Aldosterone influences free intracellular calcium in human mononuclear leukocytes in vitro

Mineralocorticoid receptors have been detected in human mononuclear leukocytes (HML) and a physiological effector mechanism was demonstrated subsequently by which aldosterone is able to prevent the loss of intracellular sodium, potassium and cell water during incubation in an aldosterone-free medium. In the present paper, free intracellular calcium, [Ca2+]i, was measured in HML from normal subjects by Quin-2 and Fura-2 fluorescence after incubation for 1 h at 37 degrees C in RPMI-1640 medium. In fresh HML, [Ca2+]i was 54 +/- 15 nM (Fura-2, mean +/- SD, n = 26). After incubation without aldosterone, [Ca2+]i in HML was 118 +/- 27 nM (Quin-2, n = 11) and 50 +/- 13 nM (Fura-2). After incubation with 1.4 (Fura-2) or 2.8 nM (Quin-2) aldosterone, [Ca2+]i was 139 +/- 38 nM (Quin-2, P less than 0.05 compared with value after incubation without aldosterone) and 57 +/- 11 nM (Fura-2, P less than 0.00001). The Kd-value for dose-response curve was 0.4 nM. The effect of aldosterone was antagonized by N-ethyl-isopropylamiloride, but not by canrenoate, canrenone, cycloheximide and actinomycin D. It was absent in a sodium-free buffer. Corticosterone and hydrocortisone were active as agonists. These results show that aldosterone exerts an effect on the [Ca2+]i in HML in vitro which could be involved in hemodynamic responses to mineralocorticoids if also present in cardiovascular tissues.     

Governance of arteriolar oscillation by ryanodine receptors

To investigate the role of ryanodine receptors in glomerular arterioles, experiments were performed using an isolated perfused hydronephrotic kidney model. In the first series of studies, BAYK-8644 (300 nM), a calcium agonist, constricted afferent (19.6 +/- 0.6 to 17.6 +/- 0.5 microm, n = 6, P < 0.01) but not efferent arterioles. Furthermore, BAYK-8644 elicited afferent arteriolar oscillatory movements. Subsequent administration of nifedipine (1 microM) inhibited both afferent arteriolar oscillation and constriction by BAYK-8644 (to 19.4 +/- 0.5 microm). In the second group, although BAYK-8644 constricted afferent arterioles treated with 1 microM of thapsigargin (19.7 +/- 0.6 to 16.8 +/- 0.6 microm, n = 5, P < 0.05), it failed to induce rhythmic contraction. Removal of extracellular calcium with EGTA (2 mM) reversed BAYK-8644-induced afferent arteriolar constriction (to 20.0 +/- 0.5 microm). In the third series of investigations, ryanodine (10 microM) but not 2-aminoethoxyphenyl borate (100 microM) abolished afferent arteriolar vasomotion by BAYK-8644. In the fourth series of experiments, in the presence of caffeine (1 mM), the stronger activation of voltage-dependent calcium channels by higher potassium media resulted in greater afferent arteriolar constriction and faster oscillation. Our results indicate that L-type calcium channels are rich in preglomerular but not postglomerular microvessels. Furthermore, the present findings suggest that either prolonged calcium influx through voltage-dependent calcium channels (BAYK-8644) or sensitized ryanodine receptors (caffeine) is required to trigger periodic calcium release through ryanodine receptors in afferent arterioles.     

Changes in luminal flow rate modulate basal and bradykinin-stimulated cell [Ca2+] in aortic endothelium.

Hemodynamic forces influence many endothelial cell functions. The coupling between hemodynamic forces and cell function could be mediated by mechano-sensitive ion channels present in the plasma membrane of endothelial cells. Because one of these channels is permeable to Ca2+, we tested whether hemodynamic forces influence endothelial cell Ca2+ ([Ca2+]i). Bovine aortic endothelial cells were grown inside cylindrical glass tubes, loaded with fura-2, and perfused at different pressures and flow rates on the stage of a fluorescence microscope. Decreasing flow from 110 to 2 ml.min-1 raised [Ca2+]i from 57 +/- 11 to 186 +/- 29 nM (mean +/- SEM, p less than 0.01) by increasing the entry of extracellular Ca2+ into the cytoplasm. Increasing flow from 2 to 110 ml.min-1 transiently decreased [Ca2+]i from 62 +/- 3 to 33 +/- 5 nM (p less than 0.01) apparently due to reduced Ca2+ entry and concomitant extrusion by the plasma membrane Ca(2+)-ATPase. The rise in [Ca2+]i induced by bradykinin was magnified during a decrease in flow; in control cells, 10(-7) M bradykinin increased [Ca2+]i by 162 +/- 26 nM, whereas [Ca2+]i increased 350 +/- 67 nM (p less than 0.05) in cells previously exposed to 110 ml.min-1. These observations suggest that flow-induced changes in [Ca2+]i might be a signal-transduction mechanism for endothelial functions responsive to hemodynamic forces and may also modulate the magnitude of hormonally mediated increases in [Ca2+]i.     

