Effect of BKca channel blockers on responses to cAMP elevating agents

The direct adenylyl cyclase activator, forskolin (1-1000 nM) induced a concentration-dependent relaxation in endothelium-denuded tissues with a log ECso of -6.42 ± 0.24 and a relaxation at 1 ^M forskolin of 89.34 ± 1.33 % (Fig. 4.9a). Relaxations were essentially unaffected by the BKca channel blocker TEA (2 mM) at any concentration tested, suggesting that BKca channels do not appear to be involved in the relaxation induced by forskolin in guinea-pig aorta. Under the same conditions as above, the effect of TEA on responses to the cell permeable cAMP analogue, dibutyryl cAMP was investigated. This agent (0.1 - 3 mM) induced a concentration-dependent relaxation with a maximal relaxation at 3 mM of 90.23 ± 5.73 % (/t=5-6. Fig. 4.9b) Relaxations were inhibited by treatment of tissues with 2mM TEA, reducing the maximum response to 50.21

lOOn _control TEA 80- § 60- 40- 9

8

7

6

forskolin (log M) control TEA 80- c o % X 60- 40- p

o'

2 0- 4 3 2

Dibutyryl cAMP (log M)

Fig. 4.9. Effect of the channel blocker, tetraethylammonium (TEA), on responses to forskolin and dibutyryl cyclic AMP. Cumulative concentration- response curves to forskolin (A, 1-1000 nM) and dibutyryl cAMP (B, 0.1-3mM) measured in the absence (control; # ) or presence of 2mM TEA (■). Guinea-pig aortic tissues were denuded of endothelium and precontracted with 6 |iM phenylephrine. TEA

was applied for 15 min prior to application of the vasorelaxants. Data are expressed as the mean ± S.E.M. of 5-6 experiments. * * P < 0.01 compared to control (two way ANOVA with Bonferroi correction).

Ot

00

Compound EC50 M axim um R elaxation

Control Test Control Test

Ilo p ro s t Glibenclamide -7.21 ± 0.09 (9 ) -7.32 ± 0.09 (6 ) 50.52 ± 6.07 (9 ) 65.1 ± 3.2 ( 6 ) * TEA -7 .2 4 ± 0.09 (1 2 ) -7.11 ± 0 .1 1 (1 2 ) 68.3 ± 5.2 (1 2 ) 31.7 ± 2 . 9 ( 1 2 ) * * Iberiotoxin -7.23 ± 0.20 (4 ) -7.14 ± 0 .1 4 (4 ) 55.8 ± 4.0 (4 ) 20.4 ± 3.5 ( 4 ) * * Cicaprost Glibenclamide -7 .5 9 ± 0.09 (1 2 ) -7.47 ± 0 .0 7 (1 2 ) 91.8 ± 8 . 4 (1 2 ) 93.0 ± 4.9 (1 2 ) TEA -7.30 ± 0.17 (5 ) -7.09 ± 0.17 (5 ) 75.6 ± 3.4 (5 ) 42.8 ± 6.2 ( 5 ) * * Iberiotoxin -7.75 ± 0.07 (5 ) -7.40 ± 0.08 (5)* 87.3 ± 2.8 (5 ) 56.4 ± 6 . 2 ( 5 ) * * Apamin -7 .6 0 ± 0.13 (8 ) -7.68 ± 0.05 (8 ) 78.2 ± 3.0 (8 ) 82.7 ± 3.0 (8 ) Neca TEA -4.02 ± 0.27 (6 ) -3 .3 4 ± 0 .1 1 ( 6 ) * 59.74 ± 4.58 (6 ) 4 8.22 ± 4.62 ( 6 ) * * Forskolin TEA -6.42 ± 0.24 (6 ) 6.40 ± 0.32 (6 ) 8 9 .3 4 ± 1.33 (6 ) 79.43 ± 3.56 (5 ) Dibutyryl cAMP TEA -3 .0 0 ± 0.05 (6 ) -2.76 ± 0 .1 1 (5 ) 90.23 ± 5.72 (6 ) 50.21 ± 2.99 ( 5 ) * *

Table 4.1. Summary of the actions of channels blockers on concentration response curves to vasorelaxants in the guinea-pig aorta. Tissues were precontracted with 6 pM PE and glibenclamide (10 pM), tetraethylammonium (2 mM), iberiotoxin (25-50

nM) or apamin (100 nM) were given at least 15 mins prior to addition of the vasorelaxants. Values are the mean ± S.E.M. of n

determinations. The concentration causing 50% of relaxation of contractions is expressed as the log EC5 0 value. Individual EC5 0 values for

control and test concentrations-response curves were determined using a sigmoidal-curve fitting routine. * P < 0.05 and * * p < <0.01 (two way ANOVA with repeated measures) compared to control. * P< 0.05 (unpaired Student's f-test) compared to control.

