The main contributors to increases in [Ca2+]i and tension are the entry of Ca2+ through voltage-dependent channels opened by depolarization or during action potential (AP) or slow-wave discharge, and Ca2+ release from store sites in the cell by the action of IP3 or by Ca(2+)-induced Ca(2+)-release (CICR). The entry of Ca2+ during an AP triggers CICR from up to 20 or more subplasmalemmal store sites (seen as hot spots, using fluorescent indicators); Ca2+ waves then spread from these hot spots, which results in a rise in [Ca2+]i throughout the cell. Spontaneous transient releases of store Ca2+, previously detected as spontaneous transient outward currents (STOCs), are seen as sparks when fluorescent indicators are used. Sparks occur at certain preferred locations--frequent discharge sites (FDSs)--and these and hot spots may represent aggregations of sarcoplasmic reticulum scattered throughout the cytoplasm. Activation of receptors for excitatory signal molecules generally depolarizes the cell while it increases the production of IP3 (causing calcium store release) and diacylglycerols (which activate protein kinases). Activation of receptors for inhibitory signal molecules increases the activity of protein kinases through increases in cAMP or cGMP and often hyperpolarizes the cell. Other receptors link to tyrosine kinases, which trigger signal cascades interacting with trimeric G-protein systems.

Adenosine and its analogues are potent inhibitors of synaptic activity in the central and peripheral nervous system. In the central nervous system (CNS), this appears to arise primarily by inhibition of presynaptic release of transmitters, including glutamate, which is possibly the major excitatory transmitter in the brain. In addition, postsynaptic effects of adenosine have been reported which would also serve to reduce neurotransmission. The mechanism by which adenosine inhibits CNS neurotransmission is unknown, although it appears to exert its effect via an A1 receptor which in some systems is negatively coupled to adenylate cyclase. In an attempt to elucidate the mechanism of inhibition, we have examined the effect of pertussis toxin (PTX) on the ability of the stable adenosine analogue (-)phenylisopropyladenosine (PIA) to inhibit glutamate release from cerebellar neurones maintained in primary culture. PTX, by ADP-ribosylating the nucleotide-binding protein Ni, prevents coupling of inhibitory receptors such as the A1 receptor to adenylate cyclase. As reported here, we found that PTX, as well as preventing inhibition of adenylate cyclase by PIA, also converts the PIA-induced inhibition of glutamate release to a stimulation. Our results suggest strongly that purinergic inhibitory modulation of transmitter release occurs by inhibition of adenylate cyclase.

1 The effects on the whole-cell carbachol-induced muscarinic cationic current (mIcat) of antibodies against the a-subunits of various G proteins, as well as the effect of a Gbg subunit, were studied in single guinea-pig ileal smooth muscle cells voltage-clamped at À50 mV. Ionized intracellular calcium concentration, [Ca 2+ ] i , was clamped at 100 nM using a 1,2-bis(2-aminophenoxyl-ethane-N,N,N 0 ,N 0 -tetraacetic acid)/Ca 2+ mixture. 2 Application of ascending concentrations of carbachol (1 -300 mM) activated mIcat (mean amplitude 0.83 nA at 300 mM carbachol; EC 50 8 mM; Hill slope 1.0). A 20 min or longer intracellular application via the pipette solution of G i3 /G o or G o antibodies resulted in about a 70% depression of the maximum response without change in the EC 50 value. In contrast, antibodies against a-subunits of G i1 , G i1 /G i2 , G i3 , G q /G 11 or G s protein over a similar or longer period did not significantly reduce mIcat. Antibodies to common Gb or infusion of the Gbg subunit itself had no effect on mIcat.3 If cells were exposed briefly to carbachol (50 or 100 mM) at early times (o3 min) after infusion of antibodies to Ga i3 /Ga o or to Ga o had begun, carbachol responses remained unchanged even after 20 -60 min; that is, the depression of mIcat by these antibodies was prevented. 4 These data show that Ga o protein couples the muscarinic receptor to the cationic channel in guinea-pig ileal longitudinal smooth muscle and that Gbg is not involved. They also show that prior activation of the muscarinic receptor presumably causes a long-lasting postactivation change of the G protein, which is not reflected in mIcat, but acts to hinder antibody binding.

