Neuropeptide Y induced increase of cytosolic and nuclear Ca²⁺in heart and vascular smooth muscle cells

Abstract

It was reported that neuropeptide Y (NPY) affects cardiac and vascular smooth muscle (VSM) function probably by increasing intracellular Ca2+. In this study, using fura-2 microfluorometry and fluo-3 confocal microscopy techniques for intracellular Ca2+ measurement, we attempted to verify whether the action of NPY receptor's stimulation in heart and VSM cells modulates intracellular Ca2+ and whether this effect is mediated via the Y1 receptor type. Using spontaneously contracting single ventricular heart cells of 10-day-old embryonic chicks and the fluo-3 confocal microscopy Ca2+ measurement technique to localize cytosolic ([Ca]c) and nuclear ([Ca]n) free Ca2+ level and distribution, 10-10 M of human (h) NPY significantly (P < 0.05) increased the frequency of cytosolic and nuclear Ca2+ transients during spontaneous contraction. Increasing the concentration of hNPY (10(-9) M) did not further increase the frequency of Ca2+ transients. The L-type Ca2+ channel blocker, nifedipine (10(-5) M), significantly (P < 0.001) blocked the spontaneous rise of intracellular Ca2+ in the absence and presence of hNPY (10(-10) and 10(-9) M). However, the selective Y1 receptor antagonist, BIBP3226 (10(-6) M), significantly decreased the hNPY-induced (10(-10) and 10(-9) M) increase in the frequency of Ca2+ transients back to near the control level (P < 0.05). In resting nonworking heart and human aortic VSM cells, hNPY induced a dose-dependent sustained increase of basal resting intracellular Ca2+ with an EC50 near 10(-9) M. This sustained increase was cytosolic and nuclear and was completely blocked by the Ca2+ chelator EGTA, and was significantly decreased by the Y1 receptor antagonist BIBP3226 in both heart (P < 0.05) and VSM (P < 0.01) cells. These results strongly suggest that NPY stimulates the resting basal steady-state Ca2+ influx through the sarcolemma and induces sustained increases of cytosolic and nuclear calcium, in good part, via the activation of the sarcolemma membrane Y1 receptor type in both resting heart and VSM cells. In addition, NPY also increased the frequency of Ca2+ transients during spontaneous contraction of heart cells mainly via the activation of the Y1 receptor type, which may explain in part the active cardiovascular action of this peptide.

Role of endothelin-1 receptors in the sarcolemma membrane and the nuclear membrane in the modulation of basal cytosolic and nuclear calcium levels in heart cells

“…A similar difference in the action of ET-1 at the sarcolemma membrane and the nuclear envelope membrane was also reported for the angiotensin II AT " receptor [9], the ET A ET-1 receptor [11] and neuropeptide Y receptors [16]. The effect of cytosolic ET-1 on the sustained basal [Ca# + ] n suggests that this peptide may exert part of its biological function intracellularly via receptors located at the nuclear membrane, which may explain the long-lasting effect of ET-1 [12,16].…”

Effects of Emodin on Ca2+ Signal Transduction of Smooth Muscle Cells in Multiple Organ Dysfunction Syndrome

“…with a laser scanning confocal microscopy as described Jacques [15]. Briefly, freshly dissociated smooth muscle cells were seeded onto glass coverslips and incubated in Fluo-3 working solution (Fluo-3 AM 7.5 mol and Pluronic F-127 0.02% dissolved in standard buffer) at 37°C under an atmosphere of 5% CO 2 .…”

Quantitative impedimetric NPY-receptor activation monitoring and signal pathway profiling in living cells

“…Development of novel NPY-receptor subtype specific therapeutics as well as diagnostics needs reliable methods for NPY-receptor detection and receptor activation monitoring. While up to now, molecular biological expression analysis by RT-PCR or antibodybased assays are limited with respect to subtype specificity, receptor activation can be determined by fluorescence or radioactive labeled ligand and receptor based assays (Lindner et al, 2008;Zwanziger et al, 2008) as well as indirect methods based on the measurement of downstream effectors like cAMP or changes in the intracellular Ca 2 þ level (Herzog et al, 1992;Jacques et al, 2000). For all these methods, there are common disadvantages like end-point determination without monitoring possibilities, the need for marking the proteins of interest or for laborious preparation of the cells and determination of activity by secondary or enhancer reactions/agents like forskolin stimulation.…”