± 2.99 % (n=5-6;

P <

0.005 two way ANOVA with repeated measures). TEA also caused a small, though not significant, shift of the concentration-response curve to the right (log EC50 of -3.00 ± 0.05

vs..

-2 .76 ± 0.11).

4.6 Discussion

The results obtained in this chapter provide good evidence that in the guinea-pig aortic smooth muscle, BKca, and not Katp channels are involved in the relaxation induced by iloprost and cicaprost. This conclusion is based on the following findings. Iberiotoxin, a highly specific blocker of large conductance

BKca channels (Glavez

et aL,

1990), markedly attenuated the vasorelaxation to both iloprost and cicaprost, significantly reducing the maximum response. To date, iberiotoxin has not been found to inhibit any other type of IC channel and remains the most selective blocker of BKca channels available. 2) Responses to both iloprost and cicaprost were similarly inhibited by 2 mM TEA. This concentration of TEA has been demonstrated to block BKca channels at or below this concentration (Nelson and Quayle, 1995). This conclusion is further supported by observations from our laboratory which demonstrated that 2 mM TEA had no effect on relaxation induced by the Katp channel opener, levcromakalim (Clapp

eta!.,

1998). In addition higher concentrations of TEA (10 mM), which begin to block Katp and delayed rectifier K^ channels (Nelson and Quayle, 1995), had no further inhibitory effect on iioprost-induced relaxation (Clapp

et al.f

1998). 3) The Katp channel blocker, glibenclamide did not inhibit responses to iloprost and cicaprost ruling out a contribution of Katp channels to this response. We and others have shown that 10 pM glibenclamide is sufficient to almost completely inhibit responses to Katp channel openers. Moreover, small conductance Ca^^-activated K^ channels do not appear to play a role, since the

SKca inhibitor, apamin failed in inhibit relaxations induced by cicaprost.

This is the first study to demonstrate a significant involvement of BKca channels in reiaxation induced by P G I2 mimetics, and the first to use the selective I P receptor agonist, cicaprost. Since the effects of BKca channel inhibitors were observed in endothelium-denuded tissues, this suggests that

smooth muscle rather than endothelial BKca channels are the target for iloprost and cicaprost. Moreover, in our previous studies we found that endothelium removal did not affect relaxations to iloprost (Clapp

et a!.^

1998), as has been observed in other studies. The situation appears different in smaller blood vessels where the endothelium appears to play a role in mediating the effects of iloprost. For example in the isolated perfused lung, responses to iloprost in the hypoxic lung, were attenuated by nitric oxide synthase inhibitor, L-NAME, as well as by BKca channel inhibitors (Dumas

at aL,

1997). The authors concluded that nitric oxide release from the endothelium was probably responsibly for activation of BKca (Dumas

at aL,

1997), consistent with the well known effects of nitric oxide on these channels. Previous studies have demonstrated a small role for BKca channels in responses to iloprost. In the rat- tail artery responses to iloprost were inhibited by iberiotoxin and TEA, although these inhibitors had no effect on maximal relaxation (Schubert

at a!.,

1997). In contrast, glibenclamide markedly inhibited vasorelaxation to iloprost, indicating a major role Katp channels. Like-wise glibenclamide inhibited responses to

iloprost in the perfused rat lung (Dumas

at a!.,

1995), as has been found in the majority of vascular studies to date (Quayle

at a!.,

1997). However, Katp channels do not appear to underlie either the relaxant effects of prostaglandin Ez (PGEz) and PGI2 analogues in aorta (Bouchard

at a!.,

1994) or

hyperpolarisation by iloprost in rat hepatic artery (Zygmunt

at a!.,

1998). Thus, depending on the vascular bed, both Katp and BKca channels appear to be

involved in mediating the effects of PGI2 and stable analogues.

