Transmembrane signalling in human monocyte/mesangial cell co-cultures: role of cytosolic Ca(2+).

BACKGROUND: Adhesion of monocytes triggers apoptosis, cytotoxicity, cytokine release, and later proliferation of cultured human mesangial cells (HMC). In the search for transmembrane signals transducing the interaction of HMC adhesion molecules with leukocyte counterreceptors, we measured variations of cytosolic Ca(2+) ([Ca(2+)](i)) in HMC and monocytes of the U937 cell line during 6-h co-cultures. METHODS: Monolayer cultures of HMC and suspensions of U937 cells were loaded with the fluoroprobe fura 2-AM and subsequently co-cultured for 6 h while separately monitoring by microfluorometry the Ca(2+)-dependent 500 nm fluorescent emission of each cell line at fixed intervals upon excitation at 340/380 nm. RESULTS: U937 and peripheral blood monocyte adhesion was followed in HMC by a slow, progressive rise of [Ca(2+)](i) from basal levels of 96+/-9 nM to 339+/-54 at 60 min and 439+/-44 nM at 3 h. The [Ca(2+)](i) elevation reached a steady state thereafter, while parallel monolayers incubated with control media maintained resting levels throughout the co-culture with stable fluoroprobe retention. Receptor sensitivity to vasoconstrictor agents, including compounds not released by monocytes, such as angiotensin II, was rapidly downregulated in HMC co-cultured with U937 cells. No [Ca(2+)](i) changes could be elicited by the octapeptide or by the TxA(2) analogue, U-46619, as early as 30 min after exposure to U937 cells. No [Ca(2+)](i) changes were observed in U937 cells throughout the co-culture. Conditioned media from monocytes and from co-cultured HMC+U937 cells had no effect on [Ca(2+)](i) of HMC. Ca(2+) entry leading to fura 2 saturation was still inducible by Ca(2+) ionophores, such as ionomycin and 4-Br-A23187, which also inhibited the responses to vasoconstrictors. Ca(2+)-free solutions prevented the [Ca(2+)](i) rise as well as subsequent receptor inactivation, implicating Ca(2+) influx through store-operated Ca(2+) channels (SOC), a major pathway for Ca(2+) entry in these cultured cells. Ca(2+) influx was confirmed by Mn(2+)-quenching of fura 2. CONCLUSIONS: In HMC, early changes in [Ca(2+)](i) signal for monocyte adhesion in a co-culture model of glomerular inflammation. This signalling mechanism may mediate the functional responses elicited in glomerular cells by leukocytes, including downregulation of receptors for vasoactive agents.     

Effect of high glucose on basal intracellular calcium regulation in rat mesangial cell

BACKGROUND: A number of cellular mechanisms are critically dependent on intracellular Ca(2+) homeostasis. A sustained increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) is capable of activating a number of potentially harmful processes including phenotype change to secretory type, dysregulated cell proliferation, and cell injury and death. Mesangial cells (MCs) play an important role in the pathophysiology of diabetic nephropathy. METHODS: We evaluated the effect of high glucose on basal [Ca(2+)](i) in the unstimulated state and identified its contributing pathways. MCs were isolated and cultured from Sprague-Dawley rats. [Ca(2+)](i) was measured by fluorometric technique with fura-2AM. RESULTS: In a dose-dependent manner, superfusion of MCs with Tyrode's solution containing high glucose (30 and 50 mM) induced a delayed spontaneous increase in [Ca(2+)](i), which was not found in those with normal (5.5 mM) glucose or mannitol. The high glucose-induced increase in [Ca(2+)](i)()occurred through transmembrane influx of extracellular Ca(2+) and was blocked by SKF96365, an inhibitor of store-operated Ca(2+) influx. Na(+)-Ca(2+) exchanger (NCX) activity, a major channel regulating basal [Ca(2+)](i), and the clearing ability of intracellular Ca(2+) were depressed after MCs were cultured in high-glucose medium. Western blot analysis revealed the decreased expression of a 70-kD NCX protein in MCs cultured in high-glucose medium. CONCLUSIONS: A high-glucose concentration induced a spontaneous increase in basal [Ca(2+)](i) of MCs without stimulation. There was a decrease in the activity of NCX in the high-glucose condition, which seems to occur at the level of protein expression. The present results provide a novel insight into the mechanisms of diabetic nephropathy in that intracellular Ca(2+) homeostasis is an important secondary messenger and a mediator in hormonal signaling.     

Overexpression of the Na/Ca exchanger shapes stimulus-induced cytosolic Ca(2+) oscillations in insulin-producing BRIN-BD11 cells.

