Jatinder Ahluwalia

Pharmacology of receptor operated calcium entry in human neutrophils

In neutrophils, increases in intracellular calcium [Ca(2+)](i) provide a crucial link between inflammatory mediators and inflammatory responses. The modulation of [Ca(2+)](i) fluxes in non-excitable cells such as neutrophils has been studied for more than 25 years yet remains to be resolved. In these cells, the Ca(2+) influx can occur through at least two mechanisms, as follows: one dependent on the state of filling of the endoplasmic reticulum Ca(2+) stores, termed store operated calcium entry (SOCE), and the other less studied mechanism in neutrophils which is not dependent on the state of the Ca(2+) stores but is regulated by receptor occupation, termed receptor operated calcium entry (ROCE). Over the past ten years, the molecular components of SOCE have been extensively characterized, but in neutrophils, the molecular components of ROCE have only recently been explored. In this review, we discuss recent research findings that have demonstrated an important role for ROCE in human neutrophils. In addition, an overview of pharmacological approaches used to discriminate between ROCE and SOCE will be discussed. The elucidation of the molecular components of ROCE may well provide important pharmacological targets for the development of novel anti-inflammatory drugs.

Discrimination between receptor- and store-operated Ca2+ influx in human neutrophils

Ca(2+) and Sr(2+) entry pathways activated by pro-inflammatory agonists FMLP, LTB(4) and PAF have been compared to thapsigargin in human neutrophils. 2-APB (10microM) increased Ca(2+) influx and to a greater extent in agonist than in thapsigargin stimulated neutrophils. This action of 2-APB was specific to Ca(2+) because 2-APB did not augment Sr(2+) entry in agonist and thapsigargin stimulated neutrophils. This suggests that Ca(2+) and Sr(2+) entry can be used to discriminate between receptor and non-receptor (store)-operated Ca(2+) influx. Our data show for the first time that Pyr3 whilst partially inhibiting agonist induced Ca(2+) influx almost completely abolished Ca(2+) influx after thapsigargin stimulation.

Actions of calcium influx blockers in human neutrophils support a role for receptor-operated calcium entry

The action of two potent store operated Ca2+ entry (SOCE) inhibitors, ML-9 and GdCl3 on Ca2+ fluxes induced by the pro-inflammatory agonists FMLP, PAF, LTB(4) as well as the receptor-independent stimulus thapsigargin has not been documented in human neutrophils. In this study, ML-9 enhanced both release and subsequent Ca2+ influx in response to agonists whereas it enhanced Ca2+ release by thapsigargin, but inhibited Ca2+ influx. In contrast, 1muM GdCl3 completely inhibited Ca2+ influx in response to thapsigargin, but only partially blocked Ca2+ influx after agonist stimulation. These results strongly suggest a major role for receptor-operated Ca2+ influx in human neutrophils.

Students Turned Off by Turnitin? Perception of Plagiarism and Collusion by Undergraduate Bioscience Students

Abstract Research on undergraduate bioscience students and the incidence of plagiarism is still in its infancy and a key problem arises in gauging the perception of undergraduate students on plagiarism and collusion in biosciences subjects because of the lack of empirical data. The aim of this study was to provide qualitative data on the perceptions of plagiarism and collusion of final year Pharmacology students. Data with respect to student ethnicity, age and gender are discussed. The results from the pilot study suggested that students did not find Turnitin (UK) easy to use neither did they perceive it as a useful learning tool. This study demonstrated that the overall majority of the students would like an alternative way to detect for plagiarism and collusion. Understanding these perspectives of undergraduate bioscience students can assist educators in developing new strategies when encountering plagiarism and collusion in final year undergraduate biosciences.

Characterisation of electron currents generated by the human neutrophil NADPH oxidase.

Electron transport by the human neutrophil NADPH oxidase is an important microbicidal weapon for phagocytes. The electron current (I(e)) generated by the neutrophil NADPH oxidase is poorly characterised due to the lack of appropriate electrophysiological data. In this study, I fully characterise the neutrophil generated I(e) when the NADPH oxidase is activated by NADPH and GTPgammaS. The neutrophil I(e) was markedly voltage-dependent in the entire voltage range in comparison to those electron currents measured after chloride was removed from the external bath solution. The difference in I(e) measured in chloride free conditions was not due to a change in the activation kinetics of voltage-gated proton channels. The I(e) depolarises the neutrophil plasma membrane at a rate of 2.3 Vs(-1) and this depolarisation was opposed when voltage-gated proton channels are activated. 3 mM ZnCl(2) depolarised the membrane potential to +97.8+/-2.5 mV (n=4), and this depolarisation was abolished after NADPH oxidase inhibition.

Relationship between calcium release and NADPH oxidase inhibition in human neutrophils.

