Altered calcium and sodium metabolism in red blood cells of hypertensive man: assessment by ion-selective electrodes

Free intracellular calcium [Ca2+]i, sodium [Na+]i and potassium [K+]i were assessed in freeze-thawed human red blood cells (RBC) by ion-selective electrodes. After metabolic depletion by 30 mM 2-desoxy-glucose, [Ca2+]i increased faster and to significantly higher values in RBC from 16 patients with mild to moderate essential hypertension (mean diastolic blood pressure 111 +/- 10 mmHg) than in the RBC of 24 normotensives. The rate of [Ca2+]i increase was 7.0 +/- 3.6 versus 3.7 +/- 4.0 mumol/h/l cells (P less than 0.01) for the first 24 h and 8.1 +/- 4.8 versus 6.4 +/- 3.5 mumol/h/l cells for the following 24 h. [Na+]i before and after 24 h incubation was significantly higher in hypertensives, whereas basal [Ca2+]i and [K+]i before and after incubation were the same in both groups. After Ca loading by ionophore A 23187, the maximum rate of [Ca2+]i extrusion was not significantly lower in intact RBC from hypertensives than in those from normotensives (59.5 +/- 7.8 versus 87.9 +/- 18.1 mumol/min/l cells). These results indicate disturbances in RBC Ca metabolism similar to those observed earlier for Na and K. If generalized, the defect could lead to raised [Ca2+]i in smooth muscle and sympathetic nerve tissue, thus causing increased vascular tone and probably catecholamine release with subsequent arterial hypertension.     

Basal and stimulated cytosolic platelet calcium in essential hypertension

It was reported that in essential hypertension, basal platelet free cytosolic (Ca)i measured with the fluorescent dye Quin 2, is elevated, but increases normally after thrombin stimulation. These data, were interpreted to suggest that plasma membrane fluxes are altered but the release of internal Ca2+ stores is intact. Previous studies have shown that Quin 2 inhibits Ca2+ release from internal stores following thrombin (T)-stimulation. Thus, we reassessed resting and T-stimulated platelet (Ca)i using the fluorescent dyes Fura 2 or Quin 2 in 11 subjects, 5 controls and 6 hypertensives. Mean basal (Ca)i with Quin 2 in controls was 138 +/- 15 nM vs 114 +/- 11 nM in hypertensives, (NS). By contrast, in the same platelet preparation (Ca)i with Fura 2 was higher in hypertensives than controls, 217 +/- 27 nM vs 120 +/- 4 nM, P less than .05. Blood pressure was correlated to (Ca)i obtained with Fura 2, R = 0.55. Thrombin 0.5 U/ml added to platelets in Ca-free media caused a multiphasic rise in (Ca)i with Fura 2. Although the absolute rise in (Ca)i in controls (592 +/- 104 nm) vs hypertensives (512 +/- 60 nm) did not differ, the % rise was less in hypertensives. Thus, 1) in the same population a higher resting (Ca)i was detected in platelets from essential hypertensives with Fura 2, but not with Quin 2; and 2) Ca2+ release from internal stores is altered in hypertension. Thus, Fura 2 is superior to Quin 2 in evaluating platelet (Ca)i.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytosolic free calcium in platelets: relationships to blood pressure and indices of systemic calcium metabolism

Relationships between cytosolic free calcium ([Ca2+]i) in platelets, indices of systemic calcium metabolism and blood pressure were examined in 86 subjects; 29 patients with untreated and 29 patients with treated essential hypertension, six patients with borderline hypertension and 22 healthy reference subjects. In order to analyse interactions between the variables, multivariate statistical analyses were employed. The patients with untreated hypertension had higher [Ca2+]i values in non-activated platelets (P = 0.04) and lower levels of plasma ionized calcium (P = 0.02) than the reference subjects. In multivariate models analysing platelet [Ca2+]i mean blood pressure (MBP), plasma ionized calcium, serum parathyroid hormone (PTH) and body mass index (BMI), the relationship between platelet [Ca2+]i and blood pressure was attenuated (P = 0.13), whereas the inverse relationships between plasma ionized calcium and MBP (P = 0.01) and between platelet [Ca2+]i and serum PTH (P = 0.06) seen in univariate analyses persisted. According to the multivariate models the [Ca2+]i value explained only 5% of the MBP variability. Thus, the data from this investigation do not support a close relationship between basal platelet [Ca2+]i and blood pressure. The inverse relationship between plasma ionized calcium and blood pressure, independent of platelet [Ca2+]i and serum PTH, suggests a direct interaction between plasma ionized calcium and blood pressure regulation.     

