the absence of the TEA-induced increase in [Ca2+]i in skin fibroblast cell lines does not identify all AD patients.

Endothelin 1 is overexpressed in human cirrhotic liver and exerts multiple effects on activated hepatic stellate cells.

BACKGROUND & AIMS: Endothelin (ET) 1 could be involved in the regulation of hepatic microcirculation and in the development of portal hypertension. The expression and distribution of ET-1 in normal and cirrhotic liver tissue and its effects on hepatic stellate cells (HSCs), liver-specific pericytes, were investigated. METHODS: ET-1 expression in liver tissue was analyzed using in situ hybridization and immunohistochemistry. Secretion of ET-1 by HSC was evaluated by radioimmunoassay. Changes in intracellular Ca2+ concentration and cell contraction were studied using digital video imaging. Specific binding of ET-1 was evaluated using self- and cross-displacement curves. RESULTS: ET-1 expression was markedly enhanced in cirrhotic liver tissue, where activated HSCs were shown to be major sites of ET-1 synthesis, as confirmed by studies performed on cultured human HSC. ET-1 exerted several biological actions on HSC, including mitogenicity, activation of mitogen-activated protein kinase, and a rapid increase in intracellular Ca2+ coupled with reversible cell contraction. All these effects appeared to be mediated by ETA receptors. Finally, the relative prevalence of ETA and ETB binding sites changed with the progressive phenotypical modulation of HSC. CONCLUSIONS: ET-1 may act as a paracrine and autocrine factor for activated HSC and contribute to the increased resistance to portal flow in cirrhotic liver.

Iloprost antagonizes the increase in internal calcium concentration induced by alpha-thrombin in human platelets: a study of desensitization.

We studied the interaction between the synthetic prostacyclin analog iloprost and the aggregating agent alpha-thrombin by measuring the internal calcium ion concentration ([Ca(2+)]i) of human fura-2-loaded platelets. Iloprost (0.003-100 micrograms/l) did not modify the resting calcium level; when added 2 minutes before exposure of the platelets to a submaximally active concentration of alpha-thrombin (10 U/l), iloprost dose-dependently antagonized the increase in [Ca(2+)]i. To evaluate if iloprost retained this antagonistic effect even after a prolonged contact, which is well known to cause a "desensitization" phenomenon, platelets were preincubated with iloprost (35 micrograms/l) for 3 hours. After washout, the effect of newly added iloprost (0.01-100 micrograms/l) on the alpha-thrombin-induced increase in [Ca(2+)]i was tested. Iloprost was still able to antagonize the increase in [Ca(2+)]i induced by alpha-thrombin in "desensitized" platelets; however, the dose-inhibitory response curve was significantly shifted to the right when compared with that obtained in control platelets (i.e., platelets preincubated for 3 hours with iloprost's solvent), and the resulting IC50 was significantly higher: 1.78 versus 0.2 micrograms/l (p < 0.001). Since the maximal inhibitory effect of iloprost could also be reached under these experimental conditions, we conclude that iloprost retains its ability to antagonize the increase in [Ca(2+)]i induced by alpha-thrombin in desensitized platelets.

The mitogenic effect of platelet-derived growth factor in human hepatic stellate cells requires calcium influx.

Platelet-derived growth factor (PDGF) is a key mitogen for hepatic stellate cells (HSC) and has been shown to be implicated in liver tissue repair and fibrogenesis. In this study the relationship between PDGF-induced intracellular Ca2+ concentration ([Ca2+]i) increase and mitogenesis in cultured human HSC was evaluated. In high-density cell cultures (80-90% subconfluence), PDGF induced a significant increase in [Ca2+]i, characterized by a short-lasting peak phase, which was followed by a long-lasting plateau phase. The plateau phase was abolished in the absence of extracellular Ca2+. However, in low-density cell cultures (30-40% subconfluence), the plateau phase was absent or markedly less pronounced. In parallel sets of experiments, PDGF was significantly less effective in inducing mitogenesis in low-density cell cultures than in high-density cell cultures and was totally ineffective in the absence of extracellular Ca2+. These results suggest that 1) spatial and time dynamics of PDGF-induced [Ca2+]i increase are dependent on cell density and 2) PDGF-induced mitogenesis requires extracellular Ca2+ influx.

