Báo cáo Y học: Irregular spiking in free calcium concentration in single, human platelets Regulation by modulation of the inositol trisphosphate receptors

Irregular Spiking In Free Calcium Concentration In Single, Human Platelets

Tên đề tài: Irregular spiking in free calcium concentration in single, human platelets

Tác giả: Roosje M. A. van Gorp, Marion A. H. Feijge, Wim M. J. Vuist, Martin B. Rook and Johan W. M. Heemskerk

Lĩnh vực: Biochemistry and Human Biology, Medical Physiology

Nội dung tài liệu: Nghiên cứu này khám phá cơ chế điều hòa sự tăng/giảm đột ngột của nồng độ canxi tự do (Ca²⁺) trong các tiểu cầu đơn lẻ, đặc biệt tập trung vào vai trò của các thụ thể inositol trisphosphate (InsP3). Các thí nghiệm sử dụng kỹ thuật ghi hình tỷ lệ huỳnh quang trên các tiểu cầu đã được xử lý bằng aspirin và Fura-2 cho thấy các phản ứng tăng/giảm Ca²⁺ bất thường, với đỉnh và khoảng thời gian giữa các đỉnh thay đổi. Nghiên cứu chỉ ra rằng các hợp chất nhạy cảm với nhóm thiol như thimerosal và U73122 có thể gây ra các phản ứng tương tự ngay cả khi không có sự tạo thành InsP3. Các kết quả cho thấy các thụ thể InsP3 đóng vai trò quan trọng trong tín hiệu Ca²⁺ bất thường này, và tín hiệu này có thể bị điều hòa bởi nhiều yếu tố, bao gồm nồng độ InsP3, tác động tăng cường của Ca²⁺ thông qua cơ chế CICR, sự biến đổi nhóm thiol của thụ thể InsP3, và sự điều hòa giảm bởi cAMP.

Mục lục chi tiết:

• Irregular spiking in free calcium concentration in single, human platelets
• Regulation by modulation of the inositol trisphosphate receptors
• Fluorescence ratio imaging indicates that immobilized, aspirin-treated platelets, loaded with Fura-2, respond to inositol 1,4,5-trisphosphate- (InsP3)-generating agonists such as thrombin by high-frequency, irregular rises in cytosolic [Ca2+]; with spikes that vary in peak level and peak-to-peak interval.
• This differs from the regular [Ca2+]i oscillations observed in other, larger cells.
• We found that the thiol-reactive compounds thimerosal (10 µm) and U73122 (10 µm) evoked similar irregular Ca2+ responses in platelets, but in this case in the absence of InsP3 generation.
• Thrombin-induced spiking was acutely abolished by inhibiting phospholipase C or elevating intracellular cAMP levels, while spiking with sulfhydryl reagents was only partially blocked by cAMP elevation.
• Confocal laser scanning microscopy using fluo-3-loaded platelets indicated that, with all agonists or conditions, the irregular spikes were almost instantaneously raised in various regions within a single platelet.
• When using saponin-permeabilized platelets, we found that InsP3-induced Ca2+ release from stores was stimulated by modest Ca2+ concentrations, pointing to a mechanism of InsP3-dependent Ca2+-induced Ca2+ release (CICR).
• This process was completely inhibitable by heparin.
• The Ca2+ release by InsP3, but not the CICR sensor, was negatively regulated by cAMP elevation.
• Thimerosal treatment did not release Ca2+ from intracellular stores, but markedly potentiated the stimulatory effect of InsP3.
• In contrast, U73122 caused a heparin/cAMP-insensitive Ca2+ leak from stores that differed from those used by InsP3.
• Taken together, these results demonstrate that InsP3 receptor channels play a crucial role in the irregular, spiking Ca2+ signal of intact platelets, even when induced by agents such as thimerosal or U73122 which do not stimulate InsP3 formation.
• The irregular Ca2+ release events appear to be subjected to extensive regulation by: (a) InsP3 level, (b) the potentiating effect of elevated Ca2+ on InsP3 action via CICR, (c) InsP3 channel sensitization by sulfhydryl (thimerosal) modification, (d) InsP3 channel-independent Ca2+ leak with U73122, and (e) down-regulation via cAMP elevation.
• The observation that individual Ca2+ peaks were generated in various parts of a platelet at similar intervals and amplitudes points to effective cooperation of the various stores in the Ca2+-release process.
• Keywords: Ca2+-induced Ca2+ release; cyclic AMP; cytosolic Ca2+; inositol trisphosphate; platelets.
• Most vertebrate cells respond to specific agonists by repetitive spiking or oscillation in cytosolic [Ca2+]; as a consequence of regenerative release of Ca2+ from stores into the cytosol through inositol 1,4,5-trisphosphate (InsP3) or ryanodine receptor channels, located in the membrane of the endoplasmic or sarcoplasmic reticulum, respectively [1].
• For large cells such as oocytes and HeLa cells, evidence has been collected that local clusters of InsP3 receptors in the reticular membrane function as discrete Ca2+ release sites.
• Such local spots, being spaced at intervals of tens of micrometers apart, are taken responsible for so-called elementary Ca2+ release events [2-4].
• At low concentrations, InsP3 may trigger individual release sites, which results in the appearance of local Ca2+ ‘puffs’, i.e. of brief Ca2+ release events of usually low amplitude.
• Higher InsP3 concentrations cause a summation in amplitude or frequency mode of these release events, and lead to recruitment of neighbouring release sites.
• As a consequence, global increases in [Ca2+]; can develop that propagate through the entire cell as Ca2+ oscillations or waves.
• These whole-cell Ca2+ responses are usually regular in shape, such in contrast to the local Ca2+ puffs which are heterogeneous in both amplitude and time of appearance.
• In a variety of cells, the InsP3 receptor channels play crucial roles in eliciting [Ca2+]; oscillations and puffs [1-4].
• Three different InsP3 receptor isoforms are presently recognized with subtle differences in the regulation of Ca2+ channel opening.
• Characteristic for the type 1 InsP3 receptors is a biphasic effect of cytosolic Ca2+ on the channel activity, with Ca2+ stimulating the Ca2+ release