Abnormal platelet and lymphocyte calcium handling in prehypertensive rats

We have reported that the basal and stimulated cytosolic free calcium concentrations [( Ca2+]i) are elevated in platelets isolated from 12-14-week-old spontaneously hypertensive rats (SHR) as compared with normotensive Wistar-Kyoto (WKY) rats. To determine whether altered cell calcium metabolism precedes the development of overt hypertension, we measured [Ca2+]i under resting and stimulated conditions in blood platelets and thymic lymphocytes isolated from 4-week-old prehypertensive SHR and WKY rats. Blood pressure was similar in both groups (SHR 95 +/- 8 versus WKY rats 92 +/- 7 mm Hg). Basal [Ca2+]i in platelets was higher in SHR than WKY rats (63.4 +/- 3.9 versus 54.8 +/- 3.1 nM, p less than 0.003). Also the [Ca2+]i response to thrombin was greater in SHR than WKY rats in both the presence and absence of extracellular calcium. For lymphocytes, although no difference was detected in basal [Ca2+]i, the concanavalin A-induced peak [Ca2+]i was higher for SHR than WKY rats in both calcium-containing and calcium-free media. These results suggest that agonist-stimulated calcium influx and calcium discharge from intracellular stores are enhanced in both platelets and lymphocytes of 4-week-old SHR. We conclude that abnormalities in calcium metabolism in two different cell types precede the development of overt hypertension in the SHR.     

Increased basal and thrombin-induced free calcium in platelets of essential hypertensive patients

Intracellular free calcium, [Ca2+]i, was studied in platelets of essential hypertensive subjects and normotensive controls under basal conditions and after stimulation with epinephrine, norepinephrine, angiotensin II, ouabain, and thrombin, using the fluorescent calcium indicator quin 2. Basal [Ca2+]i was significantly higher in hypertensive subjects (n = 32) than in normotensive controls (n = 30; 167.4 +/- 5.0 vs 143.2 +/- 3.1 nmol/L; p less than 0.001). Epinephrine, norepinephrine, angiotensin II, and ouabain had no effect on platelet calcium, whereas thrombin induced a dose-dependent increase in [Ca2+]i in both the presence and absence of extracellular calcium. This [Ca2+]i increase in the presence of extracellular calcium, which depends mainly on calcium influx, was significantly higher (p less than 0.05) in platelets of hypertensive subjects at all thrombin concentrations (ranging from 0.025-0.1 U/ml), while the [Ca2+]i increase in the absence of extracellular calcium, which depends only on release from intracellular stores, was similar in hypertensive subjects and controls. These results suggest that, in essential hypertension, there is not only increased platelet resting [Ca2+]i but also an increase in agonist-mediated calcium influx, which appears to indicate a cell membrane abnormality in the platelets of subjects with essential hypertension.     

Effects of thrombin on intracellular calcium and pH in human and murine platelets

The aim of this study was to compare the effect of thrombin (Thr) on cytosolic calcium [Ca2]+i and intracellular pH [pH]i in human and murine platelets. Rich-platelet suspensions from both species were loaded with Fura-2 (2 microM) or BCECF (0.75 microM) by incubation with their respective acetoxymethyl esters to measure cytosolic calcium [Ca2+]i or intracellular pH [pH]i, respectively. Suspensions were challenged with increasing concentrations of Thr, from 0.1 to 10 IU/ml. Basal [Ca2+]i in human platelets was 98 +/- 6 and 99.1 +/- 9 nM in rat platelets (n = 20). Thr increased [Ca2+]i, EC50 was 1.1 +/- 0.04 in human and 0.97 +/- 0.06 IU/ml in rat platelets (n = 7). Extracellular Mg2+ (4 or 8 mM) abolished Thr response on [Ca2+]i. [pH]i in human was 7.09 +/- 0.08 and 7.11 +/- 0.04 in rat platelets. Thr induced alkalinization of platelets in both species. Our results indicate that the potency of Thr to change [Ca2+]i and [pH]i was similar in both species, allowing for comparisons between human and murine platelets and to extrapolate results from an animal model to human pathology.     

