P2Y receptor-mediated Ca2+ signalling in cultured rat aortic smooth muscle cells.

1. ATP, UTP, ADP and ADP-beta-S elicited Ca2+ -signals in cultured aortic smooth muscle cells although ADP, UDP and ADP-beta-S gave approximately 40% of the maximal response seen with ATP and UTP. Adenosine, AMP or alpha,beta-methylene-ATP had no effect. These responses were attributed to P2Y2/4 and P2Y1 receptors, which we assumed could be selectively activated by UTP and ADP-beta-S respectively. 2. The response to UTP was reduced (approximately 50%) by pertussis toxin, whilst this toxin had no effect upon the response to ADP-beta-S. This suggests P2Y2/4 receptors simultaneously couple to pertussis toxin-sensitive and -resistant G proteins whilst P2Y1 receptors couple to only the toxin-resistant proteins. 3. Repeated stimulation with UTP or ADP-beta-S caused desensitization which was potentiated by 12-O-tetradecanoyl phorbol-13-acetate (TPA) and attenuated by staurosporine. 4. TPA completely abolished sensitivity to ADP-beta-S but the response to UTP had a TPA-resistant component. In pertussis toxin-treated cells, however, TPA could completely abolish sensitivity to UTP and so the TPA-resistant part of this response seems to be mediated by pertussis toxin-sensitive G proteins. 5. Loss of sensitivity to UTP did not occur when pertussis toxin-treated cells were repeatedly stimulated with this nucleotide, suggesting that pertussis toxin-sensitive G proteins mediate this effect. The toxin did not, however affect desensitization to ADP-beta-S.

Nucleotide-evoked calcium signals and anion secretion in equine cultured epithelia that express apical P2Y2 receptors and pyrimidine nucleotide receptors.

1. Experiments with a spontaneously transformed equine epithelial cell line showed that certain nucleotides increased intracellular free calcium ([Ca2+]i) in cells plated on glass coverslips. The rank order of potency was ATP UTP > 5-Br-UTP, whilst UDP and ADP were ineffective. The response thus appears to be mediated by P2Y2 receptors. 2. Nucleotides also increased short circuit current (Isc) in cells grown into epithelial monolayers and the rank order of potency was UDP> UTP > 5-Br-UTP > ATP > ADP. The increase in [Ca2+]i and the rise in ISC thus have different pharmacological properties. Cross-desensitization experiments indicated that, as well as P2Y2 receptors, the monolayer cultures express at least one additional receptor population that allowed nucleotides to increase ISC. 3. The UDP-evoked increase in ISC was essentially abolished in BAPTA-loaded epithelia suggesting that this response is dependent upon increased [Ca2+]i. Moreover, experiments in which ISC and [Ca2+]i were measured simultaneously showed that the UDP- and ADP-evoked increases in ISC were accompanied by increases in [Ca2+]i. 4. When grown under conditions which favour the development of a polarized phenotype, these epithelial cells thus appear to express [Ca2+]i-mobilizing receptors sensitive to UDP and ADP that are not present in non-polarized cells on coverslips.

Predicting small molecule fluorescent probe localization in living cells using QSAR modeling. 1. Overview and models for probes of structure, properties and function in single cells.

Small molecule fluorochromes (synonyms: biosensors, chemosensors, fluorescent probes, vital stains) are widely used to investigate the structure, composition, physicochemical properties and biological functions of living cells, tissues and organisms. Selective entry and accumulation within particular cells and cellular structures are key processes for achieving these diverse objectives. Despite the complexities, probes routinely are applied using standard protocols, often without experimenter awareness of what factors that control accumulation and localization. The mechanisms of many such selective accumulations, however, now are known. Moreover, the influence of physicochemical properties of probes on their uptake and localization often can be defined numerically, hence predicted, using quantitative structure activity relations (QSAR) models with its required numerical structure parameters (or "descriptors"). The state of the art of this approach is described. Available QSAR models are summarized for uptake into cells and localization in the cytosol, endoplasmic reticulum, generic biomembranes, Golgi apparatus, lipid droplets, lysosomes/endosomes, mitochondria, eukaryotic nuclei (histones and DNA), plasma membrane, and ribosomal RNA (cytoplasmic and nucleolar). Integration of such core models to both aid understanding and troubleshooting of current fluorescent probes and to assist the design of novel probes is outlined and illustrated using case examples. Limitations and generic problems arising with this approach and comments on application of such approaches to xenobiotics other than probes, e.g., drugs and herbicides, together with a brief note about an alternative approach to prediction, are given.

The role of GPCR dimerisation/oligomerisation in receptor signalling.