In response to glucose, mouse beta-cells display slow oscillations of the membrane potential and cytosolic free Ca(2+) concentration ([Ca(2+)](i)), whereas rat beta-cells display a staircase increase in these parameters. Mouse and rat islet cells differ also by their level of Na/Ca exchanger (NCX) activity. The view that the inward current generated by Na/Ca exchange shapes stimulus-induced electrical activity and [Ca(2+)](i) oscillations in pancreatic beta-cells was examined in insulin-producing BRIN-BD11 cells overexpressing the Na/Ca exchanger. BRIN-BD11 cells were stably transfected with NCX1.7, one of the exchanger isoforms identified in the beta-cell. Overexpression could be assessed at the mRNA and protein level. Appropriate targeting to the plasma membrane could be assessed by microfluorescence and the increase in Na/Ca exchange activity. In response to K(+), overexpressing cells showed a more rapid increase in [Ca(2+)](i) on membrane depolarization as well as a more rapid decrease of [Ca(2+)](i) on membrane repolarization. In response to glucose and tolbutamide, control BRIN cells showed large amplitude [Ca(2+)](i) oscillations. In contrast, overexpressing cells showed a staircase increase in [Ca(2+)](i) without such large oscillations. Diazoxide-induced membrane hyperpolarization restored large amplitude [Ca(2+)](i) oscillations in overexpressing cells. The present data confirm that Na/Ca exchange plays a significant role in the rat beta-cell [Ca(2+)](i) homeostasis, the exchanger being a versatile system allowing both Ca(2+) entry and outflow. Our data suggest that the current generated by the exchanger shapes stimulus-induced membrane potential and [Ca(2+)](i) oscillations in insulin-secreting cells, with the difference in electrical activity and [Ca(2+)](i) behavior seen in mouse and rat beta-cells resulting in part from a difference in Na/Ca exchange activity between these two cells.     

Dynamics of calcium clearance in mouse pancreatic beta-cells

Pancreatic beta-cells maintain glucose homeostasis by their regulated Ca(2+)-dependent secretion of insulin. Several cellular mechanisms control intracellular Ca(2+) levels, but their relative significance in mouse beta-cells is not fully known. We used photometry to measure the dynamics of cytosolic Ca(2+) ([Ca(2+)](i)) clearance after brief, depolarization-induced Ca(2+) entry. Treatment with thapsigargin or cyclopiazonic acid, inhibitors of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) pumps, nearly doubled the peak and slowed the decay of the depolarization-induced Ca(2+) transients. The remaining thapsigargin-insensitive decay was slowed further by inhibition of the plasma membrane Ca(2+)-ATPase (PMCA) and plasma membrane Na(+)/Ca(2+) exchanger (NCX) via alkalization of the bath solution, by adding lanthanum, or by substitution of Na(+) with Li(+). Mitochondrial Ca(2+) uptake contributed little to clearance in thapsigargin-pretreated cells. Together, the SERCA, PMCA, and NCX transport mechanisms accounted for 89 to 97% of clearance in normal solutions. We developed a quantitative model for the dynamic role of removal mechanisms over a wide range of [Ca(2+)](i). According to our model, 50 to 64% of initial Ca(2+) removal is via the SERCA pump, whereas the NCX contributes 21-30% of the extrusion at high [Ca(2+)](i), and the PMCA contributes 21-27% at low [Ca(2+)](i).     

Na+ dependence of extracellular Ca2+-sensing mechanisms leading to activation of an outwardly rectifying Cl- channel in murine osteoclasts