Platelet cytosolic proton and free calcium concentrations in essential hypertension

Alterations in the metabolism of intracellular messengers, such as calcium and cyclic adenosine 5'-phosphate (cAMP), have been reported in essential hypertension. Since intracellular pH (pHi) participates in the control of fundamental cell functions, we looked for changes in platelet cytosolic H+ concentration [( H+]i) in hypertension and investigated whether or not its impaired metabolism is linked to the calcium handling abnormalities. The fluorescent pH indicator BCECF has been used to evaluate intracellular H+ concentration in platelets, unstimulated ex vivo, from normotensive (n = 20) and hypertensive patients (n = 20). Cytosolic [H+] was 20% lower in hypertensive than in normotensive subjects (49.5 +/- 3.4 and 61.8 +/- 2.2 nmol/l cells, respectively, P less than 0.005; mean pHi values were 7.21 and 7.33, respectively). Platelet cytosolic H+ and free Ca2+ concentrations ([Ca2+]i) were determined in parallel in 15 normotensive and 15 hypertensive patients. [Ca2+]i was found to be 19% higher (P less than 0.01), and [H+]i 22% lower (P less than 0.02), in the hypertensive patients compared with the normotensive subjects. Platelet pHi and [Ca2+]i were increased simultaneously in some hypertensive patients. These results are compatible with the hypothesis of an in vivo activation of platelets in hypertension. If a similar alkalinization exists in smooth muscle cells, it may participate in cell proliferation and in an enhanced sensitivity to agonists, two parameters thought to be involved in blood pressure elevation.     

Protein phosphorylation and intracellular free calcium in platelets of patients with essential hypertension.

Platelet intracellular free calcium concentration [Ca2+]i from patients with essential hypertension has been found to be elevated, but the intracellular effects of this increase are still unclear. As protein phosphorylation is an important regulatory step in cell activation and increased protein phosphorylation has been demonstrated in platelets from hypertensive animals, we investigated protein phosphorylation and [Ca2+]i in platelets from patients with essential hypertension and age-matched normotensives. We measured the 32P incorporation into a 20 kDa protein and a 47 kDa protein in 17 hypertensive patients and 20 normotensive, age-matched subjects. The [Ca2+]i was measured with the fluorescent dye fura-2. Protein phosphorylation and [Ca2+]i were assessed in unstimulated platelets and after exposure of the cells to 0.1 and 0.25 U/mL thrombin at 20, 60, and 300 sec. In addition we assessed the activity of protein kinase C by incubating the platelets with phorbol-ester TPA at 20, 60, and 300 sec. Basal phosphorylation of the two proteins was not different between the two groups. After exposure of the platelets to thrombin 32P, incorporation into the 20 kDa protein and the 47 kDa protein was significantly increased in platelets from hypertensive patients at all times. Furthermore, the specific stimulation of protein kinase C with TPA resulted in a significantly higher phosphorylation of the 47 kDa protein, whereas the 20 kDa protein was not phosphorylated after incubation with TPA for 1 min. Basal [Ca2+]i was higher in platelets from hypertensive patients (124 +/- 7 nmol/L v 104 +/- 5 nmol/L, P less than .05), although there was a wide overlap between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+i:K+i ratio and total intracellular calcium in essential and renal hypertension

The calcium ion (Ca2+) and potassium ion (K+) content in the ashed material from red blood cells was determined by flame photometry in 61 essential hypertensives, 11 renal hypertensives and in 47 normotensive controls, and intracellular K+ concentration was measured in the haemolysate. The ratio between Ca2+ and K+ content in ashed red blood cells (Ca2+i:K+i) was 2.07 +/- 0.91 X 10(-3) in normotensives, 4.91 +/- 2.17 X 10(-3) in essential hypertensives (P less than 0.01) and 3.48 +/- 2.04 X 10(-3) in renal hypertensives (P less than 0.05). Intracellular K+ concentration was 94.3 +/- 3.1 mmol/l in normotensives, 94.7 +/- 3.8 mmol/l in essential hypertensives and 93.8 +/- 3.9 mmol/l in renal hypertensives. Therefore intracellular total Ca2+ concentration is increased in the red blood cells from essential hypertensives and, to a lesser extent, in the red blood cells from renal hypertensives. The use of Ca2+i:K+i ratios in red blood cells may thus be useful in assessing cellular Ca2+ content in hypertension.     