Vasopressin-induced calcium increases in smooth muscle cells from spontaneously hypertensive rats

Cytosolic free Ca2+ concentrations [( Ca2+]i) were measured in smooth muscle cells (SMC) from spontaneously hypertensive rats (SHR) and age and sex matched Wistar-Kyoto rats (WKY). Resting levels of [Ca2+]i were 114 +/- 6 nM and 116 +/- 5 nM in SMC from WKY and SHR, respectively. Angiotensin II (AII) induced a dose-dependent large increases in [Ca2+]i in SMC. There were no significant differences in resting or AII-stimulated levels of [Ca2+]i when SMC from WKY and SHR were compared. Arg-vasopressin (AVP) caused a similar but smaller [Ca2+]i increase than AII in SMC. AVP caused larger [Ca2+]i increases in SMC from SHR than in SMC from WKY. Although concentrations of AVP higher than those ordinarily detected in plasma were necessary to obtain different responses between SHR and WKY, these differences may be related to the pathogenesis of hypertension.     

Calcium homeostasis in a clonal pituitary cell line of mouse corticotropes.

Calcium homeostasis was studied following a depolarization-induced transient increase in [Ca2+]i in single cells of the clonal pituitary cell line of corticotropes, AtT-20 cells. The KCl-induced increase in [Ca2+]i was blocked in (i) extracellular calcium-deficient solutions, (ii) external cobalt (2.0 mM), (iii) cadmium (200 microM), and (iv) nifedipine (2.0 microM). The mean increase in [Ca2+]i in single cells in the presence of an uncoupler of mitochondrial function [carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, FCCP, 1 microM] was 54 +/- 13 nM (n = 9). The increase in [Ca2+]i produced by FCCP was greater either during or following a KCl-induced [Ca2+]i load. However, FCCP did not significantly alter the clearance of calcium during a KCl-induced rise in [Ca2+]i. Fifty percent of the cells responded to caffeine (10 mM) with an increase in [Ca2+]i (191 +/- 24 nM; n = 21) above resting levels; this effect was blocked by ryanodine (10 microM). Thapsigargin (2 microM) and 2,5 di(-t-butyl)-1,4 hydroquinone (BuBHQ, 10 microM) produced increases in [Ca2+]i (47 +/- 11 nM, n = 6 and 22 +/- 4 nM, n = 8, respectively) that increased cell excitability. These results support a role for mitochondria and sarco-endoplasmic reticulum calcium stores in cytosolic [Ca2+]i regulation; however, none of these organelles are primarily responsible for the return of [Ca2+]i to resting levels following this KCl-induced [Ca2+]i load.     

The effects of insulin-like growth factor binding protein-3 (IGFBP-3) on T47D breast cancer cells enriched for IGFBP-3 binding sites