An elevation in the extracellular Ca(2+) concentration ([Ca(2+)](o)) is a key signal for bone remodeling by inhibiting the resorbing activity of osteoclasts. The [Ca(2+)](o)-sensing responses include a variety of morphological and functional changes, but the underlying mechanisms are yet to be defined. This study was aimed at investigating the [Ca(2+)](o)-sensing mechanisms leading to the activation of the Cl(-) channel in murine osteoclasts. A rise in either Ca(2+) or Gd(3+) activated an outwardly rectifying Cl(-) (OR(cl)) channel reversibly and dose-dependently, which was characterized by rapid activation kinetics, little inactivation, and blockage by DIDS. The concentration required for a half-maximal response was estimated to be >20-30 mmol/L for Ca(2+). Intracellular dialysis with an ATP-free pipette solution or application of an actin destabilizer, cytochalasin D, decreased the [Ca(2+)](o)-activated OR(cl) current. Substitution of extracellular Na(+) by an impermeable cation, N-methyl-D-glucamine(+), inhibited the [Ca(2+)](o)-activated OR(cl) channel, suggesting that the activation depended on extracellular Na(+). A blocker for the Na(+)-Ca(2+) exchanger, 2'4'-dichlorobenzamil hydrochloride (DCB), inhibited the [Ca(2+)](o)-activated OR(cl) channel as well. Although 10 mmol/L Ca(2+) activated the OR(cl) current only slightly at a standard intracellular pH (7.3), decreasing pH by dialyzing cells with an acidic pipette solution (pH 6.6) enhanced the [Ca(2+)](o)-activated OR(cl) current. This potentiation by cell acidosis was eliminated by amiloride, a blocker for the Na(+)-H(+) exchanger. Zinc ion (0.1 mmol/L) and a polycation, neomycin (0.2 mmol/L), activated the OR(cl) current at intracellular pH 6.6, whereas the effects of those cations were negligible at intracellular pH 7.3. These results suggest that [Ca(2+)](o)-sensing mechanisms, leading to activation of the OR(cl) channel in murine osteoclasts, are regulated by ATP and actin cytoskeletal organization, and are sensitized greatly by cell acidosis. Contributions of Na(+)-dependent transporters in this activating process are examined in the context of a possible intermediate signal of cell swelling caused by Na(+) influx.     

Modulation of angiotensin II-mediated signalling by heparan sulphate glycosaminoglycans.

BACKGROUND: Heparin and angiotensin-converting enzyme inhibitors can be used as a therapeutic option in diabetic nephropathy (DN). Although the mode of action is poorly understood, both agents may retard the progression of DN. Previously, we demonstrated that angiotensin II (Ang II) has an inhibitory effect on the production of heparan sulphate proteoglycan (HSPG) in mesangial cells (MCs). We have now studied the influence of heparin on the Ang II-induced intracellular Ca(2+) release and activation of nuclear factor kappa B (NF-kappaB). METHODS: Human MCs were isolated from renal cortex and cultivated to measure Ca(2+) influx and NF-kappaB activation. RESULTS: Stimulation of MCs with 100 nM Ang II resulted in a rapid increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)), followed by a decline to baseline level. The addition of heparin resulted in an oscillatory pattern of Ca(2+) influxes upon Ang II stimulation. Whereas the rapid increase in [Ca(2+)](i) was most likely due to release from intracellular stores, oscillations in [Ca(2+)](i) were dependent on the presence of extracellular Ca(2+). Heparin alone did not induce Ca(2+) influx. Both the initial increase and the subsequent oscillations in [Ca(2+)](i) could be blocked by losartan. In MCs with chemically or enzymatically altered membrane-associated heparan sulphate glycosaminoglycan (HS-GAG), Ang II stimulation resulted in [Ca(2+)](i) oscillations. Interestingly, in these cells, the addition of heparin or GAG completely prevented [Ca(2+)](i) oscillations. Heparin inhibited NF-kappaB activation in Ang II-stimulated MCs that expressed either normal or chemically altered GAG. CONCLUSIONS: These findings suggest that alterations in HS-GAG chemistry or metabolism under pathological conditions, such as DN, may have direct functional consequences for the local effect of Ang II.     

Erythropoietin modulates angiotensin II- or noradrenaline-induced Ca(2+) mobilization in cultured rat vascular smooth-muscle cells.

BACKGROUND: It has been reported that human recombinant erythropoietin (rHuEpo) modulates the sensitivity of the cardiovascular system to angiotensin II (Ang II) or noradrenaline (NA). In the present study, we explored the effect of rHuEpo on the responsiveness of Ang II- or NA-induced cytosolic free calcium ([Ca(2+)]i) mobilization in cultured rat vascular smooth-muscle cells (VSMC). METHODS: [Ca(2+)]i concentrations in VSMC were measured by using the calcium-sensitive fluorescent dye fura-2. RESULTS: The addition of rHuEpo (250 U/ml) alone induced elevation in [Ca(2+)]i, which remained significantly elevated above basal level for at least 60 min in the presence of extracellular Ca(2+). Pre-incubation with specific protein kinase C (PKC) inhibitor calphostin C (1 micromol/l) significantly reduced the peak and the sustained elevations of [Ca(2+)]i. Pre-treatment with rHuEpo for 60 min increased both basal [Ca(2+)]i and the changes in [Ca(2+)]i by Ang II or NA in a dose-dependent manner in the presence of extracellular Ca(2+). The synergistic effects of rHuEpo with Ang II or NA were also retained when VSMC were bathed in the Ca(2+)-free medium after the pre-incubation of rHuEpo. Conversely, they were diminished in the presence of extracellular Ca(2+) combined with intracellular Ca(2+) release inhibitor 8-(NN-diethylamino)octyl-1,3,4,5-trimethoxybenzoate (TMB-8). The synergistic effects of rHuEpo were also diminished by PKC depletion or by PKC inhibitor. CONCLUSIONS: These observations suggest that rHuEpo has synergistic effects on Ang II- or NA-induced [Ca(2+)]i mobilization, particularly on intracellular Ca(2+) release, in VSMC. This may be a potential mechanism contributing to hypertension associated with rHuEpo therapy.     