Ionophore bromo-A23187 reveals cellular calcium stores in single pituitary somatotropes

Free cytosolic calcium concentration, [Ca2+]i, in single rat pituitary cells can be measured with the fluorescent, calcium-sensitive probe fura-2 and digital image analysis. A reverse hemolytic plaque assay (RHPA) identifies somatotropes in the mixed population of pituitary cells. Previous studies showed that growth hormone releasing factor (GRF) stimulates growth hormone (GH) release from pituitary somatotropes by increasing the influx of calcium into the cell. Somatostatin reduced [Ca2+]i and inhibits hormone release presumably by closing calcium channels in the membrane. The calcium-ionophore bromo-A23187 rapidly increased [Ca2+]i from a baseline of 226 +/- 38 nM to a peak of 842 +/- 169 nM (mean +/- SEM) which was reached 30 s after exposure to the drug. This spike was followed by a sustained phase of elevated [Ca2+]i approximately 370 nM. When somatostatin (SRIF) (10 nM) was combined with ionophore treatment, the initial rise was preserved. However, the second phase was abolished and SRIF lowered [Ca2+]i to 57 +/- 7 nM. Depolarizing the cellular membrane with high extracellular potassium (60 mM) increased cytosolic calcium as well (797 +/- 178 nM); however, this was not affected by the addition of SRIF (988 +/- 71 nM). KCl depolarization in calcium-free medium (+1.5 mM EGTA) provoked no rise in cytosolic calcium. In contrast, after ionophore, the initial spike was preserved while the sustained phase of elevated [Ca2+]i was abolished. We conclude from these data that (1) membrane depolarization and ionophore treatment lead to an influx of calcium into the cytosol of normal pituitary somatotropes. (2) SRIF inhibits calcium influx induced by ionophore but not influx after depolarization with high potassium concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular calcium concentration and growth hormone secretion in individual somatotropes: effects of growth hormone-releasing factor and somatostatin

The cytosolic free calcium concentration and cumulative GH release were measured simultaneously in normal pituitary cells. This was made possible by a novel combination of fluorescence microscopy using the calcium indicator fura-2 and a reverse hemolytic plaque assay. GRF (10 nM) rapidly increased the intracellular free calcium concentration ([ Ca2+]i) from a basal level of 234 +/- 17 nM (mean +/- SE) to a peak value of 480 +/- 61 nM 1 min after stimulation. This GRF-induced calcium rise was totally abolished in calcium-free medium or in the presence of calcium channel blockers cobalt chloride (2 mM) and verapamil (100 microM). When somatostatin (SRIF; 1 nM) was added after basal recordings, cytosolic calcium decreased to 96 +/- 23 nM in identified somatotropes. [Ca2+]i returned to baseline upon the removal of SRIF inhibition. This rebound was higher when a sequential treatment of SRIF followed by GRF was applied. Exposing cells to a combination of GRF (10 nM) plus SRIF (1 nM) resulted in a decrease in [Ca2+]i identical to that caused by SRIF treatment alone. Despite the 10-fold excess of GRF, SRIF not only inhibited hormone secretion, but also totally overcame the GRF-induced rise of [Ca2+]i. In summary, stimulation by GRF increases cytosolic calcium in normal somatotropes. This increase is proposed to be due to the influx of calcium through membrane ion channels. In contrast, SRIF decreases [Ca2+]i. This might explain the cAMP-independent effects of this peptide. The effect of SRIF dominates over that of GRF with respect to both changes in [Ca2+]i and hormone release. Changes in the GH secretory rate are, therefore, accompanied by parallel changes in [Ca2+]i, both of which are primarily regulated by SRIF.     

