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

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.

Direct effect of Ca2+-calmodulin on cGMP-activated Ca2+-dependent Cl-channels in rat mesenteric artery myocytes.

Recently a novel cGMP-activated Ca2+-dependent Cl- channel has been described in rat mesenteric artery smooth muscle cells. In the present work we have investigated the actions of calmodulin (CaM) on single channel cGMP-activated Ca2+-dependent Cl- current (ICl(cGMP,Ca) in inside-out patches. When 1 microm CaM was applied to the intracellular surface of inside-out patches bathed with 10 microm cGMP and 100 nm [Ca2+]i there was approximately a 10-fold increase in channel open probability (NPo). This effect of CaM was not observed with lower [Ca2+]i and 100 nm [Ca2+]i with 1 microm CaM did not activate Cl- channels in the absence of cGMP. The unitary conductance, reversal potential and mean open time of the single-channel currents were similar in the absence or presence of CaM. With 10 microm cGMP and 100 nm [Ca2+]i the relationship between NPo and CaM concentration was well fitted by the Hill equation yielding an equilibrium constant for CaM of about 1.9 nm and a Hill coefficient of 1.7. With 1 microm CaM (+10 microm cGMP) the relationship between [Ca2+]i and NPo was also fitted by the Hill equation which yielded an apparent equilibrium constant of 74 nm [Ca2+]i and a Hill coefficient of 4.8. When [Ca2+]i was increased from 300 nm to 1 microm there was a decrease in NPo. The potentiating effect of CaM was markedly reduced by the selective CaM binding peptide Trp (5 nm) but not by the Ca2+/CaM-dependent protein kinase II (CaMKII) inhibitor autocamtide II related inhibitory peptide (AIP). It is concluded that CaM potentiates the activity of single channel ICl(cGMP,Ca) by increasing the probability of channel opening via a CaMKII-independent mechanism.

Single cGMP-activated Ca(+)-dependent Cl(-) channels in rat mesenteric artery smooth muscle cells.

The present study describes the single channel properties of a novel cGMP-activated Ca(2+)-dependent Cl(-) channel in rat mesenteric artery smooth muscle cells. Single channel currents were recorded in cell-attached patches in the presence of 8 Br cGMP in response to the addition of caffeine or noradrenaline and in both outside-out and inside-out patches when the internal patch surface was bathed in cGMP and Ca(2+). The channels were permeable to Cl(-) ions with an anion permeability sequence of SCN(-) (1.7) > Cl(-) (1.0) > I(-) (0.6). Single channel mean open probability (NP(o)) was independent of voltage and the channels displayed three conductance levels of 15, 35 and 55 pS. cGMP was required for channel activation and the single channel NP(o) increased sharply with raised [Ca(2+)](i), maximal activation occurring at a [Ca(2+)](i) of about 100 nM. The relationship between NP(o) and cGMP concentration was voltage independent and could be fitted by the Hill equation giving a K(d) of about 3 microM and a Hill coefficient (n(H)) of 3. cGMP- and Ca(2+)-dependent channel currents were inhibited by 10 microM ZnCl(2) but niflumic acid, an inhibitor of Ca(2+)-activated Cl(-) channels, had no effect. Inhibition of cGMP-dependent protein kinase activity by the cGMP-dependent protein kinase inhibitor KT5823 or replacement of ATP by AMP-PNP reduced NP(o), while activation of cGMP-dependent protein kinase by guanosine 3', 5'-cyclic monophosphate, beta-phenyl-1, N(2)-etheno-8-bromo-sodium salt (8 Br PET cGMP) produced a significant increase in single channel NP(o). It is likely that these single channel currents underlie the noradrenaline-activated inward current important for vasomotion in these resistance arteries.

Properties of a constitutively active Ca2+-permeable non-selective cation channel in rabbit ear artery myocytes.