Previously we have described a constitutively active, Ca2+ -permeable, non-selective cation channel in freshly dispersed rabbit ear artery myocytes which has similar properties to some of the canonical transient receptor potential (TRPC) channel proteins. In the present work we have compared the properties of constitutive channel activity with known properties of TRPC proteins by investigating the effect of selective anti-TRPC antibodies and pharmacological agents on whole-cell and single cation channel activity. Bath application of anti-TRPC3 antibodies markedly reduced channel activity in inside-out patches and also produced a pronounced reduction of both current amplitude and variance of constitutively active whole-cell cation currents whereas anti-TRPC1/4/5/6/7 antibodies had no effect on channel activity. In the presence of antigenic peptide, anti-TRPC3 antibodies had no effect on whole-cell or single cation channel activity. Bath application of flufenamic acid, Gd 3+ , La 3+ and Ca 2+ inhibited spontaneous channel activity in outside-out patches with IC 50 values of 6.8 μM, 25 nM, 1.5 μM and 0.124 mM, respectively, which are similar values to those against TRPC3 proteins. Immunocytochemical studies combined with confocal microscopy showed expression of TRPC3 proteins in ear artery myocytes, and these were predominately distributed at, or close to, the plasma membrane. These data provide strong evidence that native constitutively active cation channels in rabbit ear artery myocytes have similar properties to TRPC3 channel proteins and indicate that these proteins may have an important role in mediating this conductance.

A voltage-gated Na + current was characterised in freshly dissociated mouse portal vein (PV) smooth muscle myocytes. The current was found superimposed upon the relatively slow L-type Ca 2+ current and was resistant to conventional Ca 2+ channel blockers but was abolished by external Na + replacement and tetrodotoxin (TTX, 1 µM). The molecular identity of the channel responsible for this conductance was determined by RT-PCR where only the transcripts for Na + channel genes SCN7a, 8a and 9a were detected. The presence of the protein counterparts to the SCN8a and 9a genes (NaV 1.6 and NaV 1.7 , respectively) on the individual smooth muscle myocytes were confirmed in immunocytochemistry, which showed diffuse staining around a predominantly plasmalemmal location. TTX inhibited the action potential in individual myocytes generated in the current clamp mode but isometric tissue tension experiments revealed that TTX (1 and 5 µM) had no effect on the inherent mouse PV rhythmicity. However, the Na + channel opener veratridine (10 and 50 µM) significantly increased the length of contraction and the interval between contractions. This effect was not influenced by pre-incubation with atropine, prazosin and propranolol, but was reversed by TTX (1 µM) and completely abolished by nicardipine (1 µM). Furthermore, preincubation with the reverse-mode Na + -Ca 2+ exchange blocker KB-R7943 (10 µM) also inhibited the veratridine response. We have established for the first time the molecular identity of the voltage-gated Na + channel in freshly dispersed smooth muscle cells and have shown that these channels can modulate contractility through a novel mechanism of action possibly involving reverse mode Na + -Ca 2+ exchange.