Increased platelet cytosolic free calcium concentration in essential hypertension

Cytosolic free calcium concentration [Ca2+] was studied in platelets of hypertensive patients with the use of the fluorescent indicator Quin 2/AM. Cytosolic free Ca2+ was significantly higher in platelets of hypertensive patients than in those of normotensive subjects (241 +/- 9 versus 192 +/- 7 nmol/l, n = 58 and 57, respectively P less than 0.001). When all 115 subjects were included, there was a significant correlation between cytosolic free Ca2+ and systolic or diastolic blood pressure (r = 0.262, P less than 0.0025 and r = 0.251, P less than 0.0025, respectively). Intracellular Quin 2 concentration was measured to evaluate the formaldehyde production (a product of Quin 2/AM hydrolysis which has been described as reducing the adenosine triphosphate (ATP) production). The Quin 2 concentrations in platelets of the two groups of subjects were observed to be similar (0.41 +/- 0.03 versus 0.38 +/- 0.03 mmol/l, n = 8 and 7 for hypertensives and normotensives, respectively). The effects of prostaglandin E1 (PGE1), an adenylate cyclase stimulator, on cytosolic free Ca2+ were studied. The presence of 10(-7) mol/l PGE1 lowered the Ca2+ in platelets of hypertensive patients only, suppressing the difference between the two groups.     

血小板游离Ca~(2+)、胰岛素抗性与原发性高血压

为探讨原发性高血压胰岛素抗性与细胞钙代谢障碍的关系,我们测定了18例非肥胖性原发性高血压患者静息和凝血酶激发下血小板游离Ca~(2+)、空腹及口服75g葡萄糖负荷后血糖和血清胰岛素水平,并与13例正常血压者对照。原发性高血压患者静息和凝血酶激发下血小板Ca~(2+)均高于正常对照组,葡萄糖负荷后,血糖和血清胰岛素也高于正常对照组,静息血小板Ca~(2+)与血压及胰岛素反应曲线下面积正相关。结果提示,细胞钙代谢障碍和胰岛素抗性可能相互影响,共同参与高血压病的发病机制。     

Effect of amlodipine and cilazapril treatment on platelet Ca2+ handling in spontaneously hypertensive rats.

Abnormal Ca2+ handling and enhanced aggregation response have been reported in platelets from spontaneously hypertensive rats (SHR) and patients with essential hypertension, and thought to be involved in the progression of target organ damage of hypertension. It is important to examine whether antihypertensive therapy can improve the abnormal platelet response in hypertension. We investigated the effect of antihypertensive treatment such as amlodipine and cilazapril on Ca2+ handling and aggregation response in SHR platelets. Four-week-old male SHR were divided into three groups. Each group was treated with amiodipine (A: 10 mg/kg/day), cilazapril (C: 10 mg/kg/day) or vehicle (V) for 8 weeks by gavage. At 12-week-old, platelet [Ca2+]i was measured with fura-2 in each group of SHR and age-matched Wistar-Kyoto rats (WKY) as normal control. Systolic blood pressure in amlodipine and cilazapril treated groups were similar with WKY and significantly lower than vehicle treated group (A: 124 +/- 9, C: 126 +/- 9, WKY: 122 +/- 10 and V: 180 +/- 9 mmHg, respectively). The basal [Ca2+]i in the three groups of SHR were similar and higher than WKY (A: 47 +/- 1.7, C: 47 +/- 1.2, V: 48 +/- 3.9 and WKY: 40 +/- 4.0 nmol/l, respectively). There were no significant differences in thrombin (0.1 U/ml)-stimulated [Ca2+]i rise in the presence or absence of extracellular Ca2+ among the three groups of SHR and these were higher than WKY. Intracellular Ca2+ discharge capacity, assessed by the ionomycinstimulation was similar in the all groups. Thrombin-induced maximum platelet aggregation responses in the three groups of SHR were similar and higher than WKY. The antihypertensive treatment of Ca2+ antagonist or ACE inhibitor gave no change in intraplatelet Ca2+ metabolism in SHR. These results support the hypothesis that an abnormal Ca2+ handling in SHR platelet is genetically determined and not improved by hypotensive therapy.     