In smooth muscle, non-selective cation conductances contribute to agonist-evoked depolarisation and contraction, and in the present study using patch-pipette techniques we describe the properties of a constitutively active cation channel. With whole-cell recording in K+-free conditions, there was a spontaneous current with a reversal potential (Er) that was altered by replacement of external Na+ by an impermeant cation, but not when external Cl- was replaced by an impermeant anion. The tonic cation inward current could be carried by Ca2+ ions and was greatly enhanced when the external Ca2+ concentration was reduced. In outside-out patches there was spontaneous cation channel activity that could be resolved into three conductance states of about 15, 25 and 40 pS, all with the same Er as the whole-cell current. Kinetic analysis revealed that there were two open times of about 1 and 5 ms and that the currents displayed bursting kinetics with burst durations of approximately 5 ms and 25 ms. Removal of external Ca2+ ions increased the probability of channel opening (Po) sixfold, which was associated with an increase in the longer burst duration. Bath application of the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol increased Po, but phorbol 12,13-dibutyrate, which stimulates protein kinase C (PKC), reduced channel activity. In contrast, the PKC inhibitor chelerythrine increased the activity of channel currents. It is concluded that in rabbit ear artery myocytes there is a constitutively active Ca2+-permeable cation channel that is regulated by external Ca2+ ions and suppressed by tonic PKC activity. It is proposed that this mechanism may contribute to the resting membrane conductance and basal Ca2+ influx in this particular arterial preparation.

Multiple conductance states of single Ca2+-activated Cl- channels in rabbit pulmonary artery smooth muscle cells.

Ca2+-activated Cl- channels contribute to agonist-evoked contraction and spontaneous activity in some smooth muscle preparations. Patch pipette techniques were used to study the properties of single Ca2+-activated Cl- channels in freshly dispersed rabbit pulmonary artery myocytes. In the cell-attached recording mode, two conductance states of 3.5 and 1.8 pS were recorded either spontaneously or in response to increasing [Ca2+]i. With inside-out patches, the 3.5 pS channel current predominated at 50 nM [Ca2+]i, but at 500 nM [Ca2+]i most channels opened to the 1.8 pS level and an additional 1.2 pS channel conductance was resolved. At 1 microM [Ca2+]i all of the Cl- channels opened either to the 1.8 pS or 1.2 pS level. In 0 [Ca2+]i, no channel activity was observed at -100 mV to +100 mV, but with 10-250 nM [Ca2+]i the total single channel open probability (NP(o)) increased with depolarisation. This voltage dependence was not seen at higher values of [Ca2+]i. The plot of NPo vs. [Ca2+]i yielded Ca2+ affinity constants of 8 and 250 nM and Hill slopes of 1.3 and 2.3 at +100 and -100 mV, respectively. The distribution of open times was fitted by two exponentials of about 5 and 30 ms, which were neither voltage nor Ca2+ dependent. Replacement of external Cl- by I- shifted the reversal potential by about -30 mV and lengthened the longer of the two mean open times without significant effects on other kinetic parameters. Based on these data, a model for the activation of Ca2+-activated Cl- channels is proposed.

Dual effect of blocking agents on Ca2+-activated Cl(-) currents in rabbit pulmonary artery smooth muscle cells.

The effects of the Cl- channel antagonists, niflumic acid (NFA), dichloro-diphenylamine 2-carboxylic acid (DCDPC) and diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) on Ca2+-activated Cl- current (I(Cl(Ca))) evoked by adding fixed intracellular calcium concentrations ([Ca2+]i) to the pipette solution were studied in rabbit pulmonary artery myocytes. With 250 and 500 nM [Ca2+]i bath application of NFA (100 microM) increased inward current at negative potentials, but inhibited outward current at positive potentials. On wash out of NFA, I(Cl(Ca)) was greatly enhanced at all potentials. When external Na+ ions were replaced by N-methyl-D-glucamine (NMDG+) NFA still enhanced I(Cl(Ca)) at negative potentials but the increase of I(Cl(Ca)) on wash out was blocked. When the mean reversal potential (E(r)) of I(Cl(Ca)) was shifted to negative potentials by replacing external Cl- with SCN-, NFA increased inward current but blocked outward current suggesting that the effect of NFA is dependent on current flow. Inclusion of NFA in the pipette solution had no effect on I(Cl(Ca)). Voltage jump experiments indicated that I(Cl(Ca)) displayed characteristic outward current relaxations at +70 mV and inward current relaxations at -80 mV that were abolished by NFA. DCDPC (100 microM) produced similar effects to NFA but 1 mM DIDS produced inhibition of I(Cl(Ca)) at both positive and negative potentials and there was no increase in current on wash out of DIDS. These results suggest that NFA and DCDPC, but not DIDS, simultaneously enhance and block I(Cl(Ca)) by binding to an external site, probably close to the mouth of the chloride channel.

One-way cross-desensitization between P2X purinoceptors and vanilloid receptors in adult rat dorsal root ganglion neurones.

