Platelet Free Calcium Research Articles

Parathyroidectomy lowers blood pressure independently of changes in platelet free calcium

Several lines of evidence indicate that the parathyroid gland plays a role in blood pressure regulation. To examine whether the attenuation of the development of hypertension by parathyroidectomy is associated with modifications in cellular calcium metabolism, intracellular free calcium was determined in fura-2-loaded platelets of spontaneously hypertensive rats after parathyroidectomy. Parathyroidectomy decreased systolic blood pressure (146 +/- 8 versus control 160 +/- 5 mmHg, n = 8 and 9, respectively; P less than 0.05) and serum ionized calcium (0.81 +/- 0.08 versus 1.28 +/- 0.04 mmol/l; P less than 0.01). However, no effect of parathyroidectomy on basal intracellular free calcium was detected (63.1 +/- 5.5 versus 65.0 +/- 5.1 nmol/l). Furthermore, there were no differences in the intracellular free calcium responses to thrombin in the presence and absence of external calcium, intracellular calcium discharge capacity or calcium extrusion from the cytosol. These results indicate that parathyroidectomy lowers blood pressure independently of alterations in platelet calcium handling.

Elevated platelet-free calcium in uraemia

Bleeding complications in uraemia are not uncommon. The pathogenesis of haemorrhage in uraemia is still a matter of controversy and the pattern of bleeding suggests a defect of primary haemostasis. Platelet aggregation and biochemistry, including calcium levels, have been studied; however, the results are controversial. We have examined platelet aggregation, platelet-free calcium and calmodulin in platelet-rich plasma because of the significant role of calcium and calmodulin in regulating platelet and other cells' functions. Platelet aggregation in uraemic subjects was similar to that of controls. Platelet basal free cytosolic calcium and platelet calcium in response to 10 microM Ca++ ionophore A23187 in eight subjects with uraemia were 117 +/- 33 nM and 2025 +/- 398 nM (mean +/- SEM) respectively. By contrast in seven matched healthy controls basal calcium and ionophore-stimulated calcium values were 47 +/- 14 nM and 1354 +/- 414 nM, significantly less than in the patients with uraemia (P less than 0.05). The sensitivity of uraemic platelets to A23187 was similar to that of controls. Calmodulin activity in platelet-rich plasma of 12 subjects with uraemia showed no significant difference from that of controls [1.86 +/- 0.29 micrograms/ml (mean +/- SEM) and 2.0 +/- 0.37 micrograms/ml (mean +/- SEM) respectively]. We conclude that despite elevation of platelet calcium in uraemia, which may be due to a plasma factor such as parathyroid hormone, platelet aggregation is normal and bleeding in uraemia is more likely to be due to other factors, including the effect of reduced haematocrit on platelet endothelial interaction. Disturbances in platelet calcium cannot explain the bleeding manifestations in uraemia but warrant further investigation in order to identify the pathogenic mechanisms responsible.

Blood pressure, left ventricular mass and intracellular calcium in primary hyperparathyroidism

1. Blood pressure, left ventricular mass and platelet cytosolic free calcium concentrations were measured in 23 patients with untreated primary hyperparathyroidism, 30 normotensive control subjects and 23 control subjects matched for age, sex and blood pressure. In 12 patients measurements were repeated after parathyroidectomy. 2. Patients with primary hyperparathyroidism had significantly elevated blood pressures (139 +/- 6/86 +/- 3 mmHg, mean +/- SEM) compared with control subjects (125 +/- 2/78 +/- 1 mmHg), but high values persisted after hypercalcaemia was corrected. 3. Despite chronic extracellular hypercalcaemia, intracellular free calcium levels were lower in patients with hyperparathyroidism than in controls matched for age, sex and blood pressure (median concentrations 81.5 nmol/l vs 93 nmol/l, 95% confidence interval 0.1 to 20.1; P less than 0.05) and values tended to increase after parathyroidectomy. 4. Left ventricular mass index was increased in the primary hyperparathyroid group as compared with control subjects matched for age, sex and blood pressure (123 g/m2 vs 100 g/m2, 95% confidence interval -36.1 to -3.1; P = 0.03). Parathyroidectomy resulted in a small reduction of the left ventricular mass index (123.5 g/m2 vs 104 g/m2, 95% confidence interval 46.5 to 2.5; P = 0.1) but no change in blood pressure. 5. Hypertension and left ventricular hypertrophy in primary hyperparathyroidism are associated with relatively low levels of free calcium in platelets.