Prostaglandin F2 alpha-induced calcium transient in ovine large luteal cells: II. Modulation of the transient and resting cytosolic free calcium alters progesterone secretion

A previous study demonstrated that prostaglandin F2 alpha (PGF2 alpha) stimulates a transient increase in cytosolic free Ca2+ levels [( Ca2+]i) in ovine large luteal cells. In the present study, the magnitude of the PGF2 alpha (0.5 microM)-induced calcium transient in Hanks' medium (87 +/- 2 nM increase above resting levels) was reduced (P less than 0.05) but not completely eliminated in fura-2 loaded large luteal cells incubated in Ca2(+)-free or phosphate- and carbonate-free medium (10 +/- 1 nM, 32 +/- 6 nM, above resting levels; respectively). Preincubation for 2 min with 1 mM LaCl3 (calcium antagonist) eliminated the PGF2 alpha-induced calcium transient. The inhibitory effect of PGF2 alpha on secretion of progesterone was reduced in Ca2(+)-free medium or medium plus LaCl3. Resting [Ca2+]i levels and basal secretion of progesterone were both reduced (P less than 0.05) in large cells incubated in Ca2(+)-free medium (27 +/- 4 nM; 70 +/- 6% control, respectively) or with 5 microM 5,5'-dimethyl bis-(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (40 +/- 2 nM; 49 +/- 1% control; respectively). In addition, secretion of progesterone was inhibited (P less than 0.05) by conditions that increased (P less than 0.05) [Ca2+]i; that is LaCl3 ([Ca2+]i, 120 +/- 17 nM; progesterone, 82 +/- 8% control) and PGF2 alpha ([Ca2+]i, 102 +/- 10 nM; progesterone, 82 +/- 3% control). In small luteal cells, resting [Ca2+]i levels and secretion of progesterone were reduced by incubation in Ca2(+)-free Hanks ([Ca2+]i, 28 +/- 2 nM; progesterone, 71 +/- 6% control), however, neither LaCl3 nor PGF2 alpha increased [Ca2+]i levels or inhibited secretion of progesterone. The findings presented here provide evidence that extracellular as well as intracellular calcium contribute to the PGF2 alpha-induced [Ca2+]i transient in large cells. Furthermore, whereas an adequate level of [Ca2+]i is required to support progesterone production in both small and large cells, optimal progesterone production in large cells depends upon an appropriate window of [Ca2+]i.     

Effects of ischemia and hypercarbic acidosis on myocyte calcium transients, contraction, and pHi in perfused rabbit hearts

The time courses of changes in pHi and cytosolic calcium were compared in isolated perfused rabbit hearts with the use of the calcium-sensitive fluorescent indicator indo-1 and the pH indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Cell-permeant forms of these indicators were loaded into myocytes by arterial infusion or by direct infusion into the extravascular space. Indo-1 fluorescence was recorded from the epicardial surface of the left ventricle at an excitation wavelength of 360 nm and emission wavelengths of 400 and 550 nm. BCECF fluorescence was recorded at an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Calibration procedures were developed for each indicator that allowed [Ca2+]i and pHi to be quantified during ischemia. Global ischemia decreased contractility and caused a rapid increase in both the systolic and end-diastolic levels of the calcium transients. Ninety seconds of ischemia increased peak systolic [Ca2+]i from 609 +/- 29 to 1,341 +/- 159 nM, while end-diastolic [Ca2+]i increased from 315 +/- 25 to 553 +/- 52 nM. The observed increase in diastolic [Ca2+]i, was shown not to arise from indo-1-loaded endothelial cells. The initial increase in [Ca2+]i was followed by a gradual decline and then a secondary rise occurring between 5 and 15 minutes of ischemia. In contrast, ischemia caused a monotonic decrease in pHi from a baseline of 7.03 +/- 0.06 to 6.83 +/- 0.02 after 2 minutes, 6.32 +/- 0.1 after 10 minutes, and 6.11 +/- 0.04 after 15 minutes. Perfusion of hearts with acidified (hypercarbic) saline increased the systolic and diastolic levels of the calcium transients, but only when pHi fell below a threshold value, which was more acidic than values achieved during the first 2 minutes of ischemia (6.83 +/- 0.03). Lesser degrees of acidification caused a decrease in contractility but did not affect the calcium transients. Effects of pHi on the calcium transients were not due to altered calcium sensitivity of indo-1. These results suggest that cytosolic acidification may contribute to the increase in [Ca2+]i during the first 15 minutes of global ischemia, but the [Ca2+]i increase during the first 2 minutes is mediated by other factors.     