1. Capsaicin and ATP can activate ligand-gated cation channels in nociceptive rat dorsal root ganglion (DRG) neurones. We have studied cross-desensitization between these two agents in rat isolated DRG neurones using the whole-cell voltage-clamp technique. 2. ATP (10 microM) activated an inward current in DRG neurones at a holding potential of -60 mV. ATP evoked 'fast' responses that underwent rapid activation and desensitization, 'slow' responses that activated and desensitized more slowly, or responses that displayed a mixture of these two characteristics. The time course of the response to ATP was not related obviously to capsaicin sensitivity. 3. Prior application of capsaicin (0.5 microM) increased the proportion of cells displaying only fast responses to ATP (10 microM) suggesting that cross-desensitization had occurred between capsaicin and the slow component of the ATP response. Prior desensitization to ATP had no apparent effect on the inward current response to capsaicin (0.5 microM). 4. Cross-desensitization between capsaicin and ATP was Ca2+ dependent. 5. Changing the membrane holding potential (Vh) to +40 mV for brief period before applying ATP at -60 mV had a similar effect to capsaicin, i.e. the proportion of cells displaying only fast responses to ATP was increased significantly. This effect of depolarization was not Ca2+ dependent. 6. The heterogenity of responses to ATP is probably due to co-expression of homomeric P2X3 receptors and heteromeric receptors comprising P2X3 subunits with other P2X subunits. We propose that the change in time course of the ATP response produced by prior desensitization to capsaicin is due to selective cross-desensitization with the heteromeric P2X receptors.

A study of the voltage dependence of capsaicin-activated membrane currents in rat sensory neurones before and after acute desensitization.

1. Responses to capsaicin in isolated sensory neurones have been shown to desensitize in a Ca2+- and voltage-dependent manner. We have studied desensitization of capsaicin-activated currents in cultured adult rat dorsal root ganglion (DRG) neurones over a range of membrane potentials using whole-cell patch-clamp techniques. 2. Acute desensitization of responses to capsaicin (0.5 microM) was significantly less when the holding potential (Vh) was +40 mV rather than -60 mV. This was not due only to reduced Ca2+ entry as the response to capsaicin was desensitized by the same amount whether prior exposure to capsaicin was at -60 or +40 mV. The I-V relationship for capsaicin-induced current, determined using a voltage step protocol, was outwardly rectifying and during the acute phase of desensitization the degree of outward rectification increased. 3. Acute desensitization and the increase in outward rectification that accompanied desensitization were inhibited when cells were dialysed with the rapid Ca2+ chelator BAPTA. Addition of a pseudosubstrate inhibitor of the Ca2+-calmodulin-dependent enzyme calcineurin (CI, 100 microM) prevented the increase in outward rectification although it did not cause a significant decrease of acute desensitization. 4. Removal of external Ca2+ or Mg2+ did not reverse the increase in outward rectification of capsaicin-activated current after Ca2+-dependent desensitization had occurred. This indicates that a voltage-dependent block of the capsaicin-activated ion channel by Ca2+ or Mg2+ was not responsible for the observed changes in the properties of the capsaicin-activated conductance.

Capsazepine block of voltage-activated calcium channels in adult rat dorsal root ganglion neurones in culture.

1. We have found that capsazepine, a competitive antagonist at the vanilloid (capsaicin) receptor, blocks voltage-activated calcium currents in sensory neurones. 2. The block of calcium current was slow to develop with a half time of about one minute at 100 microM and lasted for the duration of the experiment. The rate of block of calcium current was strongly concentration-dependent. 3. The EC50 for the blocking effect at 0 mV was 7.7 +/- 1.4 microM after 6 min exposure to capsazepine. The EC50 at equilibrium was estimated to be 1.4 +/- 0.2 microM. 4. The block of calcium current showed some voltage-dependence but there was no indication of any selectivity of action for a calcium channel subtype. The characteristics of the blocking action of capsazepine on the residual current of cells which were pretreated with either omega-conotoxin or nimodipine were similar to control. 5. The data suggest that capsazepine, in addition to its competitive antagonism of vanilloid receptors, has a non-specific blocking action on voltage-activated calcium channels which should be taken into account when interpreting the effects of this substance on intact preparations in vitro or in vivo.

ATP and beta,gamma-methylene ATP produce relaxation of guinea-pig isolated trachealis muscle via actions at P1 purinoceptors.