Na/Ca exchanger overexpression induces endoplasmic reticulum-related apoptosis and caspase-12 activation in insulin-releasing BRIN-BD11 cells

Ca(2+) may trigger programmed cell death (apoptosis) and regulate death-specific enzymes. Therefore, the development of strategies to control Ca(2+) homeostasis may represent a potential approach to prevent or enhance cell apoptosis. To test this hypothesis, the plasma membrane Na/Ca exchanger (NCX1.7 isoform) was stably overexpressed in insulin-secreting tumoral cells. NCX1.7 overexpression increased apoptosis induced by endoplasmic reticulum (ER) Ca(2+)-ATPase inhibitors, but not by agents increasing intracellular calcium concentration ([Ca(2+)](i)), through the opening of plasma membrane Ca(2+)-channels. NCX1.7 overexpression reduced the rise in [Ca(2+)](i) induced by all agents, depleted ER Ca(2+) stores, sensitized the cells to Ca(2+)-independent proapoptotic signaling pathways, and reduced cell proliferation by approximately 40%. ER Ca(2+) stores depletion was accompanied by the activation of the ER-specific caspase (caspase-12), and the activation was enhanced by ER Ca(2+)-ATPase inhibitors. Hence, Na/Ca exchanger overexpression, by depleting ER Ca(2+) stores, triggers the activation of caspase-12 and increases apoptotic cell death. By increasing apoptosis and decreasing cell proliferation, overexpression of Na/Ca exchanger may represent a new potential approach in cancer gene therapy. On the other hand, our results open the way to the development of new strategies to control cellular Ca(2+) homeostasis that could, on the contrary, prevent the process of apoptosis that mediates, in part, beta-cell autoimmune destruction in type 1 diabetes.     

Sepsis and burn complicated by sepsis alter cardiac transporter expression

Sepsis alone and burn complicated by sepsis produce significant cardiac dysfunction. Since calcium handling by the cardiomyocyte is essential for cardiac function, one mechanism for cardiac abnormalities may be calcium dyshomeostasis. We hypothesized that sepsis and burn plus sepsis alter cardiac calcium transporter expression. Sprague-Dawley rats were divided into: (1) control, (2) sepsis (intratracheal S. Pneumoniae, 4x10(6) CFU), and (3) burn (40% TBSA) plus sepsis. Myocyte [Ca(2+)](i) and [Na(+)](i) were quantified with Fura-2 AM and SBFI, respectively. Western blot analysis of rat hearts used antibodies against the sarcoplasmic reticular Ca(2+) ATPase (SERCA), the L-type calcium channel, the Na(+)/Ca(2+) exchanger or the Na(+)/K(+) ATPase. RESULTS: Sepsis in the presence and absence of burn trauma increased [Ca(2+)](i) and [Na(+)](i). SERCA expression was decreased in the sepsis and burn plus sepsis groups while calcium channel expression was transiently increased in these sepsis groups. Na(+)/Ca(2+) exchanger expression exhibited a biphasic pattern of altered expression. Sepsis and burn plus sepsis reduced Na(+)/K(+) ATPase protein levels. These data suggest that altered transporter expression produce cardiomyocyte calcium and sodium loading and may contribute to sepsis-mediated cardiac contractile dysfunction.     

K+ channel currents in rat ventral prostate epithelial cells

BACKGROUND: Electrophysiological function of the normal prostate has not been extensively studied. In particular, ion channel currents and their regulation have not been studied in freshly-isolated prostate cells. METHODS: Rat prostate secretory epithelial (RPSE) cells were isolated by collagenase treatment. Columnar epithelial cells were used for nystatin-perforated, whole-cell voltage clamp, and the intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using fura-2. RESULTS: Step-like depolarizing pulses (900 msec) starting from - 90 mV induced outwardly rectifying K(+) currents without inactivation. ACh (10 microM) or ATP (100 microM) increased the outward current and hyperpolarized the cell membrane potential. Ionomycin (0.1 microM), a Ca(2+) ionophore, induced a similar increase in the outward current. TEA (5 mM), charybdotoxin (50 nM), and iberiotoxin (30 nM) inhibited the effect of ACh (or ATP) on the outward current, whereas apamin (100 nM) had no effect. The [Ca(2+)](i) of RPSE cells was increased by ACh, ATP, and UTP. CONCLUSIONS: RPSE cells have iberiotoxin-sensitive Ca(2+)-activated K(+) channels that may play an important role in the exocrine secretions of the prostate.     