Relation of mitochondrial and cytosolic free calcium to cardiac myocyte recovery after exposure to anoxia.

Mitochondrial calcium overload has been suggested as a marker for irreversible injury in the ischemic heart. A new technique is used to measure dynamic changes in mitochondrial free calcium concentration ([Ca2+]m) in electrically stimulated (0.2 Hz) adult rat cardiac myocytes during exposure to anoxia and reoxygenation. Cells were incubated with indo-1 AM, which distributes in both the cytosol and mitochondria. After Mn2+ quenching of the cytosolic signal, cells were exposed to anoxia, and the residual fluorescence was monitored. [Ca2+]m averaged 94 +/- 3 nM (n = 16) at baseline, less than the baseline diastolic cytosolic free calcium concentration ([Ca2+]c, 124 +/- 4 nM, n = 12), which was measured in cells loaded with the pentapotassium salt of indo-1. [Ca2+]m and [Ca2+]c rose steadily only after the onset of ATP-depletion rigor contracture. At reoxygenation 35 minutes later, [Ca2+]c fell rapidly to preanoxic levels and then often showed a transient further rise. In contrast, [Ca2+]m showed only a slight transient fall and a secondary rise at reoxygenation. At reoxygenation, cells immediately either recovered, demonstrating partial relengthening and retaining their rectangular shape and response to stimulation, or they hypercontracted to rounded dysfunctional forms. Recovery occurred only in cells in which [Ca2+]m or [Ca2+]c remained below 250 nM before reoxygenation. Early during reoxygenation, [Ca2+]m remained higher in cells that hypercontracted (305 +/- 36 nM) than in cells that recovered (138 +/- 9 nM, p less than 0.05), whereas [Ca2+]c did not differ between the two groups (156 +/- 10 versus 128 +/- 10 nM, respectively; p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary aldosteronism in normokalemic patients with adrenal incidentalomas

OBJECTIVE: Since primary aldosteronism has been reported in asymptomatic incidental adrenal masses (adrenal incidentalomas, AI), the aim of our study was to detect primary aldosteronism in normokalemic patients with AI and to verify whether a raised plasma aldosterone (ALD)/plasma renin activity (PRA) ratio may be useful for diagnosis. DESIGN: One-hundred and twenty-five normokalemic patients with solid AI (90 hypertensives and 35 normotensives) and 82 essential hypertensives (EH) were studied. Upright ALD and PRA determination was performed in all cases while patients with abnormal ALD/PRA ratios were submitted to confirmatory tests (saline infusion and captopril tests) for diagnosis of primary aldosteronism. METHODS: ALD and PRA were measured by specific radioimmunoassays. RESULTS: PRA values in AI hypertensives (1.05+/-0.13 ng/ml/h) were lower than in AI normotensives (1.14+/-0.14 ng/ml/h, P<0.05) and in EH (1.68+/-0.15 ng/ml/h, P<0.0001). The ALD/PRA ratio in AI hypertensives (46.4+/-5.1) was higher than in AI normotensives (30.7+/-5.8, P<0.03) and in EH (33.2+/-3.5). Four patients with EH and 2 AI normotensive patients had elevated ALD/PRA ratios but normal responses to the suppressive tests, thus excluding diagnosis of primary aldosteronism. Eight patients with AI and hypertension had a high ALD/PRA ratio, and 7 of these were further studied: in 5 patients diagnosis of primary aldosteronism was well-established by dynamic tests, adrenal vein sampling or by surgery. CONCLUSIONS: Primary aldosteronism in normokalemic patients with incidentally discovered adrenal masses was detected in 4 of all cases and in at least 5.5% of those with hypertension. Consequently, these patients, particularly if hypertensive, need to be routinely studied to exclude this hormonal disease. Evaluation of the ALD/PRA ratio seems to be a simple and reliable test for diagnosis.     

