Jean-Louis BÉNy

To investigate whether in isolated porcine ciliary processes, stimulation of the nitric oxide (NO)-guanylate cyclase (GC)-3',5'-cyclic guanosine monophosphate (cGMP) pathway modulates ciliary epithelial transmembrane potential. Changes in transmembrane potential induced by the two NO donors, sodium nitroprusside (SNP; 100 microM) and S-nitroso-N-acetyl-penicillamine (SNAP; 100 microM), or by the cGMP-analogue 8-para-chlorophenylthioguanosine-3', 5'-cyclic guanosine monophosphate (8-pCPT-cGMP; 100 microM) were measured with microelectrodes in the presence or in the absence of the GC-inhibitor 1-H-(1,2,4)oxadiazole(4,3-alpha)quinoxalin-1-1 (ODQ; 10 microM). The effect of 8-pCPT-cGMP was also assessed in the presence of the anion channel inhibitors niflumic acid (100 microM), diisothiocyanatostilbene-2,2' disulfonic acid (DIDS; 1 mM), anthracene-9-carboxylic acid (9-AC; 1 mM), or the K+ channel blocker tetraethylammonium chloride (TEA; 10 mM). cGMP production was measured by immunoassay. Significant membrane depolarizations (P < 0.05-0.001; n = 5-8) were induced by SNP (6 +/- 1 mV; mean +/- SEM), SNAP (8 +/- 1 mV), or 8-pCPT-cGMP (13 +/- 1 mV). In presence of ODQ, the effect of SNP and SNAP were significantly inhibited (-2 +/- 0 mV and 0 +/- 0 mV, respectively; P < 0.05; n = 5-6), but not depolarizations elicited by 8-pCPT-cGMP. These were prevented (P < 0.05-0.01; n = 5) by niflumic acid (1 +/- 1 mV), DIDS (1 +/- 1 mV), or 9-AC (5 +/- 1 mV), but not by TEA (12 +/- 2 mV). The increase in cGMP production induced by SNP (9.5-fold) was inhibited by ODQ (P < 0.001; n = 6). Activation of the NO-GC-cGMP pathway modulates epithelial transmembrane potential in isolated porcine ciliary processes.

Epoxyeicosatrienoic acids (EETs) have been described as endothelium‐derived hyper‐polarizing factors (EDHFs), based on their stimulatory effects on smooth muscle K⁺channels. In order to reveal a putative autocrine effect of EETs on endothelial channels, we have studied the effects of the four EET regioisomers (5,6‐EET, 8,9‐EET, 11,12‐EET and 14,15‐EET) on the high‐conductance, Ca²⁺‐dependent K⁺ (BK_Ca) channel recorded in inside‐out patches of primary cultured pig coronary artery endothelial cells. Currents were recorded in the presence of either 500 nm or 1 μm free Ca²⁺ on the cytosolic side of the membrane. In 81% of experiments, EETs at < 156 nm, applied on the cytosolic side of the membrane, transiently increased BK_Ca channel open state probability (P₀) without affecting its unitary conductance, thus providing evidence for direct action of EETs, without involvement of a cytosolic transduction pathway. The four EET regioisomers appeared to be equally active, multiplying the BK_Ca channel P_o by a mean factor of 4.3 ± 0.6 (n= 15), and involving an increase in the number and duration of openings. The EET‐induced increase in BK_Ca channel activity was more pronounced with low initial P_o. When the BK_Ca channel was activated by 500 nm Ca²⁺, application of EETs increased the initial P_o value of below 0.1 by a factor of 5. When the channel was activated by 1 μm Ca²⁺, application of EETs increased the initial P_o value by a factor of 3. Our results show that EETs potentiate endothelial BK_Ca channel activation by Ca²⁺. The autocrine action of EETs on endothelial cells, which occurs in the same concentration range as their action on muscle cells, should therefore fully participate in the vasoactive effects of EETs, and thus be taken into account when considering their putative EDHF function.

To assess the effects of K(+)-channel blockers on bradykinin-induced relaxations in porcine ciliary artery. Vascular isometric forces were measured with a myograph system. Ciliary vascular rings were precontracted with thromboxane A2 analog (U 46619, 10(-7) M) to assess dose-dependent (10(-10)-3 x 10(-6) M) bradykinin-induced relaxation after addition of one of the following: the nitric oxide (NO) synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) or inactive enantiomer (D-NAME, 10(-4) M); the nonspecific K(+)-channel blocker tetra-ethylammonium (TEA, 10(-2) M); or the ATP-sensitive K(+)-channel blocker glibenclamide (10(-5) M). The effect of TEA on relaxations to the NO donor, sodium nitroprusside (SNP, 10(-10)-10(-4) M) was investigated. The membrane potential of vascular smooth muscle cells (VSMC) was recorded after exposure to bradykinin (2.5 x 10(-7) M). Endothelium-dependent relaxations to bradykinin (maximal [max], 99% +/- 3%) were strongly inhibited by L-NAME (max, 39% +/- 4%, P < 0.01) and partially by TEA (max, 62% +/- 3%, P < 0.01) or glibenclamide (max, 77% +/- 4%, P < 0.01). Administration of glibenclamide plus L-NAME further suppressed bradykinin-induced relaxation (max, 23% +/- 6%; P < 0.01), whereas TEA and L-NAME (max, 6% +/- 2%; P < 0.01) abolished the relaxation. SNP relaxations were unaffected by TEA. Bradykinin had no effect on the membrane potential of VSMC. In porcine ciliary artery, the endothelium-dependent relaxations to bradykinin are primarily mediated by NO and involve K(+)-channels. As only relaxations to bradykinin, but not those mediated by SNP, were inhibited by TEA, this implies that K(+)-channel blockers most likely affect the bradykinin-evoked NO production or release by the endothelium.

In porcine coronary artery, bradykinin-induced endothelium-dependent vasodilatations are associated with simultaneous endothelium as well as endothelium-dependent smooth muscle cell (SMC) hyperpolarizations. In contrast, in porcine ciliary artery bradykinin evokes endothelium-dependent relaxations, but no change in SMC membrane potential. This study addresses the question of whether the lack of bradykinin-induced SMC hyperpolarization is also associated with an absence of endothelial hyperpolarization in porcine ciliary artery. With a microelectrode to impale cells in arterial strips, a 12-mV transient bradykinin-induced hyperpolarization was measured in endothelial cells. Bradykinin evoked no SMC hyperpolarization deep in the media. Only occasionally, a slight 4-mV hyperpolarization could be recorded in some SMC next to the endothelium. The endothelial intracellular injection (through the recording electrode) of the fluorescent tracers, lucifer yellow or ethidium bromide, showed the existence of a heterocellular dye coupling between endothelial cells and SMC. These observations in porcine ciliary artery demonstrate that the lack of bradykinin-induced endothelium-dependent SMC hyperpolarization is not due to an absence of endothelial cell hyperpolarization, but most likely to an insufficient electrotonic propagation space constant from endothelial cells to SMC, despite the presence of a dye coupling between these cells.