This study investigated the effects of cholecystokinin-octapeptide (CCK-8) on pancreatic juice flow and its contents, and on cytosolic calcium (Ca2+) and magnesium (Mg2+) levels in streptozotocin (STZ)-induced diabetic rats compared to healthy age-matched controls. Animals were rendered diabetic by a single injection of STZ (60 mg kg(-1), I.P.). Age-matched control rats obtained an equivalent volume of citrate buffer. Seven weeks later, animals were either anaesthetised (1 g kg(-1) urethane; IP) for the measurement of pancreatic juice flow or humanely killed and the pancreas isolated for the measurements of cytosolic Ca2+ and Mg2+ levels. Non-fasting blood glucose levels in control and diabetic rats were 92.40 +/- 2.42 mg dl(-1) (n = 44) and >500 mg dl(-1) (n = 27), respectively. Resting (basal) pancreatic juice flow in control and diabetic anaesthetised rats was 0.56 +/- 0.05 ul min(-1) (n = 10) and 1.28 +/- 0.16 ul min(-1) (n = 8). CCK-8 infusion resulted in a significant (p < 0.05) increase in pancreatic juice flow in control animals compared to a much larger increase in diabetic rats. In contrast, CCK-8 evoked significant (p < 0.05) increases in protein output and amylase secretion in control rats compared to much reduced responses in diabetic animals. Basal [Ca2+]i in control and diabetic fura-2-loaded acinar cells was 109.40 +/- 15.41 nM (n = 15) and 130.62 +/- 17.66 nM (n = 8), respectively. CCK-8 (10(-8)M) induced a peak response of 436.55 +/- 36.54 nM (n = 15) and 409.31 +/- 34.64 nM (n = 8) in control and diabetic cells, respectively. Basal [Mg2+]i in control and diabetic magfura-2-loaded acinar cells was 0.96 +/- 0.06 nM (n = 18) and 0.86 +/- 0.04 nM (n = 10). In the presence of CCK-8 (10(-8)) [Mg2+]i in control and diabetic cells was 0.80 +/- 0.05 nM (n = 18) and 0.60 +/- 0.02 nM (n = 10), respectively. The results indicate that diabetes-induced pancreatic insufficiency may be associated with derangements in cellular Ca2+ and Mg2+ homeostasis.     

Oscillations of intracellular calcium induced by vasopressin in individual fura-2-loaded mesangial cells. Frequency dependence on basal calcium concentration, agonist concentration, and temperature

Intracellular free calcium concentration ([Ca2+]i) was measured in fura-2-loaded single rat mesangial cells by dual wavelength spectrofluorometry. Stimulation with arginine vasopressin (AVP) caused an initial sharp rise of [Ca2+]i followed by repetitive spikes. The frequency of the oscillations was dependent on the concentration of AVP. At 0.1, 1.0, 10.0, and 100.0 nM AVP, the frequencies of oscillations were 0.17 +/- 0.05 (n = 6), 0.32 +/- 0.05 (n = 6), 0.49 +/- 0.05 (n = 6), and 0.48 +/- 0.05 min-1 (n = 5), respectively. Reduction in extracellular [Ca2+] reduced the frequency of AVP-induced oscillations but did not abolish the oscillations. The frequency of calcium oscillations, upon stimulation with 1.0 nM AVP, was directly correlated with the basal [Ca2+]i prior to stimulation. Oscillation frequency increased with increasing temperature. An Arrhenius plot between 24 and 37 degrees C indicated a strong temperature dependency of the oscillations with a Q10 of 3.0. Protein kinase C stimulation by active phorbol esters inhibited AVP-induced calcium oscillations but not the initial [Ca2+] response to AVP. These observations are consistent with a model incorporating a feedback loop linking [Ca2+]i to the mechanism of [Ca2+]i increase. Ca(2+)-induced Ca2+ release may be involved, whereby inositol 1,4,5-trisphosphate (inositol 1,4,5-P3) formation releases Ca2+ from an inositol 1,4,5-P3-sensitive pool, with subsequent Ca2+ uptake and release from an inositol 1,4,5-P3-insensitive pool.     

Thapsigargin increases cytoplasmic free Ca2+ without influencing steroidogenesis in chicken granulosa cells.

The effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) and progesterone production were determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye Fura-2. Thapsigargin stimulated a 4.6 +/- 0.2-fold increase in [Ca2+]i from a resting level of 55 +/- 6 nM up to 233 +/- 23 nM (n = 8) in 100% of the cells tested (n = 86). However, two different response patterns were observed. Dependent on the cell populations, a maximally effective concentration of thapsigargin (100 nM) stimulated either a rapid (within 16 +/- 2 s) transient increase in [Ca2+]i or a slowly (99 +/- 20 s) developing and sustained increase in [Ca2+]i. Both [Ca2+]i responses were concentration (0.001-1 microM)-dependent with an EC50 around 40 nM. The transient [Ca2+]i response occurred in the absence of extracellular Ca2+ and was unaffected by pretreating the cells with the Ca2+ channel blockers methoxyverapamil (50 microM) or lanthanum (1 mM). The plateau phase of the sustained [Ca2+]i response returned to resting level in the absence of extracellular Ca2+, but remained elevated in the presence of methoxyverapamil (50 microM) or lanthanum (1 mM). Despite its ability to cause transient or prolonged increases in [Ca2+]i, thapsigargin (0.001-1 microM) did not affect basal or luteinizing hormone-stimulated progesterone production by chicken granulosa cells.     