Adenosine 5'-triphosphate (ATP), beta, gamma-methylene ATP and alpha, beta-methylene ATP produced relaxation of carbachol-precontracted isolated trachealis muscle from the guinea-pig in the presence of indomethacin (2.8 microM) and the adenosine uptake inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI; 300 nM). The potency order for ATP and analogues was: beta, gamma-methylene ATP = ATP > alpha, beta-methylene ATP = uridine 5'-triphosphate (UTP) = 2-methylthio ATP. Adenosine and 5'-N-ethylcarboxamidoadenosine (NECA) also caused relaxation. Relaxations to ATP, beta, gamma-methylene ATP, adenosine and NECA were not inhibited by the P2 purinoceptor antagonist suramin (100 microM), but were inhibited by the P1 purinoceptor antagonist 8-sulphophenyltheophylline (140 microM). NBTI significantly potentiated adenosine and ATP but not beta, gamma-methylene ATP or NECA. The data are compatible with the idea that beta, gamma-methylene ATP could interact directly with P1 purinoceptors while ATP acts indirectly at P1 purinoceptors via conversion to adenosine.

P2-purinoceptors mediating spasm of the isolated uterus of the non-pregnant guinea-pig.

1. The isolated uterus of the non-pregnant guinea-pig has been suggested to contain P1-, and possibly P2-purinoceptors mediating spasm. The presence of P1-purinoceptors has been confirmed and these receptors have been further characterized. 2. In the presence of the adenosine uptake inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI, 300 nM) and a pA100 concentration of the P1-purinoceptor antagonist 8-sulphophenyltheophylline (140 microM), the potency order of agonists as spasmogens was: 2 methylthio ATP >> alpha,beta methylene ATP = UTP = ATP >> beta,gamma methylene ATP. This order is not consistent with any single recognised P2-purinoceptor subtype. 3. Indomethacin (1 microM) treatment abolished responses to 2 methylthio ATP, alpha,beta methylene ATP and UTP, while spasm to ATP was significantly inhibited. When the endometrial and circular smooth muscle cell layers were removed, spasmogenic responses to ATP, 2 methylthio ATP, alpha,beta methylene ATP and UTP were significantly reduced. 4. 2-methylthio ATP was able to cause desensitization to itself, but not to UTP, indicating that these agonists act at different receptor sites. 5. The P2-purinoceptor antagonist, suramin antagonized 2 methylthio ATP with a PA2 of 5.9 +/- 0.3. Suramin was also an antagonist of ATP and UTP. In the case of ATP, the antagonism was not dependent on suramin concentration, while for UTP the interaction appeared to be non-equilibrium. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 microM) had no effect on spasm to ATP, UTP or 2 methythio ATP. 6. In the presence of indomethacin, responses to ATP were unaffected by 8-sulphophenyltheophylline (140 microM) or by suramin (100 microM), but PPADS (10 microM) antagonized ATP. 7. These results suggest that the isolated uterus of the non-pregnant guinea-pig contains a mixture of P2-purinoceptors. P2U- (or UTP-selective pyrimidinoceptors) and P2Y-purinoceptors appear to be present, probably mainly located on the endometrial or circular smooth muscle layer. Activation of these receptors leads to spasm via increases in prostanoid generation. There appears also to be a third class of non-P2X-, non p2Y-purinoceptor present, at which ATP is an agonist and PPADS is an antagonist, located on the longitudinal smooth muscle, activation of which causes spasm independent of changes in prostanoids.

The purinoceptors of the guinea-pig isolated taenia caeci.

The guinea-pig taenia caeci contains both P1 and P2 purinoceptors mediating relaxation. The P2 purinoceptors have been further characterized using an experimental approach designed to minimise complicating factors. In the presence of the adenosine uptake inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI, 300 nM) and a pA100 concentration of the P1 purinoceptor antagonist 8-sulphophenyltheophylline (140 microM), the potency order of agonists was: 2-methylthio-ATP >> adenosine 5'-triphosphate (ATP) = alpha, beta-methylene ATP > beta, gamma-methylene ATP >> uridine 5'-triphosphate. Suramin antagonized ATP (pA2 = 5.52 +/- 0.17, Schild plot slope = 0.67 +/- 0.08) and 2-methylthio-ATP (pA2 = 5.78 +/- 0.30, Schild plot slope = 1.37 +/- 0.39) while responses to 5'-N-ethylcarboxamidoadenosine (NECA) were unaffected. The findings suggest that suramin, while it is selective for P2 relative to P1 purinoceptors, is not a true competitive antagonist. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) antagonized ATP in isolated guinea-pig vas deferens, but had no effect on responses to ATP in guinea-pig taenia caeci indicating it is selective for P2X relative to P2Y purinoceptors.