Purinergic receptor signaling at the basolateral membrane of macula densa cells

Purinergic receptors are important in the regulation of renal hemodynamics; therefore, this study sought to determine if such receptors influence macula densa cell function. Isolated glomeruli containing macula densa cells, with and without the cortical thick ascending limb, were loaded with the Ca(2+) sensitive indicators, Fura Red (confocal microscopy) or fura 2 (conventional video image analysis). Studies were performed on an inverted microscope in a chamber with a flow-through perfusion system. Changes in cytosolic calcium concentration ([Ca(2+)](i)) from exposed macula densa plaques were assessed upon addition of adenosine, ATP, UTP, ADP, or 2-methylthio-ATP (2- MeS-ATP) for 2 min added to the bathing solution. There was no change in [Ca(2+)](i) with addition of adenosine (10(-7) to 10(-3) M). UTP and ATP (10(-4) M) caused [Ca(2+)](i) to increase by 268 +/- 40 nM (n = 21) and 295 +/- 53 nM (n = 21), respectively, whereas in response to 2MesATP and ADP, [Ca(2+)](i) increased by only 67 +/- 13 nM (n = 8) and 93 +/- 36 nM (n = 14), respectively. Dose response curve for ATP (10(-7) to 10(-3) M) added in bath showed an EC(50) of 15 microM. No effect on macula densa [Ca(2+)](i) was seen when ATP was added from the lumen. ATP caused similar increases in macula densa [Ca(2+)](i) in the presence or absence of bath Ca(2+) and addition of 5 mM ethyleneglycotetraacetic acid (EGTA). Suramin (an antagonist of P2X and P2Y receptors) completely inhibited ATP-induced [Ca(2+)](i) dynamics. Also, ATP-Ca(2+) responsiveness was prevented by the phospholipase C inhibitor, U-73122, but not by its inactive analog, U-73343. These results suggest that macula densa cells possess P2Y(2) purinergic receptors on basolateral but not apical membranes and that activation of these receptors results in the mobilization of Ca(2+).     

Inhibition of mitochondrial Na+-Ca2+ exchanger increases mitochondrial metabolism and potentiates glucose-stimulated insulin secretion in rat pancreatic islets

The mitochondrial Na(+)-Ca(2+) exchanger (mNCE) mediates efflux of Ca(2+) from mitochondria in exchange for influx of Na(+). We show that inhibition of the mNCE enhances mitochondrial oxidative metabolism and increases glucose-stimulated insulin secretion in rat islets and INS-1 cells. The benzothiazepine CGP37157 inhibited mNCE activity in INS-1 cells (50% inhibition at IC(50) = 1.5 micro mol/l) and increased the glucose-induced rise in mitochondrial Ca(2+) ([Ca(2+)](m)) 2.1 times. Cellular ATP content was increased by 13% in INS-1 cells and by 49% in rat islets by CGP37157 (1 micro mol/l). Krebs cycle flux was also stimulated by CGP37157 when glucose was present. Insulin secretion was increased in a glucose-dependent manner by CGP37157 in both INS-1 cells and islets. In islets, CGP37157 increased insulin secretion dose dependently (half-maximal efficacy at EC(50) = 0.06 micro mol/l) at 8 mmol/l glucose and shifted the glucose dose response curve to the left. In perifused islets, mNCE inhibition had no effect on insulin secretion at 2.8 mmol/l glucose but increased insulin secretion by 46% at 11 mmol/l glucose. The effects of CGP37157 could not be attributed to interactions with the plasma membrane sodium calcium exchanger, L-type calcium channels, ATP-sensitive K(+) channels, or [Ca(2+)](m) uniporter. In hyperglycemic clamp studies of Wistar rats, CGP37157 increased plasma insulin and C-peptide levels only during the hyperglycemic phase of the study. These results illustrate the potential utility of agents that affect mitochondrial metabolism as novel insulin secretagogues.     