Erythropoietin induced transmembrane calcium influx in essential hypertension.

The effects of erythropoietin (EPO) on cytosolic free calcium concentration ([Ca2+]i) in platelets of 20 essential hypertensive patients (HT) and of 25 normotensive subjects (NT) were investigated using the fura2 technique. In resting platelets [Ca2+]i were not significantly higher in HT compared to NT (74.3 +/- 7.8 nM vs 59.8 +/- 7.0 nM, mean +/- SEM). Addition of EPO significantly increased [Ca2+]i in HT compared to NT (13.8 +/- 5.3 nM vs 0.9 +/- 1.9 nM, p less than 0.01). EPO increased the amount of calcium in intracellular stores. This was confirmed independently using thrombin-induced changes of [Ca2+]i in a calcium-free medium and using chlorotetracycline as a marker of stored calcium. After preincubation with EPO thrombin-induced changes of [Ca2+]i were significantly lower in HT compared to NT (306.1 +/- 30.0 nM vs 407.7 +/- 35.7 nM, p less than 0.05). In a calcium-free medium after preincubation with EPO thrombin-induced changes of [Ca2+]i were significantly lower in HT compared to NT (54.7 +/- 11.8 nM vs 100.9 +/- 10.5 nM, p less than 0.05) indicating lower storage capacity in HT. It is concluded that elevated response to EPO may provide a powerful tool to evaluate diagnosis and underlying pathophysiological mechanisms in essential hypertension.     

Interaction between endogenously produced carbon monoxide and nitric oxide in regulation of renal afferent arterioles

Heme oxygenases (HO-1 and HO-2) catalyze the conversion of heme to carbon monoxide (CO), iron, and biliverdin. CO causes vasorelaxation via stimulation of soluble guanylate cyclase (sGC) and/or activation of calcium-activated potassium channels. Because nitric oxide (NO) exerts effects via the same pathways, we tested the interaction between CO and NO on rat afferent arterioles (AAs) using the blood-perfused juxtamedullary nephron preparation. AAs were superfused with either tricarbonyldichlororuthenium (II) dimer, known as CO releasing molecule (CORM-2), 10 micromol/l CO solution, or 15 micromol/l chromium mesoporphyrin (CrMP, HO inhibitor). AAs were also superfused with 1 mmol/l N(omega)-nitro-L-arginine (L-NNA) to inhibit NO synthase (NOS) or 10 micromol/l 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one to inhibit sGC, and then CrMP was superfused during NOS inhibition or sGC inhibition. Treatment with 150 and 300 micromol/l CORM-2 or with CO (10 micromol/l) significantly dilated AAs (22.0 +/- 0.9 and 22.8 +/- 0.9 vs. 18.3 +/- 0.9 microm, n = 5, P < 0.05; and 26.0 +/- 1.4 vs. 18.8 +/- 0.7 microm, n = 5, P < 0.05). In untreated vessels, HO inhibition did not alter AA diameter (17.5 +/- 0.7 vs. 17.2 +/- 0.6 microm, n = 7, P > 0.05); however, during inhibition of NO production, which constricted arterioles to 14.6 +/- 1.2 microm, n = 6, P < 0.05, concurrent HO inhibition led to further vasoconstriction (11.7 +/- 1.6 microm, n = 6, P < 0.05). CORM-2 attenuated the L-NNA-induced vasoconstriction. Inhibition of sGC caused significant constriction (15.7 +/- 0.4 vs. 18.8 +/- 0.4 microm, n = 6, P < 0.05). HO inhibition during sGC inhibition did not cause further change in AAs (15.5 +/- 0.7 microm, n = 6). We conclude that endogenously produced CO does not exert a perceptible influence on AA diameter in the presence of intact NO system; however, when NO production is inhibited, CO serves as an important renoprotective reserve mechanism to prevent excess afferent arteriolar constriction.     

Thienopyridines: effects on cultured endothelial cells.

