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(Circulation Research. 2001;88:852.)
© 2001 American Heart Association, Inc.

Editorial

Calcium Spots

Elementary Signals in Response to Mechanical Stress in Vascular Endothelial Cells

Hideo Tanaka, Tetsuro Takamatsu

From the Department of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine, Kyoto, Japan.

Correspondence to Dr Tetsuro Takamatsu, Professor of Pathology and Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-Ku, Kyoto 6 02-0841, Japan. E-mail ttakam{at}basic.kpu-m.ac.jp

Key Words: endothelium • mechanotransduction • calcium • lysophosphatidic acid • confocal microscopy

	Introduction

Top Introduction References

 
Blood flow–mediated mechanical force, ie, shear stress, plays an important role in regulation of vascular function and development of various cardiovascular diseases.^{1 2 3} It directly stimulates vascular endothelial cells to mobilize intracellular Ca²⁺ ([Ca²⁺]_i), resulting in production of endothelium-derived vasoactive substances, such as nitric oxide and prostacyclin, which cause vasodilation by acting on smooth muscle cells.^{1 2 3} In this process, the mechanosensitive (MS) channel is an important pathway mediating the shear stress–induced increase in [Ca²⁺]_i.^{4 5 6 7} In addition, a specific vasoactive agonist, ATP, has been regarded as an important shear transducer of the endothelial [Ca²⁺]_i mobilization in response to fluid flow.^{8 9 10 11} Which initiates and mainly contributes to the [Ca²⁺]_i mobilization, MS channel or the shear transducer? The mechanism underlying the flow-induced [Ca²⁺]_i mobilization is controversial. Furthermore, little is known about the spatiotemporal properties of [Ca²⁺]_i mobilization triggered by MS channels or by the shear transducers.

In this issue of Circulation Research, Ohata et al¹² demonstrate novel spatiotemporal changes in [Ca²⁺]_i in response to fluid flow in cultured bovine aortic endothelial cells under the application of lysophosphatidic acid (LPA),^{13 14} a bioactive phospholipid. Using real-time confocal microscopy equipped with a multipinhole Nipkow disk–type scanner, it was shown that superfusion of the cells with LPA at physiologically relevant concentrations and flow rates produced spot-like elevations of [Ca²⁺]_i, ie, Ca²⁺ spots, which were localized to a circular area (<4 µm diameter), followed by gradual and concentric spread throughout the cells. The Ca²⁺ spots develop sporadically but exhibit a distinct spatiotemporal pattern from Ca²⁺ sparks, the elementary [Ca²⁺]_i release events from intracellular stores in heart muscle cells,¹⁵ or vascular smooth muscle cells.¹⁶ The local increase in [Ca²⁺]_i develops in a stepwise and repetitive manner under constant flow, and both the percentage of cells responding to fluid flow and the average level of increase in [Ca²⁺]_i are strongly dependent on the concentration of LPA (0.1 to 10 µmol/L) as well as the flow rates corresponding to in vivo arterial blood flow and shear stress. Pharmacological analyses revealed that the Ca²⁺ spots originate from Ca²⁺ influx from the extracellular space, because the response was abolished by Ca²⁺-free, EGTA (0.1 mmol/L)-containing superfusate, whereas it was not affected by pretreatment of the cells with thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺ uptake pump. Also, the subsequent concentric propagation of [Ca²⁺]_i rise is considered to be attributable simply to passive diffusion, because thapsigargin does not affect the propagation. In addition, Ohata et al¹² demonstrated that the [Ca²⁺]_i response was mediated by the Gd³⁺-sensitive MS channels.⁷ The responses are negatively regulated by activation of cGMP-dependent protein kinase (PKG), because 8-Br-cGMP attenuates the response, and also because the combined application of 8-Br-cGMP and a PKG inhibitor, KT5823, does not. These observations are in agreement with the recent study by Yao et al,⁷ which demonstrated that the flow-induced [Ca²⁺]_i mobilization via the MS Ca²⁺-permeable cation channels is negatively regulated by PKG. Yao et al⁷ proposed that this signaling pathway functions as a negative feedback mechanism of [Ca²⁺]_i mobilization mediated by nitric oxide–induced activation of PKG.

Ohata et al¹² assessed the functional importance of the LPA-induced Ca²⁺ spot by comparing the properties of Ca²⁺ spots with those of ATP-induced Ca²⁺ waves, a well-established mechanism of [Ca²⁺]_i mobilization related to the shear stress–induced response. Ohata et al¹² clearly demonstrated that the spatiotemporal patterns of [Ca²⁺]_i rise induced by LPA are different from those induced by ATP in that the rate of [Ca²⁺]_i rise in the Ca²⁺ spots is much higher than that in ATP-induced Ca²⁺ waves. In sharp contrast to LPA, ATP induced a gradual and spatially homogeneous increase in [Ca²⁺]_i in the majority of cells, with almost identical time courses regardless of the flow rate. Stepwise elevation of the fluid flow rate unexpectedly decreased the peak level of [Ca²⁺]_i induced by ATP. This is relevant to the observation that ATP-induced [Ca²⁺]_i waves are abolished by thapsigargin, indicating a distinct origin (ie, intracellular Ca²⁺ stores) for the [Ca²⁺]_i mobilization. Thus, Ca²⁺ spots differ fundamentally from ATP-induced Ca²⁺ waves.