Cyclosporine A amplifies Ca2+ signaling pathway in LLC-PK1 cells through the inhibition of plasma membrane Ca2+ pump

Cyclosporine A (CsA), a neutral, highly hydrophobic cyclic peptide with 11 amino acids, is currently the most widely used immunosuppressive drug for preventing graft rejection and autoimmune diseases. Despite its efficacy, the use of CsA is limited by severe side effects, mainly nephrotoxicity and arterial hypertension. Single cell microfluorimetry was used to evaluate the role of CsA on Ca(2+) signaling pathway in intact cells of the porcine proximal tubule-like cell line LLC-PK1; the assay of the in vitro activity of the plasma membrane Ca(2+) pump (PMCA) was carried out through the preparation and isolation of membranes. The addition of CsA to incubation medium at doses ranging from 0.1 to 2 microM did not change the basal level of intracellular calcium ([Ca(2+)](i)), whereas it affected the [Ca(2+)](i) response to thapsigargin (TG), a powerful inhibitor of microsomal Ca(2+) pump. In control studies, 5 microM TG produced a biphasic response: [Ca(2+)](i) peaked with a 60-s lag, and it then declined to a plateau of elevated [Ca(2+)](i), which remains above basal. However, it became evident that CsA strengthened the Ca(2+) response to TG because the addition of 5 microM TG to cells exposed to 400 nM CsA did not affect the peak response to TG, but it markedly affected the subsequent sustained phase ([Ca(2+)](i) = 156 +/- 4.84 versus 130 +/- 3.28 nmol, mean +/- SEM, n = 6, P < 0.001). In membrane preparations, 200 nM CsA brought about, in the presence of 10 microM calmodulin (CaM), a significant decrease of plasma membrane Ca(2+) pump (PMCA) activity (46.96 +/- 0.26 versus 53.48 +/- 1.96 nmol x mg of protein(-1) x min(-1), n = 6, P < 0.02), a value similar to that obtained in the presence of equimolar amounts of cyclosporine H (CsH), a non-immunosuppressive analogue of CsA. These findings suggest that in this cell line CsA affects the Ca(2+) export pathway through the reduction of the PMCA activity with consequent amplification and strengthening of [Ca(2+)](i) response after exposure to agents that trigger intracellular Ca(2+) release. The increased cell sensitivity during Ca(2+) signaling events ensuing from the impairment of this "defense system" may be regarded as one of the basic mechanisms involved in the development of the side effects induced by CsA.     

Differential effects of sera from normotensive and hypertensive pregnant women on Ca(2+) metabolism in normal vasular smooth muscle cells

The clinical features of preeclampsia have been traditionally ascribed to a generalized vascular endothelial cell dysfunction. The present study investigates the effect of sera from preeclamptic women and normal pregnancy on the metabolism of intracellular Ca(2+) concentration ([Ca(2+)](i)) in normal cultured vascular smooth muscle cells (VSMC). Sera were obtained from normotensive pregnant women (NTP) (n = 17), preeclamptic women (PE) (n = 15), pregnant women with chronic (essential) hypertension (pregnant EHT) (n = 8), non-pregnant women with essential hypertension (non-pregnant EHT) (n = 12), and age-matched non-pregnant normotensive women (NNP) (n = 18). Serum (10%) was applied to both primary cultures of rat aortic smooth muscle cells and to the A-10 vascular muscle cell line. Levels of [Ca(2+)](i) were determined fluorometrically. After a 4-h incubation with serum, basal [Ca(2+)](i) was not significantly altered. However, compared with normal pregnant sera, PE sera markedly reduced hormonally induced Ca(2+) transients. Thus, following acute stimulation of rat VSMC (primary cultures) with 10(-8)M angiotensin II, peak [Ca(2+)](i) responses (% increment over baseline) were 443 +/- 22, 184 +/- 18, 259 +/- 12, 274 +/- 23, and 255 +/- 15% in NTP, PE, pregnant EHT, non-pregnant EHT, and NNP, respectively (P <0.01 PE versus NTP, P <0.05 PE versus NNP and pregnant and non-pregnant EHT). These effects of sera on [Ca(2+)](i) were qualitatively reproduced in platelets obtained from healthy volunteers. Also, depolarization-activated Ca(2+) influx in VSMC was affected by the different sera groups in a manner similar to that seen with hormonally induced [Ca(2+)](i) responses. The altered [Ca(2+)](i) changes by PE sera disappeared 5 wk after delivery. The effect of the different sera groups on hormonally triggered Ca(2+) transients in normal VSMC, as well as the normalization of [Ca(2+)](i) responses after delivery, suggest the presence of a circulating serum factor in PE. Inasmuch as [Ca(2+)](i) is the major determinant of VSMC tone, it is possible that consequent to the attenuation of [Ca(2+)](i) responses, this putative circulating factor counterbalances the intense vasoconstriction in PE.     