A Prospective Study Investigating Changes in Basal Intracellular Free Calcium in Platelets of Normotensive and Hypertensive Primigravidae

A Prospective Study Investigating Changes in Basal Intracellular Free Calcium in Platelets of Normotensive and Hypertensive Primigravidae

Platelet free calcium concentrations measured with fura-2 are influenced by the transmembrane sodium gradient

The influence of the transmembrane Na + gradient on the intracellular free calcium concentration, [Ca 2+] i, was studied in Sepharose gel-filtered platelets from healthy human subjects, using the Ca-sensitive fluorescent dye, fura-2. Raising the internal Na + concentration, [Na +] i, by Na + pump inhibition with 0.05 mM ouabain, without changing external Na + did not cause a significant increase in [Ca 2+] i. Substitution of extracellular Na + by iso-osmolar sucrose induced a rapid (half-time about 2 min) and significant rise in [Ca 2+] i; this effect was ampilfied in Na-loaded platelets. Partial restitution of external Na + in these cells with increased [Ca 2+] i promoted a significant and rapid Na + concentration-dependent fall in [Ca 2+] i; little decline in [Ca 2+] i was observed if K + was used instead of Na +. These observations represent in vitro evidence for the existence of a Na Ca exchange mechanism in human platelets that may, in vivo, participate in the control of [Ca 2+] i.

Fura-2 used as a probe to show elevated intracellular free calcium in platelets of Dahl-sensitive rats fed a high salt diet

Elevated intracellular free calcium concentration [Ca 2+] i in vascular smooth muscle cells has been implicated in the pathophysiology of hypertension. Platelet [Ca 2+] i was measured using the fluorescent indicator, Fura-2, in Dahl sensitive (DS) and resistant (DR) rats given high (8% NaCl) and low (0.4% NaCl) salt diets, as well as in the spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. The aim of this study was to show whether [Ca 2+] i is elevated in salt induced hypertension. Platelet [Ca 2+] i and systolic blood pressure (SBP) were higher (p < 0.001) in DS rats given a high than low salt diets. In contrast, no changes in platelet [Ca 2+] i and SBP were observed in DR rats. In SHR, platelet [Ca 2+] i and SBP were higher (p < 0.001) than in the WKY rats. Platelet [Ca 2+] i correlated with SBP in all groups of rats (r=0.929; P < 0.001, n=38). The parallel increase in SBP and [Ca 2+] i in the DS high salt rats and the SHR suggests that an increased [Ca 2+] i is involved in the pathophysiology of hypertension in the two models which differ with respect to the pathogenesis of their hypertension. This increase in [Ca 2+] i therefore seems to reflect an abnormality in [Ca 2+] i handling in hypertension regardless of its cause.

The role of antihypertensive drugs in counteracting adverse influence on large arteries

Studies in humans have shown that chronic elevation of blood pressure induces early alterations in the large arteries, consisting mainly of increased diameter, increased pulse wave velocity, and decreased arterial compliance. Acute reduction of blood pressure, per se, does not necessarily correct these alterations homogeneously. For instance, cadralazine reduced brachial artery diameter and urapidil decreased pulse wave velocity, without accompanying changes in forearm arterial compliance. In contrast, acebutolol, isosorbide dinitrate, nicardipine, and nitrendipine, for the same acute decrease in blood pressure, improved forearm arterial compliance by a concomitant decrease in pulse wave velocity and increase in arterial diameter. To determine whether or not these vascular effects persist with long-term acebutolol therapy, nine hypertensive patients were treated for a period of 3 months. Forearm hemodynamic and cytosolic free calcium concentrations in platelets were simultaneously assessed before and after treatment. Compared with placebo baseline values, chronic acebutolol therapy significantly decreased mean arterial pressure ( p < 0.01), pulse wave velocity ( p < 0.01), and platelet free calcium concentration ( p < 0.05); forearm arterial compliance was increased ( p < 0.01), but brachial artery diameter did not change. Platelet calcium concentration correlated closely with pulse wave velocity even at constant mean arterial pressure. These findings suggest a relaxant effect of acebutolol on the smooth musculature of large arteries, which is independent of changes in blood pressure and arterial diameter, and possibly mediated by changes in cytosolic calcium levels.