It may be argued that the inhibition of relaxation induced by iloprost and cicaprost that was observed with TEA or iberiotoxin was due either to functional antagonism or non-specific effects (c.f. Huang

at a l,

1993) rather than direct blockade of K^ channels. Certainly, exposure of tissues to TEA did cause a noticeable contraction which could account for inhibition of relaxation. This seems unlikely for the following reasons. Firstly, similar effects were observed with iberiotoxin, which did not significantly affect PE contractions. This lack of effect on contraction suggests that BKca channels are not significantly opened in

the presence of PE. Secondly, TEA had no effect on relaxation induced by forskolin, thus supporting the idea that TEA was not producing a general reduction in relaxation. This difference between the effects of TEA and iberiotoxin on PE contraction could be explained if TEA was affecting other channels. For example, TEA blocks Kv and Katp channels with an IC50 value of 7

mM and 10 mM, respectively (Quayle and Nelson 1995). Clearly Katp channels

can be ruled out, although we did not test the sensitivity of responses Kv channel inhibitors.

Whilst there is wide belief that cAMP mediates the response to PGI2 and

its stable analogues, to my knowledge only one group has studied the effect of PKA inhibitors on relaxation mediated by these agents, and none using inhibitors of adenylate cyclase. In these previously reported studies, both iioprost-induced relaxation and activation of IKca the rat-tail artery were significantly inhibited by H-89 or Rp-8-CPT-cAMPs, both known inhibitors of PKA (Schubert

eta!.,

1996; Schubert

etaL,

1997). Moreover, the PKA activator, Sp- 5, 6-DCI-cBIMPS induced relaxation in this tissue which was sensitive to BKca channel inhibitors. However, niether H-89 or the adenylyl cyclase inhibitor, SQ22536 inhibited responses to iloprost in aorta in our hands. The reasons for these differences is not readily apparent, although in the study by Schubert

et

aL,

(1997), H89 failed to inhibit the effect of iloprost at 1 |liM but did 9 p M . At the higher concentration, this agent is known to affect other kinases (Davies

at

a!.,

2000), so that it cannot readily be assumed to be specific for PKA. Lack of effect of TEA on forskolin-induced relaxation, which presumably relaxes smooth muscle through activation of adenylyl cyclase (Laurenza

at a!.,

1989), may be taken as evidence for a cAMP-independent mechanism of BKca channel activation with iloprost and cicaprost. Indeed, while some of the effects of PGI2

analogues can be mimicked by either forskolin, or dibutyri cAMP (Nakhostine & Lamontagne, 1994; Dumas

at a!.,

1997), others cannot (Jackson, 1993). Thus, there appears to be some controversy as to whether cAMP is involved in mediating the K"^ channel responses of PGI2 and its mimetics or whether direct

possible that there are unidentified pathways of relaxation, based on recent findings suggesting that IP receptors can couple to multiple G-protein pathways (Schwaner

et a!.,

1995; Wise and Jones, 1996), this raises the possibility that there are unidentified pathways of relaxation.

Dibutyryl cAMP is a cell permeable cAMP analogue, which is hydrolysed at a much slower rate than the native cAMP (Miller

eta!.,

1973). Both forskolin and dibutyryl cAMP have been shown to activate channels in a variety of smooth muscle tissues, both in organ bath experiments and patch-clamp studies. Forskolin has been shown to activate both BKca and Katp channels in

porcine coronary arteries (White

e ta l,

2000), and BKca channels in the guinea-

pig basilar artery and tracheal smooth muscle cells (Hiramatsu

et a l,

1994; Song and Simard, 1995). However, whilst it appeared that forskolin activated BKca channels in rabbit coronary and cerebral arteries, potentiation of isoprenaline relaxation in rabbit aortic rings by forskolin did not appear to be mediated through BKca channels (Satake and Shibata, 1997b). In functional studies, dibutyryl cAMP has been found to induce relaxation through a BKca channel-dependent mechanism in the rat cerebral and mesenteric arteries (Paterno

e ta l,

1996; Tanaka

e ta l,

1999) and in patch-clamp studies has been shown to activate BKca channels in porcine coronary and guinea-pig basilar arteriole myocytes (Miyoshi and Nakaya, 1995; Song and Simard, 1995). However, in the rat aorta there appears to be some discrepancy between functional and patch-clamp experiments. In the former, dibutyryl cAMP was found not to mediate relaxation through BKca channels (Satake and Shibata, 1997) whilst in the later, dibutyryl cAMP was shown to activate BKca channels in isolated myocytes (Sadoshima

et a l,

1988). It may be difficult to compare these two approaches because in electrophysiological studies, cells are not usually precontracted with PE, and the intracellular environment is often disturbed. Thus, in rat aortic smooth muscle, whilst an increase dibutyryl cAMP is able to activate BKca channels, functionally, they do not play a major role in relaxation induced by cAMP in this tissue.