The aim of this study was to investigate the possible relationship between NADPH oxidase activity and changes in cytosolic Ca(2+) in response to different agonists. Treatment of neutrophils with leukotriene B4 (LTB(4)) demonstrated characteristic changes to cytoslic Ca(2+) yielding an EC(50) of 4nM. The pA(2) values for the specific LTB(4) receptor (BLT) antagonists, U-75302 and LY-255283 were 6.32 and 6.38, respectively. Similarly, neutrophils treated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and platelet activating factor (PAF) exhibited changes in cytoslic Ca(2+) in a dose dependant manner with pD(2) values of 9.0 and 9.9, respectively. The phorbol ester PMA prevented elevations in cytosolic Ca(2+) in response to LTB(4), FMLP and PAF with IC(50) values of 5.88, 1.44 and 5.71nM, respectively. In addition, potent NADPH oxidase inhibitors apocynin and diphenyleneiodonium (DPI) inhibited FMLP mediated cytosolic Ca(2+) release. These results demonstrate that inhibition of the NADPH oxidase suppresses cytosolic Ca(2+) release in FMLP activated human neutrophils.

Teaching a Dyslexic Student: A Personal View how Critical Incident Analysis can be used as an Effective Pedagogical Tool in Undergraduate Biosciences

Abstract This paper examines the concept of critical incident analysis in respect of improving the pedagogical practices in teaching dyslexic undergraduate biosciences students. Critical incident analysis is a well established pedagogical theory (Tripp, 1993) that allows reflection of a seemingly typical incident; the ‘critical incident’, so that changes and improvements can be implemented in teaching practices. In particular, it offers the opportunity to reflect upon these incidents and adopt different strategies by using the paradigms as described by Tripp and colleagues and uses these to foster improvement in teaching practice. However, the implementation of paradigms adopted by critical incident analysis and its reflective description in analysing an incident is currently lacking in undergraduate Bioscience. In view of the importance of critical incident analysis, here I describe the content and reflective pedagogical approaches adopted by critical incident analysis and incorporate its key principles into improving the methods and perception of teaching a dyslexic undergraduate student.

Swell activated chloride channel function in human neutrophils

Non-excitable cells such as neutrophil granulocytes are the archetypal inflammatory immune cell involved in critical functions of the innate immune system. The electron current generated (I(e)) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential. For continuous function of the NADPH oxidase, I(e) has to be balanced to preserve electroneutrality, if not; sufficient depolarisation would prevent electrons from leaving the cell and neutrophil function would be abrogated. Subsequently, the depolarisation generated by the neutrophil NADPH oxidase I(e) must be counteracted by ion transport. The finding that depolarisation required counter-ions to compensate electron transport was followed by the observation that chloride channels activated by swell can counteract the NADPH oxidase membrane depolarisation. In this mini review, we discuss the research findings that revealed the essential role of swell activated chloride channels in human neutrophil function.

Tamoxifen does not inhibit the swell activated chloride channel in human neutrophils during the respiratory burst.

Effective functioning of neutrophils relies upon electron translocation through the NADPH oxidase (NOX). The electron current generated (I(e)) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential in activated human neutrophils. Swelling activated chloride channels have been demonstrated in part to counteract the depolarisation generated by the NADPH oxidase I(e). In the present study, the effects of inhibitors of swell activated chloride channels on ROS production and on the swelling activated chloride conductance was investigated in activated human neutrophils. Tamoxifen (10 microM), a specific inhibitor for swell activated chloride channels in neutrophils, completely inhibited both the PMA and FMLP stimulated respiratory burst. This inhibition of the neutrophil respiratory burst was not due to the blocking effect of tamoxifen on the swelling activated chloride conductance in these cells. These results demonstrate that a tamoxifen insensitive swell activated chloride channel has important significance during the neutrophil respiratory burst.

Increased oxygen consumption observed in phorbol 12-myristate 13-acetate stimulated human cultured promonocytic U937 cell lines treated with calcitriol and retinoic acid

OBJECTIVE To investigate the effect of phorbol 12-myristate 13-acetate (PMA) and formyl-methionyl-leucyl-phenylalanine (FMLP) on oxygen consumption of differentiated and non-differentiated immune cell lines by retinoic acid and calcitriol treatment which might be useful in subsequent elicitation of immunological action during immunosuppressive states. METHODS PMA and FMLP were used to artificially stimulate reactive oxygen production in cultured promonocytic U937 cell line. Paralleled samples of the cultured cells were separately prepared with calcitriol (1, 25- dihydroxyvitamin D3) and retinoic acid followed by a 72-hour incubation period. The rate of respiratory burst was measured using the Clark oxygen electrode. RESULTS The average increase in cell concentrations per mL observed was significantly higher in retinoic acid-treated cells (9×10(6) cells/mL) when compared with calcitriol-treated samples (4×10(6) cells/mL). There was a marked increase in oxygen consumption of the calcitriol-treated cell lines against the retinoic acid-treated ones. Exposure of differentiated U937 cells to PMA and FMLP increased significantly (P<0.05) in their oxygen consumption when compared with the control. PMA calcitriol-treated cells resulted in 55% oxygen consumption more than the control while FMLP oxygen consumption increased 78% by comparison with the control. CONCLUSIONS The result demonstrated that calcitriol may serve as a physiological promoter of normal differentiation of precursor cells which may exert an immunological action. This effect could elicit a marker potential and increase immune cell activity of the host especially in immunosuppressed diseased states.