Synergistic potentiation by epinephrine of collagen or thrombin-induced calcium mobilization in human platelets

Changes in cytoplasmic free calcium in response to epinephrine in combination with other agonists were studied in human platelets loaded with the fluorescent calcium indicator, quin 2. In the presence of 1mM external calcium, epinephrine augmented the increase in cytoplasmic free calcium in response to collagen or thrombin, and this was inhibited by yohimbine. In the absence of extracellular calcium, thrombin and collagen only caused a small increase in cytoplasmic free calcium, and the response was not enhanced by epinephrine. High concentrations of epinephrine alone caused a small increase in cytoplasmic free calcium in platelets when calcium was present externally. Inhibition of cyclooxygenase by aspirin markedly reduced the calcium response to collagen and almost completely inhibited potentiation by epinephrine. The calcium response to thrombin was inhibited to a lesser extent by aspirin, and aspirin did not prevent potentiation by epinephrine. These findings suggest that augmentation of the cytoplasmic free calcium level may be involved in the potentiation of platelet responses to agonists by epinephrine, and that metabolites of arachidonic acid make an important contribution to this action of epinephrine.

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.

Changes in Sensitivity to Angiotensin II in Platelets

Our study on intracellular effects of angiotensin II in human platelets showed that: (1) angiotensin II increases intracellular free calcium in platelets via a receptor-operated mechanism and this increase is dose-dependent; (2) the effect on platelet intracellular free calcium depends on the extracellular calcium concentration; (3) ACE inhibition leads to an increased sensitivity of intracellular free calcium to angiotensin II but does not alter epinephrine-induced calcium increase; (4) nifedipine reduces the susceptibility of platelet intracellular free calcium for angiotensin II.

Mechanisms of Action of Calcium Antagonists in Hypertension

Cytosolic free calcium concentrations determine the magnitude of tension development of smooth muscle cells and are pivotal for regulation of vascular smooth muscle tone and systemic vascular resistance. Elevated free calcium concentrations have been found in platelets from hypertensive patients, and platelet free calcium concentration, possibly an index of vascular smooth muscle cell free calcium concentration, correlated with blood pressure in normotensive and hypertensive subjects. Increased systemic vascular resistance in essential hypertension depends on increased calcium influx. Calcium antagonists lower cytosolic free calcium concentrations mainly through a reduction of transmembraneous calcium influx and are potent arterial vasodilators. High blood pressure is lowered through a reduction of elevated systemic vascular resistance but, in contrast to other direct acting vasodilators, without clinically relevant sympathetic reflex activation. However, subtle changes of sympathetic nervous system activity may codetermine the acute and chronic blood pressure response. Calcium antagonists do not lead to volume retention, because of improved intrarenal hemodynamics and a diuretic effect. Interference with angiotensin and sympathetically mediated vasoconstrictor mechanisms probably also contributes to their antihypertensive effects. This favorable hemodynamic profile renders calcium antagonists suitable for monotherapy of uncomplicated hypertension where they are particularly effective in older patients and also for the therapy of hypertensive crisis.

Influence of the calcium-sensitive fluorophore, Quin 2, on platelet function

Recent investigations using Quin 2, a fluorophore used to monitor cytosolic free calcium shifts, have shown that strong agonists cause a dramatic dose-dependent increase in platelet fluorescence. However, weak agonists stimulated little or no increase in light emission of Quin 2-loaded platelets, suggesting that calcium flux is not involved in activation by these agents. The present study has sought an alternative explanation for the failure of weak stimuli to cause a rise in cytosolic free calcium in platelets containing Quin 2. Conditions used to prepare, wash, load, gel-filter, and evaluate the fluorophore- filled cells were examined for their compatibility with retention of sensitivity to activation by weak agonists. The technique used to measure shifts in cytosolic calcium with Quin 2 requires multiply washed, unstirred platelets. Under these conditions, platelets do not aggregate or secrete in response to weak agonists. Quin 2, at concentrations greater than 40 mumol/L, inhibits the response of platelets to strong agonists, and completely blocks their reaction to weak agonists. Quin 2 inhibition of platelet function appears related to high buffering capacity for free calcium, although other mechanisms cannot be ruled out. This suggestion is supported by the observation that Quin 2-induced blockade can be overcome by membrane modulation, which is a calcium-dependent process. However, since both agonists are weak, significant elevation in cytosolic calcium concurrent with functional restoration could not be demonstrated under the experimental conditions used for monitoring calcium. Thus, the conditions used to prepare platelets for Quin 2 evaluation and Quin 2 itself appear to be responsible for the failure of weak agonists to cause evidence of a calcium shift in fluorophore-loaded cells.

