Voltage-dependent calcium influx in human sperm assessed by simultaneous optical detection of intracellular calcium and membrane potential

AbstractThere are several physiological and pharmacological evidences indicating that opening of voltage dependent calcium channels play a crucial role in the induction of the acrosome reaction in mammalian sperm. In mature sperm, physiological inductors of the acrosome reaction such as ZP3, a zona pellucida protein, and the steroid hormone progesterone, induce depolarization and calcium influx, which are required for the acrosome reaction. In this paper, we describe a voltage-dependent calcium influx present in human sperm. We report an experimental procedure that allows measurement of intracellular calcium and membrane potential simultaneously using the fluorescent dyes DiSC3(5) and Fura-2. We found that in human uncapacitated sperm, depolarization induces a nifedipine-insensitive calcium influx that, in most cases, was transient. Calcium influx was observed in the range of −60 to −15 mV (the range tested). At resting membrane potential (around −40 mV), potassium addition depolarized and induced calcium influx, but when the depolarization was preceded by a hyperpolarization (induced with valinomycin), calcium influx was remarkably enhanced, suggesting that at −40 mV, channels are in a putative inactivated state. When sperm was incubated in medium without calcium, calcium restoration caused calcium influx that depended on voltage, and decayed between 1 and 2 min after depolarization. Unlike ram, mouse or bovine sperm, in which an alkalinization is required to induce calcium influx with potassium, the voltage-dependent calcium influx observed in human sperm did not require an increase in internal or external pH. However, we observed that ammonium, which increases intracellular pH, enhanced the voltage-dependent calcium influx about 90%. Furthermore, depolarization by itself caused a small increase in intracellular pH suggesting that pH can be regulated by membrane potential in human sperm

Interleukin-8 primes oxidative burst in neutrophil-like HL-60 through changes in cytosolic calcium

In response to a variety of stimuli, neutrophils release large amount of reactive oxygen species (ROS) generated by NADPH oxidase. This process known as the respiratory burst is dependent on cytosolic free calcium concentration ([Ca(2+)](i)). Proinflammatory cytokines such as interleukin-8 (IL-8) may modulate ROS generation through a priming phenomenon. The aim of this study was to determine the effect of human IL-8 on ROS production in neutrophil-like dimethylsulfoxide-differentiated HL-60 cells (not equalHL-60 cells) and further to examine the role of Ca(2+) mobilization during the priming. IL-8 at 10 nM induced no ROS production but a [Ca(2+)](i) rise (254 +/- 36 nM). IL-8 induced a strongly enhanced (2 fold) ROS release during stimulation with 1 microM of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF). This potentiation of ROS production is dependent of extracellular Ca(2+) (17.0+/-4.5 arbitrary units (A.U.) in the absence of Ca(2+) versus 56.6 +/- 3.9 A.U. in the presence of 1.25 mM of Ca(2+)). Also, IL-8 enhanced fMLF-stimulated increase in [Ca(2+)](i) (375 +/- 35 versus 245 +/- 21 nM, 0.1 microM of fMLF). IL-8 had no effect on not equalHL-60 cells in response to 1 microM of thapsigargin (472 +/- 66 versus 470 +/- 60 nM). In conclusion, Ca(2+) influx is necessary for a full induction of neutrophil priming by IL-8

Physical association of Gi(2)alpha with Interleukin-8 receptors

Interleukin-8 (IL-8), one of the major mediators of the inflammatory response, belongs to a family of chemokines that includes NAP-2 (Graphiceutrophil-Graphicctivating Graphiceptide-2) and Gro-α and whose biological activities are directed to a great extent toward neutrophils. Two distinct receptors have been described with overlapping, but not identical, binding affinities for IL-8, NAP-2, and Gro-α. This study was designed to examine the intracellular pathways activated upon the occupation of each of the IL-8 receptors (IL-8R). The formation of a physical coupling between IL-8 receptors and the α-subunit of heterotrimeric G proteins was tested in neutrophils by examining the presence of the former in anti-Gα immune precipitates. The addition of IL-8 to a suspension of human neutrophils led to a time-dependent detection of IL-8 in anti-Gi2α (raised against amino acids 159-168 (LERIAQSDYI) of Gi2α) and anti-Gtα (raised against the COOH-terminal 10 amino acids (KENLKDCGLF) of Gtα), but not anti-Gq, immunoprecipitates. Similar results were obtained in human 293 cells stably transfected with IL-8RA or IL-8RB. The peptide derived from the COOH-terminal sequence of Gt inhibited the co-immunoprecipitation of IL-8R and Gi observed in response to the anti-Gtα and anti-Gi2α antibodies. On the other hand, the Gi2α peptide only inhibited the immunoprecipitation induced by the anti-Gi2α antibody. Peptides derived from Gi1α or Gi3α had no effect in this assay. The introduction of the anti-Gi2α or anti-Gtα antibodies or their neutralizing peptides, but not the Gi1α or Gi3α peptides, into 293 IL-8RA or 293 IL-8RB cells completely blocked the calcium responses obtained upon stimulation with IL-8. These results demonstrate that the occupation of either type of IL-8 receptor leads to a physical coupling to the α-subunit of Gi2. In addition, the use of the subunit-specific peptides identified two functionally important but distinct regions of Giα, one involved in receptor/Giα interaction (KENLKDCGLF) and the other mediating downstream signal transmission (LERIAQSDYI). Finally, the results of this study also validate the use of the transfected 293 cell line as a model for the study of the signal transduction pathway(s) initiated by IL-8.Bassam B. Damaj, Shaun R. McColl, Wahib Mahana, Michael F. Crouch, and Paul H. Naccach