In cultured endothelial cells harvested from human umbilical vein (HUVEC) or bovine aorta (BAEC) the 30 min incubation with calcium ionophore A 23187 (1 microM) or ticlopidine (100 microM) caused an increase in nitrite generation in HUVEC from basal 227 +/- 37 to 372 +/- 60 or to 325 +/- 33 pmoles per 10(6) cells, respectively, and in BAEC from basal 182 +/- 17 to 378 +/- 18 or to 423 +/- 66 pmoles per 106 cells (n = 6), respectively. Calcium ionophore A 23187 (1 microM) or ticlopidine (100 microM) next to 30 min incubation with BAEC increased release of 6-keto-PGF 1alpha from basal level of 9.4 +/- 1.8 to 96.2 +/- 5.1 or to 99.5 +/- 10.2 pmoles per 10(6) cells, respectively. The pretreatment with aspirin (300 microM) cut down this rise to 4.2 +/- 0.1 pmoles per 10(6) cells (n = 8). Basal cytoplasmic calcium levels, [Ca2+]i, in immortalised HUVEC cell line - ECV304, HUVEC and BAEC were 47.7 +/- 3.3 nM (n = 53), 68.3 +/- 5.0 nM (n = 30) and 53.1 +/- 3.0 nM (n = 15), respectively. In these cultured endothelial cells calcium ionophore A 23187 (0.1 microM) produced net maximum rise in [Ca2+]i by 157 +/-27 nM (n = 16)[ ECV304], by 107 +/- 58 nM (n=4) [HUVEC], and by 231.0 +/- 41.3 nM (n = 8) [BAEC], respectively, while ticlopidine (30 microM) produced net maximum rise in [Ca2+]i by 30.0 +/- 3.2 nM (n=9)[ECV304], 48.8 +/- 15.6 nM (n = 4)[HUVEC] and 28.4 +/- 5.4 nM (n = 8)[BAEC], respectively. Effect of ticlopidine on [Ca2+]i was not only weaker than that of calcium A 23187 but also its maximum appeared after a lag period that was 2 3 times longer than that for A23187. In ECV304 clopidogrel at concentrations of 10, 30 and 100 microM produced maximum increment of [Ca2+]i by 16.5 +/- 3.8 nM (n = 7), 47.0 +/- 6.9 nM (n = 8) and 67.2 +/- 8.3 nM (n = 8), respectively. Incubation of BAEC with A23187 (microM), ticlopidine or clopidogrel (100 microM) for 2 h did not influence viability of cultured endothelial cells. We claim that thienopyridines, independently of their delayed anti-platelet properties ex vivo do release NO and PGI2 from cultured endothelial cells in vitro. The above endothelial action of thienopyridines might be mediated by a rise in [Ca2+]i, however, this possibility has not been proved.     

LY294002, but not wortmannin,increases intracellular calcium and inhibits calcium transients in bovine and human airway smooth muscle cells

To characterize the effect that a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, LY294002, has on cytosolic calcium concentrations ([Ca2+]i), bovine airway smooth muscle cells (BASMC) and cultured human bronchial smooth muscle cells (HBSMC) were loaded with fura 2-AM, imaged as single cells and [Ca2+]i measured ratiometrically. LY294002 (50 microM) increased [Ca2+]i by 294+/-76 nM (P<0.01, n=13) and 230+/-31 nM (P<0.001, n=10) in BASMC and HBSMC, respectively, and increases occurred in the absence of extracellular calcium. In contrast, after pre-treatment with thapsigargin, LY294002 no longer increased [Ca2+]i. This calcium mobilization by LY294002 was associated with a significant functional effect since LY294002 also inhibited calcium transients to carbachol (45+/-23 nM), caffeine (45+/-32 nM), and histamine (20+/-22 nM), with controls of 969+/-190, 946+/-156, and 490+/-28 nM, respectively. Wortmannin, a different PI3-kinase inhibitor, neither increased [Ca2+]i nor inhibited transients. Also, LY294002 increased [Ca2+]i in the presence of wortmannin, U-73122, and xestospongin C. We concluded that LY294002 increased [Ca2+]i, at least in part, by mobilizing intracellular calcium stores and inhibited calcium transients. The effects of LY294002 on [Ca2+]i were not dependent on wortmannin-sensitive PI3-kinases, phospholipase C, or inositol trisphosphate receptors (IP3R). For BASMC and HBSMC, LY294002 has effects on calcium regulation that could be important to recognize when studying PI3-kinases.     

Large changes in intracellular pH and calcium observed during heat shock are not responsible for the induction of heat shock proteins in Drosophila melanogaster.