Effect of the insulin mimetic L-783,281 on intracellular Ca2+ and insulin secretion from pancreatic beta-cells

L-783,281, an antidiabetic fungal metabolite that has previously been shown to activate insulin signaling in CHO cells, was tested for its effect on intracellular Ca(2+) ([Ca(2+)](i)) and insulin secretion in single mouse pancreatic beta-cells. Application of 10 micromol/l L-783,281 for 40 s to isolated beta-cells in the presence of 3 mmol/l glucose increased [Ca(2+)](i) to 178 +/- 10% of basal levels (n = 18) as measured by fluo-4 fluorescence. L-767,827, an inactive structural analog of the insulin mimetic, had no effect on beta-cell [Ca(2+)](i). The L-783,281-evoked [Ca(2+)](i) increase was reduced by 82 +/- 4% (n = 6, P < 0.001) in cells incubated with 1 micromol/l of the SERCA (sarco/endoplasmic reticulum calcium ATPase) pump inhibitor thapsigargin and reduced by 33 +/- 6% (n = 6, P < 0.05) in cells incubated with 20 micromol/l of the L-type Ca(2+)-channel blocker nifedipine. L-783,281-stimulated [Ca(2+)](i) increases were reduced to 31 +/- 3% (n = 9, P < 0.05) and 48 +/- 10% (n = 6, P < 0.05) of control values by the phosphatidylinositol 3-kinase (PI3-K) inhibitors LY294002 (25 micromol/l) and wortmannin (100 nmol/l), respectively. In beta-cells from IRS-1-/- mice, 10 micromol/l L-783,281 had no significant effect on [Ca(2+)](i) (n = 5). L-783,281 also resulted in insulin secretion at single beta-cells. Application of 10 micromol/l L-783,281 for 40 s resulted in 12.2 +/- 2.1 (n = 14) exocytotic events as measured by amperometry, whereas the inactive structural analog had no stimulatory effect on secretion. Virtually no secretion was evoked by L-783,281 in IRS-1-/- beta-cells. LY294002 (25 micromol/l) significantly reduced the effect of the insulin mimetic on beta-cell exocytosis. It is concluded that L-783,281 evokes [Ca(2+)](i) increases and exocytosis in beta-cells via an IRS-1/PI3-K-dependent pathway and that the [Ca(2+)](i) increase involves release of Ca(2+) from intracellular stores.     

Parathyroid hormone control of free cytosolic Ca2+ in the kidney

We have established a perifusion system to monitor free cytosolic calcium concentrations ([Ca2+]i) in mouse kidney slices, which presumably reflects in vivo status more accurately than renal cells in culture, by means of the fluorescent calcium indicators quin-2 and fura-2. An increase in the extracellular calcium concentrations from 0 (no added Ca2+) to 3.0 mM resulted in an increase in [Ca2+]i from 52 to 239 nM. Replacement of 118 mM of extracellular Na+ with choline, or the addition of ouabain, an inhibitor of Na+,K+-ATPase, at 10(-6) M in the perfusate caused an increase in [Ca2+]i from 161 +/- 13 to 873 +/- 78 nM (n = 10) and 161 +/- 13 to 395 +/- 68 nM (n = 4), respectively, suggesting the possible existence of a Na+,Ca2+ exchange mechanism in the kidney slice. We further examined the effects of PTH on [Ca2+]i mobilization in the kidney. Both human PTH-(1-34) and hPTH-(1-84) increased [Ca2+]i within 60 s at physiologic concentrations of 10(-11)-10(-9) M in a dose-dependent manner. On the other hand, an increase in intracellular cAMP in the slice was also detected above 3 X 10(-9) M hPTH-(1-34) [base 2.1 +/- 0.4 pmol/mg, 3.2 +/- 0.6 pmol/mg (p less than 0.05 versus control values) 5 minutes after the application of 3 X 10(-9) M hPTH-(1-34) and 17.3 +/- 4.3 pmol/mg (p less than 0.05 versus control values) 3 X 10(-8) M hPTH-(1-34), mean +/- SEM, n = 7, p less than 0.05 versus control values].(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible involvement of Na(+)-H+ exchange in the early phase of reperfusion in myocardial stunning.