Effects of L- and D-arginine and some related esters on the cytosolic mechanisms of alpha-thrombin-induced human platelet activation.

1. In Fura-2 preloaded human platelets, the increase in cytosolic calcium induced by alpha-thrombin was reduced by some L- and D-arginine ester compounds the IC50 (microM) values of which were 7.4 for TAEE, 56.9 for BAEE, 77.6 for TAME, 560 for T(d)AME, 656.3 for L-ArgOMe and 2206.7 for D-ArgOMe. alpha-tosyl-L-Arginine, L- and D-arginine were inactive. 2. The inhibitory activity of the L-arginine esters was not modified when platelets were pretreated with 100 microM N omega-monomethyl-L-arginine. 3. The L-arginine esters did not increase cyclic GMP content in platelets either in the presence or absence of indomethacin and apyrase at rest and after alpha-thrombin stimulation. 4. The kinetic parameters of platelet Na+/H+ antiporter (amiloride-inhibitable, evaluated after cytosolic nigericin-induced acidification) were modified by L- and D-arginine esters, while the native amino acids were ineffective. 5. The inhibitory effects of the L- and D-arginine esters on platelet activation appear to be mainly due to their inhibitory effect on Na+/H+ antiporter.

Effects of endothelin-1 on bovine parathyroid cells.

Endothelin-1 (ET-1) is synthesized and released by parathyroid epithelium. The effects of endothelin isopeptides were studied in clonal bovine parathyroid endothelial (BPE) cells. BPE cells did not produce ET-1, but showed ETA receptors (Kd = 0.1 +/- 0.02 nM, mean +/- SE). ET-1 (10(-8)10(-11)M) increased the intracellular calcium ion concentration ([Ca2+]i) in BPE cells, while endothelin-3 (ET-3) was ineffective. The increase in [Ca2+]i was less sustained in the absence of extracellular Ca2+ ions. Moreover ET-1 induced phospholipase C (PLC) activation, as demonstrated by the increase in inositol trisphosphate. Cell growth was not affected by ET-1 in a wide range of concentrations. The present findings demonstrate: 1) BPE cells possess ETA receptors; 2) the peptide activates PLC and increases cytosolic [Ca2+]i via both a release of Ca2+ ions from intracellular calcium pool(s) and an influx of the cation from the extracellular milieu. A possible role of ET-1 as a paracrine factor in parathyroid tissue can be hypothesized.

Fat-storing cells as liver-specific pericytes. Spatial dynamics of agonist-stimulated intracellular calcium transients.

Liver perisinusoidal fat-storing cells (FSC) show morphological and ultrastructural characteristics similar to pericytes regulating local blood flow in other organs. In the present study we have analyzed whether FSC respond to local vasoconstrictors such as thrombin, angiotensin-II, and endothelin-1 with an increase in intracellular free calcium concentration ([Ca2+]i) coupled with effective cell contraction. All agonists tested induced a rapid and dose-dependent increase in [Ca2+]i followed by a sustained phase lasting several minutes in confluent monolayers of Fura-2-loaded human FSC. Pharmacological studies performed using different Ca2+ channel blockers indicated that, at least for thrombin and angiotensin-II, the sustained phase is due to the opening of voltage-sensitive membrane Ca2+ channels. To analyze the temporal and spatial dynamics of Ca2+ release in response to these agonists, we performed experiments on individual Fura-2-loaded human FSC using a dual wavelength, radiometric video imaging system. The rise in [Ca2+]i was exclusively localized to the cytoplasm, particularly in the branching processes. Increases in [Ca2+]i more than four-fold were associated with a simultaneous and transient reduction of cell area indicating reversible cell contraction. Our results indicate that the Ca(2+)-dependent contraction of human FSC in vitro may reflect a potential role in regulating sinusoidal blood flow in vivo.