Heat shock caused significant changes in intracellular pH (pHi) and intracellular free calcium concentration [( Ca2+]i) which occurred rapidly after temperature elevation. pHi fell from a resting level value at 25 degrees C of 7.38 +/- 0.02 (mean +/- standard error of the mean, n = 15) to 6.91 +/- 0.11 (n = 7) at 35 degrees C. The resting level value of [Ca2+]i in single Drosophila melanogaster larval salivary gland cells was 198 +/- 31 nM (n = 4). It increased approximately 10-fold, to 1,870 +/- 770 nM (n = 4), during a heat shock. When salivary glands were incubated in calcium-free, ethylene glycol-bis(beta-aminoethyl ether)-N,N',N'-tetraacetic acid (EGTA)-buffered medium, the resting level value of [Ca2+]i was reduced to 80 +/- 7 nM (n = 3), and heat shock resulted in a fourfold increase in [Ca2+]i to 353 +/- 90 nM (n = 3). The intracellular free-ion concentrations of Na+, K+, Cl-, and Mg2+ were 9.6 +/- 0.8, 101.9 +/- 1.7, 36 +/- 1.5, and 2.4 +/- 0.2 mM, respectively, and remained essentially unchanged during a heat shock. Procedures were devised to mimic or block the effects of heat shock on pHi and [Ca2+]i and to assess their role in the induction of heat shock proteins. We report here that the changes in [Ca2+]i and pHi which occur during heat shock are not sufficient, nor are they required, for a complete induction of the heat shock response.     

Control of cytosolic free calcium in rat and chicken osteoclasts. The role of extracellular calcium and calcitonin

Single cell [Ca2+], studies were performed in chicken and rat osteoclasts loaded with fura-2 and exposed to a variety of treatments. Under resting conditions, basal [Ca2+]i, was 79.2 +/- 47.3 and 84.3 +/- 65.7 nM (averages +/- S.D.; n = 141 and 126) in the osteoclasts of the two species, respectively. Basal [Ca2+]i was stable in all rat and in approximately 80% of chicken osteoclasts. In the remaining 20%, spontaneous, irregular [Ca2+], fluctuations were observed (amplitude range: 50-200 nm over basal values). Increase of [Ca2+]o over the concentration of the Krebs-Ringer incubation medium (2 mM) induced rises of [Ca2+] in almost all cells investigated. [Ca2+] rises were already appreciable with 0.5 mM [Ca2+]o additions and reached high values with 4 mM additions: 390 +/- 113 and 364 +/- 214 nM [Ca2+], in rat and chicken osteoclasts, respectively (n = 122 and 101). Qualitatively, the responses to [Ca2+]o additions consisted of discrete [Ca2+]i transients, biphasic (an initial spike followed by a plateau), or monophasic (either the spike or the plateau). In a few chicken osteoclasts, the [Ca2+]i increase occurring after [Ca2+]o addition consisted of multiple, irregular fluctuations, similar to those observed in 20% of these cells under resting conditions. In individual osteoclasts subsequently exposed to multiple [Ca2+]o increase pulses, the type of the [Ca2+]i transient (mono- or biphasic) was maintained, and the size was dependent on the magnitude of the [Ca2+]o additions. Effects similar to those of [Ca2+]o were induced by the addition of Cd2+ or Ba2+ (but not La3+ or Mg2+) into the medium. The Cd2+ effect was maintained in part even in a Ca2+-free medium. Of various hormones and factors, parathormone, 1,25-dihydroxyvitamin D3, and prostaglandin E2 were inactive. In contrast, calcitonin was active in rat osteoclasts (which express numerous receptors). [Ca2+]i increases were small (19 +/- 17.9 nM; n = 21) when the hormone was administered alone; they were synergistic (severalfold potentiation) when the hormone was administered before or after [Ca2+]o. The [Ca2+]i effects of calcitonin were mimicked by 8Br-cAMP (31 +/- 26 nM; n = 12) when the nucleotide was administered alone; marked synergism when it was administered in combination with [Ca2+]o. This paper demonstrates for the first time that changes of [Ca2+]i are induced in osteoclasts by treatments with [Ca2+]o and calcitonin and can therefore be involved in intracellular mediation of the physiological effects of these two extracellular signals.     

Increased internal calcium mobilization in platelets of patients with chronic renal failure.