Thyrotropin-releasing hormone (TRH) stimulates biphasic elevation of cytoplasmic free calcium in GH3 cells. Further evidence that TRH mobilizes cellular and extracellular Ca2+

TRH stimulation appears to be coupled to PRL secretion, at least in part, by elevation of the concentration of Ca2+ free in the cytoplasm [( Ca2+]i). We employed an intracellularly trapped fluorescent probe of Ca2+, Quin 2, to measure [Ca2+]i in GH3 cells, cloned rat pituitary tumor cells. Basal [Ca2+]i in GH3 cells incubated in medium containing 1.5 mM Ca2+ was 148 +/- 8.6 nM (mean +/- SE). TRH caused a biphasic elevation of [Ca2+]i to 517 +/- 29 nM at less than 10 sec after TRH addition, followed by a decline towards the resting level over 1.5 min (first phase) and then a sustained elevation to 261 +/- 14 nM (second phase). We attempted to determine whether mobilization of cellular calcium or enhanced influx of extracellular Ca2+, or both, were involved in the elevation of [Ca2+]i during each of the two phases. In all experiments, the elevation of [Ca2+]i stimulated by TRH was compared with that induced by depolarization of the plasma membrane with high extracellular K+, which enhances Ca2+ influx. In medium with 1.5 mM Ca2+, K+-depolarization caused an elevation of [Ca2+]i to 780 +/- 12 nM. When the concentration of Ca2+ in the medium was lowered to 0.1 mM and 0.01 mM, basal [Ca2+]i was lowered to 114 +/- 3.4 and 110 +/- 11 nM, respectively. In medium with 0.1 and 0.01 mM Ca2+, peak K+ depolarization-induced elevation of [Ca2+]i was lowered to 30 +/- 3.9% and 7.3 +/- 2.0% of control, respectively. The peak second phase increase caused by TRH was reduced to 33 +/- 2.8% and 16 +/- 5.6% of control, respectively, whereas the peak first phase elevation of [Ca2+]i was lowered only to 79 +/- 5.5% and 52 +/- 10% of control in medium with 0.1 mM and 0.01 mM Ca2+, respectively. When cells were incubated in medium with 1.5 mM Ca2+ containing the Ca2+-channel blocking agents, nifedipine and verapamil, basal [Ca2+]i was not affected. Nifedipine plus verapamil, each at a maximally effective dose, lowered K+ depolarization-induced elevation of [Ca2+]i to 6.5 +/- 1.0% of control, the peak second phase increase caused by TRH to 28 +/- 4.3% of control, but the peak first phase elevation only to 64 +/- 3.7% of control. The decrease in the first phase response to TRH caused by the channel blockers appeared to be secondary to partial depletion of an intracellular, nonmitochondrial calcium pool.(ABSTRACT TRUNCATED AT 400 WORDS)     

Collagen-stimulated human platelet aggregation is mediated by endogenous calcium-activated neutral protease

To clarify the physiological role of calcium-activated neutral protease (CANP) in human platelets, we loaded the platelets with a Ca2+ -sensitive fluorescent dye, fura-2, and measured the degree of aggregation, cytosolic calcium ion concentration [( Ca2+]i), and proteolysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). At physiological concentration of Ca2+ (1 mM) in the incubation medium, [Ca2+]i was below 0.5 microM and platelet aggregation was not shown. Ionomycin (0.15 microM) or collagen (50 micrograms/ml), but not ADP (10 microM), sharply enhanced the [Ca2+]i to near 1 microM and caused the aggregation. A calcium entry blocker, verapamil, completely abolished both the [Ca2+]i rise and the aggregation. NCO-700, a membrane permeable inhibitor against cysteine proteases (including CANP), dose-dependently blocked the aggregation but did not change the [Ca2+]i transient. SDS-PAGE revealed that filamin, talin, and 70 kDa protein were specifically degraded when platelets were aggregated by ionomycin or collagen and that the proteolysis was not observed when the aggregation was blocked by verapamil or NCO-700. These data provided evidence that Ca2+ entry exceeding 0.5 microM is essential, but not sufficient per se, and that activation of cysteine protease, most likely CANP, is involved in the platelet aggregation by collagen or calcium ionophore.