Role of phospholipase D and diacylglycerol in activating constitutive TRPC-like cation channels in rabbit ear artery myocytes

Abstract

Previously we have described a constitutively active Ca2+-permeable non-selective cation channel in freshly dispersed rabbit ear artery myocytes that has similar properties to canonical transient receptor potential (TRPC) channel proteins. In the present study we have investigated the transduction pathways responsible for stimulating constitutive channel activity in these myocytes. Application of the pharmacological inhibitors of phosphatidylcholine-phospholipase D (PC-PLD), butan-1-ol and C2 ceramide, produced marked inhibition of constitutive channel activity in cell-attached patches and also butan-1-ol produced pronounced suppression of resting membrane conductance measured with whole-cell recording whereas the inactive isomer butan-2-ol had no effect on constitutive whole-cell or channel activity. In addition butan-1-ol had no effect on channel activity evoked by the diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Inhibitors of PC-phospholipase C (PC-PLC) and phospholipase A2 (PLA2) had no effect on constitutive channel activity. Application of a purified PC-PLD enzyme and its metabolite phosphatidic acid to inside-out patches markedly increased channel activity. The phosphatidic acid phosphohydrolase (PAP) inhibitor dl-propranolol also inhibited constitutive and phosphatidic acid-induced increases in channel activity but had no effect on OAG-evoked responses. The DAG lipase and DAG kinase inhibitors, RHC80267 and R59949 respectively, which inhibit DAG metabolism, produced transient increases in channel activity which were mimicked by relatively high concentrations (40 microm) of OAG. The protein kinase C (PKC) inhibitor chelerythrine did not prevent channel activation by OAG but blocked the secondary inhibitory response of OAG. It is proposed that endogenous DAG is involved in the activation of channel activity and that its effects on channel activity are concentration-dependent with higher concentrations of DAG also inhibiting channel activity through activation of PKC. This study indicates that constitutive cation channel activity in ear artery myocytes is mediated by DAG which is generated by PC-PLD via phosphatidic acid which represents a novel activation pathway of cation channels in vascular myocytes.

Figure 1. Effect of agents that inhibit phospholipase D on constitutive channel activity in cell-attached patches

A, bath application of 0.5% butan-1-ol produced a marked inhibition of constitutively active channel currents. a and b, show channel currents on a faster timescale from the corresponding positions on the above brace. B, bath application of 100 μm C2 ceramide also produced a pronounced inhibition of constitutive channel activity. C, mean data of effect of butan-1-ol (n = 8), butan-2-ol (n = 5), C2 ceramide (n = 5), D-609 (n = 6) and AACOCF₃ (n = 6) on relative peak NP_o of constitutive channel activity. Measurements were made 2 min after application of the pharmacological agent. Holding potential was –50 mV **P < 0.01.

Figure 2. Effect of butan-1-ol on resting whole-cell currents

A, bath application of 0.5% butan-1-ol reduced the amplitude of constitutively active whole-cell currents. *whole-cell configuration achieved at this time. Dashed line indicates 0 pA holding current. Holding potential was –50 mV. B, I–V relationship of whole-cell currents measured before (a) and 2 min after (b) application of 0.5% butan-1-ol. C, mean data of whole-cell currents in the presence of 0.5% butan-1-ol (n = 6) and 0.5% butan-2-ol (n = 6). **P < 0.01.

Figure 3. Effect of PC-PLD on channel activity in inside-out patches

A, bath application of 2.5 U ml⁻¹ PC-PLD increased constitutive channel activity in an inside-out patch at a holding potential of −50 mV; a and b, show channel currents on a faster timescale. B, amplitude histogram of PC-PLD-induced channel currents shown in A had three current amplitude levels which are represented by three conductance states of 16, 28 and 42 pS in pooled mean I–V relationships shown in C. Channel current amplitudes greater than the three unitary levels represent more than one channel in the patch.

Figure 4. Effect of phosphatidic acid on channel activity in inside-out patches

A, bath application of 100 μm phosphatidic acid increased constitutive channel activity in an inside-out patch at a holding potential of −50 mV; a and b, show channel currents on a faster timescale. B, amplitude histogram of phosphatidic acid-induced channel currents shown in A had three current amplitude levels which are represented by three conductance states of 15, 27 and 41 pS in pooled mean I–V relationships shown in C. In B channel current amplitudes greater than the three levels represent more than one channel in the patch.

Figure 5. Effect of DL-propranolol on constitutive, phosphatidic acid-induced and OAG-evoked channel activity

Effect of bath application of 500 μmdl-propranolol on constitutively active cation channel activity (A) and phosphatidic acid-induced channel activity (B) in different inside-out patches at a holding potential of −50 mV. C, mean data showing that co-application of 500 μmdl-propranolol significantly inhibited constitutively active (n = 6) and phosphatidic acid-induced (n = 6) channel activity but not OAG-induced channel activity (n = 6) after 5 min *P < 0.05.

Figure 6. Effect of inhibitors of DAG metabolism on constitutively active whole-cell and single channel cation currents in ear artery myocytes

A, bath application of 10 μm RHC80267 induced a transient increase in whole-cell current. Note that the increase in holding current followed by a decrease in activity are associated with, respectively, an increase and then decrease in ‘noisy’ appearance. Holding potential was −50 mV. The dashed line represents 0 pA holding current and vertical lines represent current responses to membrane potential ramps from −150 mV to +100 mV. B, mean I–V relationships of whole-cell currents in the absence and presence of 10 μm RHC80267 (n = 6). C, mean data of RHC80267-induced whole-cell currents (n = 6). Da, in a cell-attached patch bath application of 10 μm RHC80267 induced an initial increase in constitutive channel activity followed by a decrease in activity. Holding potential at −50 mV. Note in this patch that the RHC80267-induced decrease in channel activity after peak NP_o is less than control activity. Db, expanded timescale of a section of trace shown in a. Bold line represents closed level and three dashed lines represent three conductance levels. Larger amplitude openings represent more than one channel in the patch. Dc, histogram of RHC-induced channel current amplitudes showing channels opened to three current levels. Channel amplitudes greater than these levels were due to more than one channel in the patch. E, mean data of 10 μm RHC80267-induced (n = 10) and 10 μm R59949-induced channel activity (n = 7). F, pooled mean I–V relationship of RHC80267-induced channel currents showing the three current amplitude levels represented three conductances of 16, 27 and 42 pS (each point was determined from at least five patches). *P < 0.05.

Figure 7. Effect of exogenous OAG on constitutive channel activity in cell-attached patches

Aa, bath application of 5 μm OAG induced a sustained increase in constitutive channel activity whereas bath application of 40 μm OAG (Ab) induced a larger but transient increase in constitutive channel activity. B, mean data of OAG-induced channel activity either at the peak response or about 2 min after peak response. Note that 5 μm OAG induced a sustained response (n = 5) whereas 40 μm OAG produced a transient increase in channel activity (n = 6) that was converted to a sustained response by pretreatment with 3 μm chelerythrine for 2 min (n = 6). C, steady-state concentration–effect curve of OAG-induced responses on channel activity measured approximately 4–5 min after OAG application (i.e. with OAG concentration > 10 μm), after the peak increase in channel activity. The curves yielded an apparent EC₅₀ of about 2 μm and an apparent IC₅₀ of about 32 μm (each point was determined from at least five patches). **P < 0.01.