Platelet free calcium concentrations ([Ca2+]i) were measured with Fura-2 to elucidate the intracellular calcium kinetics in patients with renal disease. There were no significant differences of the resting [Ca2+]i among the control subjects (C) (n = 12), patients with chronic glomerulonephritis (CGN) (n = 8), and patients with chronic renal failure (CRF) (n = 12). In all groups, platelets [Ca2+]i were significantly increased by agonists (thrombin, adenosine diphosphate) compared with their respective basal level. Thrombin-induced [Ca2+]i rise was significantly higher in CRF (840 +/- 265 nM) than in C (600 +/- 163) and CGN (562 +/- 137). Also adenosine diphosphate elicited similar responses. In the presence of calcium chelator in the incubation buffer, the elevation of [Ca2+]i after thrombin stimulation was statistically higher in CRF (469 +/- 85 nM) than in C (275 +/- 60) and CGN (301 +/- 41). These findings suggest that platelets of CRF were capable of increasing [Ca2+]i in response to agonists, through further mobilization of calcium from the intracellular pool rather than the elevation of transmembrane calcium influx.     

Measurement of sarcoplasmic reticulum Ca2+ content in intact amphibian skeletal muscle fibres with 4-chloro-m-cresol.

Single skeletal muscle fibres were isolated from the toad (Bufo marinus) and isometric force and myoplasmic free calcium concentration ([Ca2+]i) were measured. Brief applications of 4-chloro- m-cresol (4-CmC, 0.2-5 mM) elevated [Ca2+]i reversibly in a dose-dependent manner. The lowest concentration of 4-CmC which reliably gave maximal [Ca2+]i was 2 mM and it was, therefore, used for measurement of sarcoplasmic reticulum (SR) Ca2+ content. Tetanic stimulations (100 Hz) increased [Ca2+]i from a resting level of 105 +/- 47 nM (n = 10) to 1370 +/- 220 nM (n = 6). Application of 2 mM 4-CmC produced a contracture that was 54 +/- 16% (n = 6) of the tetanic force and elevated [Ca2+]i to a peak of 3520 +/- 540 nM (n = 8). Both force and [Ca2+]i levels (resting and tetanic) were restored after 10 min of washout of 4-CmC. In skinned muscle fibres, the myofibrillar Ca(2+)-sensitivity was not changed by 4-CmC, but maximal force was reduced to 74 +/- 10% (n = 4). The magnitude of the peak of the 4-CmC-induced Ca2+ transient was not significantly changed by removal of extracellular Ca2+ nor by inhibiting the SR Ca2+ pump with 2,5-di-tert-butylhydroquinone. Treatment of intact fibres with 30 mM caffeine produced a peak Ca2+ level that was indistinguishable from 2 mM 4-CmC. These results indicate that it is possible to measure the SR Ca2+ content in the same fibre with 4-CmC without loss of normal muscle function.     

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.     

Production of NAADP and its role in Ca2+ mobilization associated with lysosomes in coronary arterial myocytes

The present study was designed to determine the production of nicotinic acid adenine dinucleotide phosphate (NAADP) and its role associated with lysosomes in mediating endothelin-1 (ET-1)-induced vasoconstriction in coronary arteries. HPLC assay showed that NAADP was produced in coronary arterial smooth muscle cells (CASMCs) via endogenous ADP-ribosyl cyclase. Fluorescence microscopic analysis of intracellular Ca2+ concentration ([Ca2+]i) in CASMCs revealed that exogenous 100 nM NAADP increased [Ca2+]i by 711 +/- 47 nM. Lipid bilayer experiments, however, demonstrated that NAADP did not directly activate ryanodine (Rya) receptor Ca2+ release channels on the sarcoplasmic reticulum. In CASMCs pretreated with 100 nM bafilomycin A1 (Baf), an inhibitor of lysosomal Ca2+ release and vacuolar proton pump function, NAADP-induced [Ca2+]i increase was significantly abolished. Moreover, ET-1 significantly increased NAADP formation in CASMCs and resulted in the rise of [Ca2+]i in these cells with a large increase in global Ca2+ level of 1,815 +/- 84 nM. Interestingly, before this large Ca2+ increase, a small Ca2+ spike with an increase in [Ca2+]i of 529 +/- 32 nM was observed. In the presence of Baf (100 nM), this ET-1-induced two-phase [Ca2+]i response was completely abolished, whereas Rya (50 microM) only markedly blocked the ET-1-induced large global Ca2+ increase. Functional studies showed that 100 nM Baf significantly attenuated ET-1-induced maximal constriction from 82.26 +/- 4.42% to 51.80 +/- 4.36%. Our results suggest that a lysosome-mediated Ca2+ regulatory mechanism via NAADP contributes to ET-1-induced Ca2+ mobilization in CASMCs and consequent vasoconstriction of coronary arteries.     

