Role of Endothelial [Ca2i in EDHFMediated Vasodilatation

The critical role for endothelial [Ca2+]i in EDHF-mediated responses initially gained wide support based on the following evidence: (1) agonists known to produce EDHF-mediated responses produced elevations of endothelial [Ca2+]i in cultured endothelial cells, (2) Ca2+ ionophores (such as A23187) produced endothelium-dependent smooth muscle hyperpolarization, and (3) removal of Ca2+ from the bathing medium reduced or eliminated endothelium-dependent smooth muscle hyperpolarization. The evidence supporting the critical involvement of endothelial Ca2+ is discussed further hereafter.

Chen and Suzuki were among the first to demonstrate the Ca2+-dependency of EDHF-mediated responses [5]. By measuring rabbit carotid artery smooth muscle membrane potential by sharp electrode, they were able to demonstrate that ACh-mediated smooth muscle hyperpolarization consisted of two components (see Figure 2). When ACh was applied to endothelium-intact arteries in PSS with a standard Ca2+ concentration ([Ca2+]o) of 2.5 mM, ACh produced a significant smooth muscle hyperpolarization that was sustained for several minutes (top trace). However, in the pres

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Figure 2 Acetylcholine (ACh)-induced hyperpolarization generated in different [Ca2+]o solutions. [Ca2+]o = 2.5mM (A), 0mM (B), and 0mM with 0.2mM EGTA(C). ACh (10-5M) was applied at the arrow in each trace. Reproduced with permission from Chen and Suzuki (1990). J. Physiol. 421, 521-534.

ence of nominally Ca2+-free (no Ca2+ added to the PSS) or Ca2+-free/EGTA solution (Ca2+ chelator added to remove any residual Ca2+), ACh produced only a transient hyperpolarization (middle and bottom traces). Application of ACh to arteries from which the endothelium was destroyed or remove produced no smooth muscle hyperpolarization. Additionally, application of the Ca2+ ionophore, A23187, promoted smooth muscle hyperpolarization that was similarly dependent upon an intact endothelium. Thus, it was concluded that endothelium-dependent hyperpolarization of the smooth muscle consisted of two components, a transient hyperpolarization that utilized intracellular store Ca2+ and a sustained hyperpolarization that was dependent on Ca2+ influx from the extracellular medium.

Early on in the study of EDHF-mediated responses, it was noted that the involvement of an EDHF-dependent mechanism varied greatly in the vessels studied. For instance, whereas some vessels appeared to utilize EDRF/NO and EDHF-mediated mechanisms, others appeared to utilize only the EDRF/NO-mediated mechanism. It was therefore questioned why EDHF-mediated responses occurred in some vessels but not others. One hypothesis that was put forth by Nagao et al. speculated that the reason was due in part to differences in endothelial [Ca2+]j attained in a particular artery [6]. The principal evidence for this conclusion was from an experiment in which A23187 was administered to rat mesenteric arteries in the absence or presence of an inhibitor of nitric oxide synthase (NOS). The authors found that A23187 was able to promote vasorelaxation in both conditions; however, it required a higher concentration of A23187 to do so in the presence of the NOS inhibitor. Thus, assuming that the increased concentration of A23187 resulted primarily in an increase in endothelial [Ca2+]i, it appeared that the EDHF-mediated response required a higher endothelial [Ca2+] than the EDRF/NO-mediated response. Thus, one possible explanation for the heterogeneity of EDHF-mediated responses was that the ability of the agonists to increase endothelial [Ca2+] varied in the different preparations.

A subsequent study by Marrelli investigated a possible Ca2+ threshold for EDHF-mediated responses more directly [7]. In that study, Marrelli selectively measured endothelial [Ca2+]j in response to agonists that have been shown to produce or not produce EDHF-mediated responses in rat cerebral arteries. It was demonstrated that EDRF/NO-mediated dilations were initiated at a threshold of 220 nM Ca2+ (resting = 145nM), whereas EDHF-mediated dilations were not initiated until 340nM Ca2+ (see Figure 3). The threshold for EDHF-mediated dilation was similar for both a receptor-dependent agonist (uridine triphosphate; UTP) or a receptor-independent mechanism to increase endothelial [Ca2+] (Br-A23187). Interestingly, the study also demonstrated that a "non-EDHF-dependent" agonist could be made to elicit an EDHF-mediated dilation if the Ca2+ response was augmented. For instance, stimulation of endothelial P2Yj receptors with 2-methylthioadenosine triphosphate (2MeS-ATP) produced maximal dilations through an exclusively EDRF/NO-dependent mechanism. Dilations were completely abolished in the presence of a NOS inhibitor. However, if the baseline endothelial [Ca2+]1 was elevated or primed with a low concentration of UTP or Br-A23187 (though not enough to elicit EDHF-mediated dilation by itself), subsequent addition of 2MeS-ATP resulted in a greater increase in endothelial [Ca2+]i and an EDHF-mediated dilation. These experiments indicated that EDHF-mediated dilations are not inherently linked to certain

Figure 3 Comparison of the endothelial [Ca2+] threshold for nitric oxide (NO)- and EDHF-mediated vasodilatation. Data are presented as the mean SE [Ca2+] required to elicit a 15% dilation. In the concentrations used and in the absence of L-NAME (nitric oxide synthase inhibitor), UTP and 2-MeS-ATP produced vasodilatation through the production of NO (gray bars). In the presence of L-NAME plus indomethacin (cyclooxyge-nase inhibitor), UTP and Br-A23187 produced a dilation through the production of EDHF (black bars). There is a significantly higher threshold for EDHF-mediated vasodilatation compared with that of NO. *P < 0.05 between the indicated groups by one-way ANOVA. Reproduced with permission from Marrelli (2001). Am. J. Physiol. Heart Circ. Physiol. 281, H1759-H1766.

Figure 3 Comparison of the endothelial [Ca2+] threshold for nitric oxide (NO)- and EDHF-mediated vasodilatation. Data are presented as the mean SE [Ca2+] required to elicit a 15% dilation. In the concentrations used and in the absence of L-NAME (nitric oxide synthase inhibitor), UTP and 2-MeS-ATP produced vasodilatation through the production of NO (gray bars). In the presence of L-NAME plus indomethacin (cyclooxyge-nase inhibitor), UTP and Br-A23187 produced a dilation through the production of EDHF (black bars). There is a significantly higher threshold for EDHF-mediated vasodilatation compared with that of NO. *P < 0.05 between the indicated groups by one-way ANOVA. Reproduced with permission from Marrelli (2001). Am. J. Physiol. Heart Circ. Physiol. 281, H1759-H1766.

receptor systems that either are or are not capable of eliciting EDHF-mediated responses. Rather, the ability of a receptor system to produce an EDHF-mediated response depends more on its potential to increase endothelial [Ca2+]r

Essentials of Human Physiology

Essentials of Human Physiology

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