Forskolin and dibutyryl cAMP are routinely used to probe for the involvement of cAMP. However in this study, the mechanism of action of both forskolin and dibutyryl cAMP appeared to be different, which clearly does not match up with the widely assumed notion that both agents induce relaxation

via

cAMP. There are several possible interpretations of this data. Firstly, it is possible that large increases in cAMP are needed to activate BKca channels in this tissue. Such levels may not be attainable

via

forskolin-induced activation of adenylyl cyclases, but may be attainable with dibutyryl cAMP, as this analogue is resistant to phosphodiesterases and would not readily be broken down. The cytosolic concentration of cAMP generated by this agent may be such that it saturates the PKA pathway thereby leading to widespread phosphorylation of the BKca channel or a closely associated protein. However, there appears to be a lack of correlation between the ability of stable cAMP analogues to induce vasorelaxation and to stimulate protein kinase A. In rat aortic rings, dibutyryl cAMP inhibited KCI- and PE induced contractions in a concentration-dependent manner, although another cell permeable analogue, 8-bromo-cAMP had little effect on tension (MacDonell and Diamond, 1994). Interestingly, when the ability of these agents to stimulate PKA was investigated, 8-bromo-cAMP greatly increased the activity of this enzyme, whereas dibutyryl cAMP had little effect on activity. In other studies, dibutyrlyl cAMP, like forskolin, was shown induce relaxation in rat aorta through decreasing cystolic Ca^^ levels and the Ca^^ sensitivity of contractile elements (Abe and Karaki, 1992) through a similar mechanism. Therefore, it is possible that dibutyryl cAMP activates BKca channels

via

a PKA-independent mechanism in guinea-pig aorta, or that cAMP can directly activate the channel. In porcine smooth muscle cells forskolin, was demonstrated to activate BKca channels in cell attached patches which was interpreted to indicate the involvement of cAMP in this response. This was further supported by studies in excised patches, where BKca channels were shown to be activated by cytoplasmic application of cAMP in the presence of protein kinase A. However, application of cAMP in the presence of Ca^^, but in the absence of PKA, was also able to activate BKca channels (Minami

et ai.,

1993). This indicated that cAMP was able to activate BKca channels both in a PKA- dependent and independent manner. Another consideration, is that cAMP generated by forskolin is unable to induce relaxation through PKA, because it is not generated in the right loci of the cell. Dibutyryl cAMP, presumed to increase cAMP globally, may be more capable of activating BKca channels. Clearly, the

putative mechanism of activation of BKca channels with dibutyryl cAMP needs further clarification using single cell techniques.

Although our results with glibenclamide indicate that Katp channels are

not involved in responses to PGI2 mimetics in guinea-pig aorta, they do

highlight a difference in the mechanism of action between iloprost and cicaprost. We found that glibenclamide significantly potentiated relaxation to iloprost, but not to cicaprost. One explanation for these differences is that cicaprost has a greater degree of selectivity toward the IP receptor whereas, iloprost is known to have additional effects on EPi and EP3 receptors, activation

of which would lead to smooth muscle contraction (Coleman

et aL,

1994; Narumiya

at a!.,

1999). Indeed iloprost is equipotent with PGE2 at binding to

the EPi receptor subtype (having a Kj of 21 and 20 nM, respectively), and is at least 20-fold more potent than PGI2 (Sheldrick et al., 1988; Kiriyama

at a!.,

1997). Cicaprost on the other hand has no effect on the EPi receptor up to the micromolar range. The observation that in tissues denuded of endothelium, preincubation of the tissue with the cyclooxygenase inhibitor, indomethacin augmented relaxation induced by iloprost and attenuated the effects of glibenclamide, suggests that iloprost may release a constricting prostanoid from the smooth muscle which is sensitive to inhibition by glibenclamide. Indeed, previous studies have demonstrated that indomethacin enhances contraction induced by PGI2 in rat aorta (Van Dam

at a!.,

1986). Moreover, it is also known

that glibenclamide can inhibit contractions elicited by the prostanoids PGE2,

In document The role of cyclic AMP and K+ channels in mediating vasorelaxation induced by prostacyclin analogues and other agonists of Gs-coupled receptors (Page 164-172)