Influence of the Calcium-Sensitive Fluorophore, Quin 2, on Platelet Function

Recent investigations using Quin 2, a fluorophore used to monitor cytosolic free calcium shifts, have shown that strong agonists cause a dramatic dose-dependent increase in platelet fluorescence. However, weak agonists stimulated little or no increase in light emission of Quin 2-loaded platelets, suggesting that calcium flux is not involved in activation by these agents. The present study has sought an alternative explanation for the failure of weak stimuli to cause a rise in cytosolic free calcium in platelets containing Quin 2. Conditions used to prepare, wash, load, gel-filter, and evaluate the fluorophore-filled cells were examined for their compatibility with retention of sensitivity to activation by weak agonists. The technique used to measure shifts in cytosolic calcium with Quin 2 requires multiply washed, unstirred platelets. Under these conditions, platelets do not aggregate or secrete in response to weak agonists. Quin 2, at concentrations greater than 40 mumol/L, inhibits the response of platelets to strong agonists, and completely blocks their reaction to weak agonists. Quin 2 inhibition of platelet function appears related to high buffering capacity for free calcium, although other mechanisms cannot be ruled out. This suggestion is supported by the observation that Quin 2-induced blockade can be overcome by membrane modulation, which is a calcium-dependent process. However, since both agonists are weak, significant elevation in cytosolic calcium concurrent with functional restoration could not be demonstrated under the experimental conditions used for monitoring calcium. Thus, the conditions used to prepare platelets for Quin 2 evaluation and Quin 2 itself appear to be responsible for the failure of weak agonists to cause evidence of a calcium shift in fluorophore-loaded cells.

Parallel Reduction of Calcium-Influx-Dependent Vasoconstriction and Platelet-Free Calcium Concentration with Calcium Entry and ??-Adrenoceptor Blockade

In essential hypertension, calcium-influx-dependent vasoconstriction is enhanced, and free calcium concentration in platelets is elevated. To investigate calcium-influx-dependent vasoconstriction as related to platelet calcium concentration blood pressure, forearm blood flow (FAF) and free calcium concentration in platelets were measured in five patients with essential hypertension before and after a 6-week treatment with nitrendipine or acebutolol. Starting from comparable basal values, the increase in FAF (ml/min/100 ml tissue) to intraarterially infused nitrendipine (20 micrograms/100 ml tissue) was 40.7 +/- 6.1 before and 30.7 +/- 12.3 after chronic nitrendipine treatment (p less than 0.05) and 41.6 +/- 6.2 before and 34.6 +/- 14.9 after chronic acebutolol treatment. The "nonspecific" vasodilator response to intraarterial sodium nitroprusside or to postischaemic reperfusion were similar before and during treatment. Platelet-free calcium concentration fell on nitrendipine (p less than 0.05) and on acebutolol (p less than 0.01) treatment. Antihypertensive treatment with calcium entry as well as beta-adrenoceptor blockade resulted in a reduction of calcium-influx-dependent vasoconstriction and a fall in free calcium concentration in platelets. Thus, increased calcium platelets may reflect increased calcium in vascular smooth muscle cells and arteriolar tone.

The cytoplasmic concentration of free calcium in platelets is controlled by stimulators of cyclic AMP production (PGD 2, PGE 1, forskolin)

Maximal stimulation of platelets with thrombin results in a rapid increase in cytoplasmic Ca 2+ (from 0.1 μM to 1–3 μM), as measured with the fluorescent intracellular Ca 2+ indicator Quin-2. Prior addition of the adenylate cyclase stimulators PGD 2, PGE 1 or forskolin inhibited the rise in cytoplasmic Ca 2+. When added after the maximal response to thrombin was attained adenylate cyclase stimulators caused a rapid fall of cytoplasmic Ca 2+ back to the original “resting” level. This effect coincides with the reversal of thrombin-induced, Ca 2+-dependent protein phosphorylation, and cytoskeleton assembly. It is suggested that cAMP-dependent reactions maintain low levels of cytoplasmic Ca 2+ by promoting transport and/or binding of Ca 2+.