We have previously shown a constitutively active and noradrenaline‐evoked Ca2+‐permeable cation channel in rabbit ear artery myocytes which is proposed to have an important role is setting resting membrane conductance and increasing excitability of vascular tissue. In the present work we compared the properties of these native constitutively active cation channels with known properties of canonical transient receptor potential (TRPC) cation channel proteins at the single channel level using freshly dispersed rabbit ear artery myocytes and patch clamp techniques. Bath application of two anti‐TRPC3 antibodies raised against distinct epitopes on the TRPC3 channel protein markedly reduced constitutive channel activity in inside‐out patches whereas antibodies raised TRPC1,4,5,6 and 7 channel proteins had no effect on channel activity. Moreover in control experiments bath application of anti‐TRPC3 antibodies preincubated with its antigenic peptide also had no effect on channel activity. Bath application of flufenamic acid, Gd3+, La3+ and Ca2+ inhibited spontaneous channel activity in outside‐out patches with IC50 values similar to those previous described for expressed TRPC3 channel proteins in cell lines. In addition immunocytochemical studies combined with confocal microscopy showed expression of TRPC3 channel proteins at, or close to, the plasma membrane. These data provide strong evidence that the native constitutively active cation channels in rabbit ear artery myocytes have similar properties to TRPC3 cation channel proteins and indicate that these proteins may have an important role in mediating this conductance.

It is now established that non-contractile cells with thin filopodia, also called vascular interstitial cells (VICs), are constitutively present in the media of many, if not all, blood vessels. The aim of this study was to determine the type of cell lineage to which arterial VICs belong using immunocytochemical, and real-time and reverse transcription PCR (RT-PCR). Using RT-PCR, we compared gene expression profiles of single VICs and smooth muscle cells (SMCs) freshly dispersed from rat middle cerebral artery. Both VICs and SMCs expressed the SMC marker, smooth muscle myosin heavy chain (SM-MHC), but did not express fibroblast, pericyte, neuronal, mast cell, endothelial or stem cell markers. Freshly isolated VICs also did not express c-kit, which is the marker for interstitial cells of Cajal in the gastrointestinal tract. Immunocytochemical labelling of contractile proteins showed that VICs and SMCs expressed SM-MHC similarly to the same degree, but VICs in contrast to SMCs had decreased expression of α-SM-actin and very low or no expression of calponin. Real-time RT-PCR was consistent with immunocytochemical experiments and showed that VICs had four times lower gene expression of calponin comparing to SMCs, which may explain VICs’ inability to contract. VICs had greater expression than SMCs of structural proteins such as non-muscular β-actin and desmin. The results obtained suggest that VICs represent a subtype of SMCs and may originate from the same precursor as SMCs, but later develop filopodia and a non-contractile cell phenotype.

1 The abilities of muscarinic agonists (arecoline, bethanechol, carbachol, methacholine, pilocarpine) to inhibit isoprenaline-induced cyclic AMP production in chopped fragments (via M 2 receptors), and to evoke cationic current (I cat ) (via M 2 receptors) or calcium store release (via M3 receptors) in enzyme-dispersed, single voltage-clamped cells from longitudinal smooth muscle of the guinea-pig small intestine were examined. 2 All muscarinic agonists (1 ± 300 mM) examined inhibited isoprenaline (1 mM)-induced accumulation of cyclic AMP, the IC 50 varying from 52 to 248 mM. However, their relative potencies to evoke this M 2 eect were not signi®cantly correlated with their ability to evoke I cat , also a M 2 eect, whether or not calcium stores were depleted; pilocarpine and McN-A343 inhibited the I cat response to carbachol. 3 Muscarinic agonists (concentration 300 or 1000 mM), except pilocarpine and McN-A343 which were ineective, evoked Ca 2+ -activated K + current (I K-Ca ) resulting from Ca 2+ store release (M 3 eect). Their eectiveness was tested by estimating residual stored calcium by subsequent application of caeine (10 mM). The relative potencies to evoke Ca 2+ store release (M 3 ) and for I cat activation (M 2 ) were closely correlated (P50.001). 4 These data might be explained if M 2 -mediated adenylyl cyclase inhibition and I cat activation involve dierent G proteins, or involve dierent populations of M 2 receptors. The observed correlation of agonist potency between I cat activation and Ca 2+ store release supports the proposal (Zholos & Bolton, 1997) that M 3 activation can potentiate M 2 -cationic channel coupling through Ca 2+ -independent mechanisms.