Histamine type I receptor occupancy increases endothelial cytosolic calcium, reduces F-actin, and promotes albumin diffusion across cultured endothelial monolayers

Considerable evidence suggests that Ca2+ modulates endothelial cell metabolic and morphologic responses to mediators of inflammation. We have used the fluorescent Ca2+ indicator, quin2, to monitor endothelial cell cytosolic free Ca2+, [Ca2+]i, in cultured human umbilical vein endothelial cells. Histamine stimulated an increase in [Ca2+]i from a resting level of 111 +/- 4 nM (mean +/- SEM, n = 10) to micromolar levels; maximal and half-maximal responses were elicited by 10(-4) M and 5 X 10(-6) M histamine, respectively. The rise in [Ca2+]i occurred with no detectable latency, attained peak values 15-30 s after addition of stimulus, and decayed to a sustained elevation of [Ca2+]i two- to threefold resting. H1 receptor specificity was demonstrated for the histamine-stimulated changes in [Ca2+]i. Experiments in Ca2+-free medium and in the presence of pyrilamine or the Ca2+ entry blockers Co2+ or Mn2+, indicated that Ca2+ mobilization from intracellular pools accounts for the initial rise, whereas influx of extracellular Ca2+ and continued H1 receptor occupancy are required for sustained elevation of [Ca2+]i. Ionomycin-sensitive intracellular Ca2+ stores were completely depleted by 4 min of exposure to 5 X 10(-6) M histamine. Verapamil or depolarization of endothelial cells in 120 mM K+ did not alter resting or histamine-stimulated [Ca2+]i, suggesting that histamine-elicited changes are not mediated by Ca2+ influx through voltage-gated channels. Endothelial cells grown on polycarbonate filters restricted the diffusion of a trypan blue-albumin complex; histamine (through an H1- selective effect) promoted trypan blue-albumin diffusion with a concentration dependency similar to that for the histamine-elicited rise in [Ca2+]i. Exposure of endothelial cells to histamine (10(-5) M) or ionomycin (10(-7) M) was associated with a decline in endothelial F- actin (relative F-actin content, 0.76 +/- 0.07 vs. 1.00 +/- 0.05; histamine vs. control, P less than 0.05; relative F-actin content, 0.72 +/- 0.06 vs. 1.00 +/- 0.05; ionomycin vs. control, P less than 0.01). The data support a role for cytosolic calcium in the regulation of endothelial shape change and vessel wall permeability in response to histamine.     

Evidence that binding of Indo-1 to cardiac myocyte protein does not markedly change Kd for Ca2+

Quantitative measurement of [Ca2+]i with the fluorescent Ca(2+)-indicators Indo-1 and Fura-2 is complicated by the possibility that the value of the dissociation constant (Kd) may be influenced by binding to intracellular proteins. We investigated this question in cultured chick ventricular myocytes by use of two different Indo-1 calibration methods. First, the Indo-1 fluorescence ratio (R) (400/500 nm) was measured in beating myocytes loaded by exposure to Indo-1/AM. Then, cells were exposed to the Ca2+ ionophore Br A-23187 and fluorescence ratio was measured in the presence of 500 nM Ca2+ (EGTA-Ca2+ buffer). Subsequently cells were permeabilized to Ca2+ by a 1 min exposure to 25 microM digitonin in the presence of 'zero' Ca2+ (10 mM EGTA) and saturating 1 mM Ca2+ to obtain Rmin, Rmax and beta. We then calculated [Ca2+]i from the formula ([Ca2+]i = Kd [( R - Rmin)/(Rmax - R)]beta). With Kd = 250 nM, calculated systolic [Ca2+]i was 750 +/- 44 nM and diastolic 269 +/- 19 nM (means +/- SEM, n = 16). The R value calculated for an assumed [Ca2+]i = 500 nM using the above formula and digitonin derived constants was very similar to the value measured using Br A-23187 (digitonin, 0.67 +/- 0.03: Br A-23187, 0.66 +/- 0.03, ns). As the Br A-23187 method is independent of the value chosen for Kd, we conclude that the Kd of 250 nM for Indo-1 measured in free solutions closely approximates the Kd for intracellular Indo-1 in these cells, and that therefore the Kd of Indo-1 for Ca2+ does not appear to be markedly affected by binding to proteins or other intracellular molecules.