Calcium overload during reperfusion after prolonged ischemia has been associated with the Na(+)-Ca2+ exchange system. It has been proposed that the promotion of Na(+)-Ca2+ exchange at reperfusion may be mediated by Na(+)-H+ exchange. To evaluate whether this hypothesis is applicable for stunned myocardium, we examined the influence of temporary suppression of Na(+)-H+ and/or Na(+)-Ca2+ exchange during early reperfusion in isolated rat hearts. Myocardial stunning was produced by global ischemia for 15 min at 37 degrees C. The initial reperfusate was given during the subsequent 10 min after ischemia, and followed by reperfusion with normal Krebs-Henseleit buffer solution for 40 min. Hemodynamic indices, creatine kinase in coronary effluent, and myocardial water content were measured during reperfusion. The functional recovery of stunned myocardium was improved with higher extracellular Na+ concentration and/or lower Ca2+ concentration of the initial reperfusate. Aortic flow recovery of group II (135 mM Na(+)-0.5 mM Ca2+) was 77.0 +/- 3.4%, which was substantially greater (P < 0.05) than that of other groups: group I (control, 135 mM Na(+)-1.5 mM Ca2+), 68.2 +/- 2.4%; group III (25 mM Na(+)-0.5 mM Ca2+), 48.7 +/- 2.9%; group IV (25 mM Na(+)-1.5 mM Ca2+), 21.6 +/- 1.5%. Administration of amiloride, an inhibitor of Na(+)-H+ exchange, in the initial reperfusate ameliorates cardiac damage and improved aortic flow recovery in a dose-dependent manner (10(-6) M, 70.1 +/- 3.7%; 10(-5) M, 77.3 +/- 1.7%; 10(-4) M, 82.0 +/- 2.1% vs control 68.2 +/- 2.4%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of the epithelial Ca(2+) channel in rabbit kidney and intestine

The epithelial Ca(2+) channel (ECaC), which is exclusively expressed in 1,25-dihydroxyvitamin D(3)-responsive tissues, i.e., kidney, intestine, and placenta, is postulated to constitute the initial step in the process of transcellular Ca(2+) transport. To strengthen this postulated function, the present study compares the segmental and cellular distribution of ECaC and the other Ca(2+) transport proteins known to be involved in transcellular Ca(2+) transport. In rabbit kidney, ECaC mRNA and protein expression were primarily present in the connecting tubule. Immunopositive staining for the ECaC protein was exclusively found at the apical domain of this tubular segment. Importantly, ECaC completely colocalized with calbindin-D(28K), Na(+)-Ca(2+) exchanger (NCX), and plasma membrane Ca(2+) -ATPase (PMCA). A minority of cells along the distal tubule lacked immunopositive staining for ECaC and the other Ca(2+) transporting proteins. These negative cells were identified as intercalated cells. In intestine, ECaC was present in a thin layer along the apical membrane of the duodenal villus tip, whereas the crypt and goblet cells were negative. Again, a complete colocalization was observed between ECaC, calbindin-D(9K), and PMCA. In contrast to the kidney, NCX could not be detected in duodenum. The present finding that ECaC completely colocalizes with the Ca(2+) transport proteins in the connecting tubule and duodenum, together with its apical localization, further substantiates the postulated function of ECaC as the gatekeeper of active Ca(2+) (re)absorption.     

Flow-induced [Ca2+]i increase depends on nucleotide release and subsequent purinergic signaling in the intact nephron

Flow induces cytosolic Ca(2+) increases ([Ca(2+)](i)) in intact renal tubules, but the mechanism is elusive. Mechanical stimulation in general is known to promote release of nucleotides (ATP/UTP) and trigger auto- and paracrine activation of P2 receptors in renal epithelia. It was hypothesized that the flow-induced [Ca(2+)](i) response in the renal tubule involves mechanically stimulated nucleotide release. This study investigated (1) the expression of P2 receptors in mouse medullary thick ascending limb (mTAL) using P2Y(2) receptor knockout (KO) mice, (2) whether flow increases induce [Ca(2+)](i) elevations in mTAL, and (3) whether this flow response is affected in mice that are deplete of the main purinergic receptor. [Ca(2+)](i) was imaged in perfused mTAL with fura-2 or fluo-4. It is shown that luminal and basolateral P2Y(2) receptors are the main purinergic receptor in this segment. Moreover, the data suggest presence of basolateral P2X receptors. Increases of tubular flow were imposed by promptly rising the inflow pressure, which triggered a marked increase of [Ca(2+)](i). This [Ca(2+)](i) response was significantly reduced in P2Y(2) receptor KO tubules (fura-2 ratio increase WT 0.44 +/- 0.09 [n = 28] versus KO 0.16 +/- 0.04 [n = 13]). Furthermore, the flow response was greatly inhibited with luminal and basolateral scavenging of extracellular ATP (apyrase 7.5 U/ml) or blockage of P2 receptors (suramin 300 microM). The flow response could still be elicited in the absence of extracellular Ca(2+). These results strongly suggest that increase of tubular flow elevates [Ca(2+)](i) in intact renal epithelia. This flow response is caused by release of bilateral nucleotides and subsequent activation of P2 receptors.