Cardiovasular pharmacologyFenamates block gap junction coupling and potentiate BKCa channels in guinea pig arteriolar cells
Introduction
Fenamates, the derivatives of N-phenylanthranilic acid, are a family of nonsteroidal anti-inflammatory drugs including flufenamic acids (FFA), meclofenamic acid (MFA) and niflumic acids (NFA). The pharmacological knowledge of fenamates has expanded over the years but remains limited, which hinders a better understanding of the mechanisms for their clinic usefulness and side effects.
Fenamates have been demonstrated to block gap junctions in rat fibroblasts and neuroblastoma cells (Juszczak and Swiergiel, 2009), antagonize a subset of chloride channels (Duran et al., 2010, Hartzell et al., 2005), and exert variable effects on transient receptor potential (TRP) channels (Hill et al., 2004, Saleh et al., 2006). The present study is a detailed investigation of FFA- and NFA-induced gap junction blockade between arteriolar cells.
Gap junctions are specialized arrays of intercellular channels which connect adjacent cells in many tissues and provide intercellular chemical and electrical communications (McCracken and Roberts, 2006, Sohl et al., 2005). Gap junction channels comprise two hemi-channels or connexons, one contributed by each cell. Connexons are composed of six protein subunits called connexins (Cx). To date, more than 20 different connexin genes have been identified as expressed in many mammalian cell types and tissues (McCracken and Roberts, 2006, Tran and Welsh, 2009). Vascular gap junctions are assembled with various combinations of Cx37, Cx40, Cx43, and Cx45 (Tran and Welsh, 2009). Cx37, Cx40, and Cx43 are typically found in endothelial cells (ECs), and predominantly Cx43, but also Cx37 and Cx45, in vascular smooth muscle cells (VSMCs) (Figueroa et al., 2006).
Fenamates have been shown to inhibit gap junctions in a monolayer of normal rat kidney fibroblasts and SKHep1 cells which over-expresses connexin43 (Cx43) (Harks et al., 2001). FFA blocks gap junctions formed of Cx26, Cx32, Cx40, Cx43, Cx46, and Cx50 in N2A cells with low selectivity—the half inhibition concentration (IC50) ranged between 20 and 60 μM (Srinivas and Spray, 2003). FFA has also been found to block mouse connexin50 (Cx50) and rat connexin46 (Cx46) hemichannels expressed in Xenopus laevis oocytes (Eskandari et al., 2002). More recently, MFA, FFA, and NFA were reported to inhibit dye coupling in a retina network of A-type horizontal cells (Pan et al., 2007). However, analogous inhibition of gap junctions and other membrane effects in native vascular tissues are yet to be shown.
Gap junctions play a key role in the development, structure, physiology and pathology of many organs, particularly the vascular system (Figueroa et al., 2004, Figueroa et al., 2006, Griffith, 2004, Jiang et al., 2005, Sandow, 2004). For instance, vascular tone and conductive vasomotion rely on gap junction-mediated coupling and synchronization of the VSMCs and ECs (Figueroa et al., 2004, Juszczak and Swiergiel, 2009, Segal, 2005). Vaso-active agents such as ACh, substance P and bradykinin cause a primary hyperpolarization in ECs and a secondary hyperpolarization in VSMCs via so-called endothelium-derived hyperpolarizing factor (EDHF) (Busse et al., 2002, Juszczak and Swiergiel, 2009). Studies of various vascular preparations have implicated nitric oxide (NO), prostaglandins, and cytochrome P450 products epoxyeicosatrienoic acids (EETs) as the EDHF (Busse et al., 2002), but the gap junction appears to be the major and universal mechanism (Griffith, 2004, Sandow, 2004).
Using whole-cell voltage-clamp techniques, we studied fenamates actions on vascular gap junctions and non-junctional channels in VSMCs in situ of acutely isolated arteriole segments from the cochlear spiral modiolar artery (SMA), anterior inferior cerebellar artery (AICA) and mesenteric artery (MA), and in dispersed VSMCs. The present investigation should contribute to better understanding of fenamates effects/side effects in clinic as well as to a better data interpretation when they are used in basic research.
Section snippets
Animals and preparations
Guinea-pigs (250–300 g) were killed by exsanguination under deep general anesthesia by intramuscular injection of an anesthetic mixture (1 ml/kg) of ketamine (500 mg), xylazine (20 mg) and acepromazine (10 mg) in 8.5 ml water. The entire length of the SMA was harvested from the cochlea. Brain arteriolar segments were harvested from branches of the AICA in the pia. The MA and its branches were harvested from upper ileum mesentery. The animal use protocol was approved by the University Animal care and
Membrane property of cells in the arterioles
Whole-cell recordings were made on VSMCs in situ of and dispersed from the SMA, AICA and MA from 59 guinea pigs. Stable recording lasted from 5 min to 6 h on in situ cells, whereas only up to 2 h in dispersed VSMCs. Data of the measurement of input conductance (Ginput) or Rinput and Cinput of the in situ and dispersed VSMCs from the three arteriole groups are summarized in Table 1. Note that Ginput was 8, 8.5, and 11.5 times higher in in situ cells than in the dispersed cells of the SMA, AICA, and
Discussion
It was shown in previous studies that fenamates block gap junction mediated current spread and dye transfer in cultured kidney cells expressing Cx43 (Harks et al., 2001, Juszczak and Swiergiel, 2009), block dye transfer between N2A cells expressing defined connexins (Srinivas and Spray, 2003), and block Cx36-related electrical synapses in retinal amacrine and on-cone bipolar cells (Veruki and Hartveit, 2009). We extend these observations by examining the effect of FFA and NFA on coupling
Acknowledgment
This work was supported by the National Natural Science Foundation of China 30900490 (to Ke-Tao Ma), 30960417(to Jun-Qiang Si), 31100829 (to Xin-Zhi Li) and 81000411 (to Li Li); and by NIH USA, NIDCD R01-DC-004716 (to Zhi-Gen Jiang). The authors thank Mr. Allan Kachelmeier, OHRC of Oregon Health and Science University, for his meticulous English language editing.
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