John Westwick

Characterization of platelet-activating factor-induced elevation of cytosolic free calcium concentration in eosinophils

In order to evaluate the role of calcium in the activation processes in eosinophils induced by platelet‐activating factor (PAF), we investigated the changes in free cytoplasmatic Ca2+ concentration using fura‐2. PAF causes a rapid and transitory rise of the intracellular free calcium ion concentration ([Ca2+]i) in purified guinea pig eosinophils of approx. 1000 nM above a basal level of 120.7 ± 36.5 nM (n = 10). The effect was dose‐related with a maximum rise at 1000 nM PAF and an EC50 of 17.4 nM and specifically inhibited by the PAF antagonist WEB 2086 with an IC50, of 95.5 nM. WEB 2086 did not affect either the leukotriene B4‐ or the fMet‐Leu‐Phe‐induced elevation of [C2+]i. The response to PAF was dependent on external Ca2+ as it was significantly inhibited by EGTA (85.6 ± 5.4%) and Ni2+ (95.8 ± 2.1 %) but not by the dihydropyridine antagonist nimodipine. We conclude that Ca2+ entry via receptor‐operated Ca2+ channels may be involved in PAF‐induced degranulation of eosinophils.

Activation of protein kinase C inhibits sodium fluoride-induced elevation of human platelet cytosolic free calcium and thromboxane B2 generation

Addition of NaF to washed platelets produces a dose-dependent and transient elevation of the intracellular free calcium concentration ([Ca++]i), thromboxane B2 (TxB2) generation and dense granule release, all of which are significantly inhibited when the extracellular calcium concentration ([Ca++]e) is reduced with EGTA. Inhibition of platelet cyclo-oxygenase by acetylsalicylic acid (ASA) does not affect NaF-induced elevation of [Ca++]i and dense granule release in the presence of 1 mM [Ca++]e. Pre-incubation of the platelets with the phorbol ester TPA produces a marked inhibition of NaF-induced elevation of [Ca++]i and TxB2 generation without affecting dense granule release. Thus, NaF may have more than one site of action. Pretreatment of the platelets with the selective protein kinase C inhibitor H7 prevents TPA induced inhibition of NaF mediated rise in [Ca++]i and TxB2 generation. Thus we propose that NaF induced calcium mobilisation is analogous to receptor-operated calcium mobilisation in platelets, as it is readily inhibited by protein kinase C activation or by the reduction of [Ca++]e and is independent of platelet cyclo-oxygenase activity.

ENDOTHELIN INDUCES AN INCREASE IN RENAL VASCULAR-RESISTANCE AND A FALL IN GLOMERULAR-FILTRATION RATE IN THE RABBIT ISOLATED PERFUSED KIDNEY

1 The effects of endothelin infusion on renal vascular resistance (RVR), glomerular filtration rate (GFR) and the interaction with locally generated endothelium‐derived relaxant factor (EDRF) were studied in the rabbit isolated perfused kidney (IPK). For comparison the effects of infusions of angiotensin II (AII) and noradrenaline (NA) were also assessed. 2 Each kidney was perfused at a constant rate of 10 ml min−1 and alterations in RVR determined by measuring changes in perfusion pressure. GFR was determined by the clearance of [51Cr]‐EDTA, using timed urine collections. 3 Endothelin (10−11‐10−9 M) produced a dose‐related increase in RVR. Endothelin was approximately 30 times more potent in molar terms than AII and 500 times more than NA at inducing a 50 mmHg increase in perfusion pressure. 4 Endothelin appeared to be a weak inducer of EDRF release in the IPK as EDRF inhibitors methylene blue (10 μm) or haemoglobin (10 μm) only slightly augmented the increase in RVR at a given concentration of endothelin. In contrast the effect of NA on RVR was significantly increased by methylene blue (10 μm) whereas that induced by AII was not affected. 5 Endothelin infusion produced a significant, dose‐dependent decrease in GFR of the IPK, contrasting with an increase in GFR during AII infusion and a minimal effect of NA on GFR. This supports evidence that AII is predominantly a constrictor of efferent glomerular arterioles and that NA constricts both afferent and efferent glomerular vessels. We suggest that the vasoconstrictive effect of endothelin in the kidney is predominantly preglomerular, which explains its effect on GFR.

Antagonism of the platelet activating factor-induced rise of the intracellular calcium ion concentration of U937 cells

1 U937 cells are a continuous line of human cells of committed monocytic origin which serve as a useful model for studying human monocytic function. The present study investigated the effect of platelet‐activating factor (Paf) on intracellular free calcium ion concentration ([Ca2+]i) in U937 cells using the calcium fluorescent probe fura‐2. 2 The naturally‐occurring stereoisomer (R)‐Paf (0.01–300 nm) and the stable, less hydrolysable racemic Paf analogue PR1501 (10 nm‐3 μm) produced dose‐related and rapid elevations of 100–1200 nm [Ca2+]i above a basal value of 135 ± 9 nm (n = 22). 3 The unnatural stereoisomer (S)‐Paf and the natural stereoisomer lyso‐(R)‐Paf had no effect on basal [Ca2+]i at 30 μm, approximately 100,000 times the concentration found to be the threshold concentration to elicit a response to (R)‐Paf. 4 Leukotriene B4 (LTB4) also induced increases in [Ca2+]i in the concentration range 28.5 nm‐2.85 μm but the responses were smaller and of shorter duration than those induced by Paf. 5 Five compounds, WEB 2086, Ro 19–3704, L‐652,731, BN 52021, and CV 3988, inhibited sub‐optimal Paf (10 nm)‐induced increase in [Ca2+]i with IC50s of 48 ± 2, 118 ± 33, 318 ± 131, 340 ± 205 and 2320 ± 183 nm respectively. All five compounds have previously been reported as specific Paf receptor antagonists, at least with respect to platelets. 6 The above compounds at 10 μm* had no effect upon the increased [Ca2+]i induced by either LTB4 or the calcium ionophore, ionomycin. 7 These results suggest that U937 cells respond to Paf at least with respect to elevated [Ca2+]i as measured by fura‐2 and that these cells may well possess a Paf receptor as suggested by the action of specific antagonists and the stereoselectivity observed with Paf.

