Kinetics of calcium ion concentration accompanying transduction of signals into neutrophils from diabetic patients and its modification by insulin.
The goal of the study was to evaluate the process of Ca(2+)-mediated transduction of signals into neutrophils from patients with type I diabetes and its modification by insulin. The study was performed with the use of isolated peripheral blood neutrophils from 20 diabetic patients and 30 healthy volunteers. Isolated granulocytes were stimulated separately by fMLP or insulin, or by both substances added to the medium in combinations: fMLP + insulin (after 20 min) or insulin + fMLP (after 20 min). fMLP evoked fast intracellular increase of free Ca(2+) concentration in neutrophils compared with the resting state (P<0.001). Similarly, the peak of fluorescence, as measured by Fluo 3 to Fura Red ratio, was significantly higher in neutrophils stimulated by insulin. Insulin did not cause any changes in intracellular Ca(2+) level when it was added to the previously fMLP-stimulated cells. Prestimulation with insulin significantly decreased fMLP-induced intracellular free Ca(2+) concentration, expressed as Fluo3/Fura Red ratio compared with fMLP alone (1.77 +/- 0.6 vs. 2.63 +/- 0.8, P<0.001). No relation between initial intracellular Ca(2+) in the resting state and the response to insulin was found. Nor was the response to fMLP alone related to intracellular Ca(2+) before stimulation. A strong correlation was observed between initial intracellular Ca(2+) after incubation with insulin and the response to fMLP (r=0.90, P<0.0001). In diabetic granulocytes, the intracellular Ca(2+) was significantly lower than in those from healthy donors in unstimulated cells (P<0.001), after fMLP stimulation (P<0.0001), in medium enriched by insulin (P<0.05), and after fMLP stimulation in insulin rich medium (P<0.001). Only in fMLP prestimulated samples, the emission of light did not differ after stimulation with insulin in granulocytes from both diabetic and healthy subjects. In conclusion, patients with type I diabetes have decreased levels of cytosolic Ca(2+) after insulin and fMLP stimulation in polymorphonuclear granulocytes. This abnormality is probably primarily responsible for the impaired neutrophilic function seen in these patients.
Demkow
U
U
Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Warsaw Medical University, Warsaw, Poland. demkow@litewska.edu.pl
Winklewski
P
P
Potapinska
O
O
Popko
K
K
Lipinska
A
A
Wasik
M
M
D000328:Adult; D002118:Calcium / Q000493:pharmacokinetics*; D020013:Calcium Signaling / Q000502:physiology*; D003922:Diabetes Mellitus, Type 1 / Q000378:metabolism* / Q000473:pathology; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007328:Insulin / Q000502:physiology*; D008875:Middle Aged; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism* / Q000473:pathology; D055815:Young Adult
D007328:Insulin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D002118:Calcium