Ohata et al¹² proposed that the Ca²⁺ spot is a novel, elementary event in the fluid flow–induced [Ca²⁺]_i rise in endothelial cells. Because the linkage between Ca²⁺ spots and the corresponding activation of MS channels was not assessed in this study, no direct evidence was provided to indicate that the Ca²⁺ spots are an elementary phenomenon. However, it is reasonable to consider that they are an elementary phenomenon, based on the observation that the peak level of increase in F/F₀ was not affected by fluid flow rate. Increase in fluid flow rate augments the averaged level of [Ca²⁺]_i by increasing the frequency of spots without modification of the amplitude in each individual Ca²⁺ spot, thus fulfilling expectations for an elementary [Ca²⁺]_i response. Before it is concluded that the Ca²⁺ spot is an elementary event, however, the following issues should be additionally addressed: whether opening of MS channels per se produces such a large Ca²⁺ spot and whether the subsequent circular diffusion of Ca²⁺ is really passive. One might think that single MS channel opening is too weak to produce such a large rise and expansion of [Ca²⁺]_i. There might be some amplification mechanisms of [Ca²⁺]_i rise with involvement of LAP in these processes. Although the localization of MS channels has not been established, electrophysiological analysis of the channels in combination with the [Ca²⁺]_i dynamics study would help us understand the functional linkage between the channel activity and [Ca²⁺]_i mobilization.

It is surprising that the Ca²⁺ spots develop sparsely and diversely from cell to cell in spite of the continuous and homogeneous application of fluid flow or mechanical stress over all the cells. Ohata et al¹² offered the explanation that this scarcity was attributable to the lower density of the MS channels and lower responsiveness (only 3% to 10%) of MS channels to the fluid flow. In addition, they postulate that the number of Ca²⁺ spots must be underestimated because of the limitations of the spatiotemporal performance of the real-time confocal system (ie, undetectable out-of-focus Ca²⁺ spots may exist); also, a large Ca²⁺ spot may mask the neighboring spots. Furthermore, Ohata et al¹² described the importance of the specific regions where mechanical force is focused depending on cell shape, distribution of the cytoskeleton, and direction of fluid flow for development of the spots. In practice, on the basis of three-dimensional surface geometry of the endothelium visualized by atomic force microscope, Davies et al¹⁷ proposed that variations of local forces defined by the cell-surface geometry contribute to the heterogeneous endothelial responses to fluid flow. We should also consider the scarcity of Ca²⁺ spots from the physiological point of view. Sensitivity to shear stress should be minor in elastic great arteries (aorta) compared with small vessels (peripheral muscular arteries or arterioles). Therefore, comparison of Ca²⁺ spots on endothelial cells among various regions in the vasculature may provide explanation for the lower density of Ca²⁺ spots in aortic endothelial cells.

Ohata et al¹² conducted experiments on confluent cultured endothelial cells, ie, under conditions that were far from physiological. The recent evolution of three-dimensional microscopy,¹⁸ such as confocal microscopy, multiphoton microscopy, and near-field microscopy, enables not only real-time imaging but also in situ imaging in living tissues or organs, such as vascular smooth muscles¹⁹ and the whole heart.²⁰ It is reasonable to assume that cell-to-cell communication of endothelial cells influences the responsiveness of the individual cells to fluid flow stress. Moreover, communication of endothelial cells with the adjacent vascular smooth muscle cells may also be important in the fluid flow–induced [Ca²⁺]_i mobilization. Therefore, in situ imaging of the vasculature would provide much deeper insight into the pathophysiological significance of the flow-mediated Ca²⁺ spots. Use of a confocal imaging system would allow us to discriminate between the endothelial cells and the smooth muscle cells, and, furthermore, the real-time imaging of the [Ca²⁺]_i dynamics in these cell layers along the z-axis would be possible if scanning were conducted using a piezoelectric actuator added to the real-time scanning system. In practice, Ohata et al²¹ previously demonstrated the confocal images of [Ca²⁺]_i in endothelial cells in situ with clear discrimination from the smooth muscle layer.

In summary, Ohata et al¹² propose a novel elementary signal of [Ca²⁺]_i mobilization as an initial response to shear stress in endothelial cells. They also propose a possible pathophysiological significance of LPA as an endogenous mediator in the fluid flow–induced regulation of endothelial function. Recently, they identified Ca²⁺ spots in lens epithelial cells²² with properties similar to those demonstrated in this study.¹² Therefore, Ca²⁺ spots may be a universal phenomenon occurring as an initial event of the [Ca²⁺]_i mobilization induced by mechanical stress.

	Footnotes

 
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

	References

Top Introduction References

 

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This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Tanaka, H. Articles by Takamatsu, T. Search for Related Content PubMed PubMed Citation Articles by Tanaka, H. Articles by Takamatsu, T. Related Collections Endothelium/vascular type/nitric oxide Cell signalling/signal transduction Ion channels/membrane transport