Cyclosporin therapy in vivo attenuates the response to vasodilators in the isolated perfused kidney of the rabbit

1 The endothelium releases a number of vasoactive compounds, including the vasodilator prostaglandins, prostacyclin (PGI2) and prostaglandin E2 (PGE2) and endothelium‐derived relaxing factor (EDRF), which play an important role in the regulation of vascular tone in the microcirculation. Nephrotoxicity is the major complication of cyclosporin (CS) therapy and is related to an increase in intrarenal vascular tone. Endothelial cell generation of PGI2 is inhibited by CS although this cannot fully explain the changes in vascular tone observed. We have investigated the possibility that EDRF‐dependent vasodilatation is also affected by CS therapy in vivo. 2 CS nephrotoxicity was induced in rabbits with CS (15 mg kg−1 per day s.c. for 20 days (n = 6)); 6 rabbits were given CS vehicle (Veh) and 9 animals were studied without any treatment. Creatinine clearance fell significantly during treatment in the CS‐treated rabbits (11.78 ± 1.5 ml min−1, mean ± s.e.mean, to 7.79 ± 1.2 after 20 days treatment) but did not change in the vehicle‐treated animals. 3 The responses to the endothelium‐dependent (acetylcholine (ACh)) and endothelium‐independent (nitroprusside (NP) and PGI2) vasodilators were assessed in indomethacin‐treated isolated perfused kidneys (IPKs) from untreated, CS‐ and Veh‐treated animals. Vascular tone was induced with a constant infusion of noradrenaline 150 nM and the perfusion rate adjusted to produce a perfusion pressure of 90 mmHg. Perfusate flow rate (22.3 ± 4.6 vs 20.4 ± 3.1 ml min−1) and glomerular filtration rate (2.04 ± 0.37 vs 1.88 ± 0.16 nl min−1) did not differ between IPKs from CS‐ and Veh‐treated animals. 4 The vasodilator response to ACh was reduced in the kidneys from CS‐treated animals compared with those from untreated and Veh‐treated animals (mean reduction 35.3 ± 2.3% compared with Veh) as were the responses to both NP (42.8 ± 3.6%) and PGI2 (27.7 ± 7.4%). 5 This suggests that CS nephrotoxicity is not mediated via an effect on endothelium‐dependent responses and that it is more likely that CS has a direct effect on vascular smooth muscle.

Inhibition by staurosporine of mitogen-induced calcium mobilisation in human T lymphoblasts

Addition of monoclonal antibodies (UCHT1) directed against the antigen receptor/CD3 complex or the addition of the mitogens concanavalin A or phytohemagglutinin to human T lymphoblasts induced an elevation of the cytosolic free calcium concentration ([Ca2+]i) as monitored by the fluorescence of the intracellular fura‐2. The rise in [Ca2+]i induced by these agents was effectively inhibited by staurosporine, an agent which inhibits protein kinase (PK)C and PKA. Conversely the PKC activator 12‐O‐tetradecanoyl phorbol‐13‐acetate decreased the delay and accelerated the rate of elevation of [Ca2+]i induced by the mitogens and UCHT1. Increasing the concentration of cAMP or cGMP in the T lymphoblasts had no effect on agonist‐induced elevation of [Ca2+]i. Our data indicate that PKC can exert a positive feedback on the level of cytosolic Ca2+ in T lymphocytes, in contrast to what has been observed with other cells.

Interleukin-8 —A Mediator of Inflammatory Lung Disease?

Chronic inflammation is a feature of the airways of patients exhibiting lung diseases such as asthma, sarcoidosis and bronchitis. This has been recognised from autopsy studies of asthmatic airways (1) and has also been shown to be evident in the airways of asthmatics who have mild asthma (2) or are asymptomatic (3). Bronchial airway hyperreactivity (BHR) to a wide range of pharmacological and physical agents is one of the most characteristic features of asthmatic airways. It is known that intensification of BHR follows antigen challenge in atopic asthmatics (4) and it has been widely presumed that inflammatory events during late-onset reactions may determine the changes in airway reactivity (5).

Human mesangial cell-derived interleukin 8 and interleukin 6: modulation by an interleukin 1 receptor antagonist.

The position and properties of the mesangial cell confer a major role for these cells in the regulation of renal function (1,2). Mesangial cells reside in the intercapillary space of the glomerulus and are embedded in an extracellular matrix. The interstitial region is unique in that entry of substances from the capillary lumen to the intercapillary space occurs without crossing a capillary basement membrane, the area is therefore well suited for a sieving function.