A wide range of techniques have been employed to examine the quaternary structure of G-protein-coupled receptors (GPCRs). Although it is well established that homo-dimerisation is common, recent studies have sought to explore the physical basis of these interactions and the role of dimerisation in signal transduction. Growing evidence hints at the existence of higher-order organisation of individual GPCRs and the potential for hetero-dimerisation between pairs of co-expressed GPCRs. Here we consider how both homo-dimerisation/oligomerisation and hetero-dimerisation can regulate signal transduction through GPCRs and the potential consequences of this for function of therapeutic medicines that target GPCRs. Hetero-dimerisation is not the sole means by which co-expressed GPCRs may regulate the function of one another. Heterologous desensitisation may be at least as important and we also consider if this can be the basis for physiological antagonism between pairs of co-expressed GPCRs. Although there may be exceptions (Meyer et al. 2006), a great deal of recent evidence has indicated that most G-protein-coupled receptors (GPCRs) do not exist as monomers but rather as dimers or, potentially, within higher-order oligomers (Milligan 2004b; Park et al. 2004). Support for such models has been provided by a range of studies employing different approaches, including co-immunoprecipitation of differentially epitope-tagged but co-expressed forms of the same GPCR, co-operativity in ligand binding and a variety of resonance energy transfer techniques (Milligan and Bouvier 2005). Only for the photon receptor rhodopsin has the organisational structure of a GPCR been studied in situ. The application of atomic force microscopy to murine rod outer segment discs indicated that rhodopsin is organised in a series of parallel arrays of dimers (Liang et al. 2003) and based on this, molecular models were constructed to try to define and interpret regions of contact between the monomers (Fotiadis et al. 2004). Only for relatively few other GPCRs are details of the molecular basis of dimerisation available but within this limited data set, recent studies on the dopamine D2 receptor suggest a means by which information on the binding of an agonist can be transmitted between the two elements of the dimer via the dimer interface (Guo et al. 2005). Although the availability of cDNAs encoding molecularly defined GPCRs has allowed high-throughput screening for ligands that modulate GPCR function, this is performed almost exclusively in heterologous cell lines transfected to express only the specific GPCR of interest. Given that the human genome contains some 400-450 genes encoding non-chemosensory GPCRs, it is clear that any individual cell of the body may express a considerable number of GPCRs. Interactions between these, either via hetero-dimerisation, via heterologous desensitisation or via the integration of downstream signals can potentially alter the pharmacology, sensitivity and function of receptor agonists and hence produce varied responses. In this article, we will use specific examples to consider the role of homo-dimerisation/oligomerisation in GPCR function and whether either direct hetero-dimerisation or heterologous desensitisation between pairs of co-expressed GPCRs affects the function of the receptor pairs.

The effect of a phorbol ester upon the cholinergic regulation of potassium permeability in the rat submandibular gland.

Acetylcholine releases calcium from cytoplasmic stores and permits an influx of calcium in salivary acinar cells. The resultant rise in [Ca2+]i causes an increase in potassium permeability which is an important part of the secretory response. We have investigated the effects of 12-O-tetradecanoyl phorbol-13-acetate, a potent activator of protein kinase C, upon this regulation of potassium permeability in superfused pieces of rat submandibular salivary gland. This compound inhibited the initial [Ca2+]O-independent component of the response of acetylcholine but had no effect upon the subsequent [Ca2+]O-dependent phase. This compound does not, therefore, appear to inhibit receptor-regulated calcium influx.

Quantitative imaging in live human cells reveals intracellular alpha(1)-adrenoceptor ligand-binding sites.

Cellular distribution and binding characteristics of native alpha(1)-adrenoceptors (ARs) were determined in a live, single, human smooth muscle cell (SMC) with confocal laser scanning microscopy and a fluorescent ligand, BODIPY-FL prazosin (QAPB). This allowed single-cell competitive ligand binding and showed that 40% of alpha(1)-AR-binding sites in native cells are intracellular. QAPB had high affinity and acted as a nonselective, competitive antagonist versus [(3)H]prazosin at cloned human alpha(1a)-, alpha(1b)-, and alpha(1d)-AR subtypes on membrane preparations and whole cells. RS100329 had 70-fold selectivity for alpha(1a)-ARs versus alpha(1b)- and alpha(1d)-ARs, validating its use to identify this subtype. In similar cells QAPB-associated fluorescence provided quantitative data analogous and comparable to [(3)H]prazosin binding in whole cells. In human, dissociated, prostatic smooth muscle cells QAPB-associated fluorescence binding exhibited specific high-affinity binding properties (FK(D) = 0.63 +/- 0.02 nM), which was 3- to 4-fold higher compared with recombinant cells (FK(D) = 2. 1-2.3 nM). Internal consistency in the data showed that affinity is greater, in general, in membrane preparations than in cells but also greater in the native prostatic tissues or cells than in equivalent recombinant receptors. Fluorescence revealed binding sites both on the plasmalemmal membrane and on intracellular compartments: at all locations RS100329 inhibited QAPB binding identifying the sites as alpha(1A)-ARs. Quantitative three-dimensional mapping of QAPB-associated fluorescence binding in native human cells showed that 40% of high-affinity-binding sites was in intracellular compartments. This provides a potential new site for physiological agonism and makes intracellular access a potential differentiator of drug action.

The effect of removing external sodium upon the cholinergic regulation of potassium permeability in the rat submandibular gland in vitro.

Fragments of rat submandibular gland were loaded with 86Rb+ and superfused so that the rate of 86Rb(+)-efflux could be quantified as an indicator of potassium permeability. Acetylcholine evoked an increase in permeability consisting of a transient, calcium-independent response and a sustained, calcium-dependent. Total removal of external sodium significantly inhibited both phases of this response. The results thus confirm that the cholinergic regulation of potassium permeability is compromised by removal of external sodium but do not support the view that this is due, exclusively, to an effect on calcium influx.

Amiloride impairs the cholinergic regulation of potassium permeability in the human sweat gland but not in the rat submandibular gland.

Potassium permeability was monitored in human sweat glands and rat submandibular glands. Acetylcholine increased permeability in both tissues and the responses consisted of transient, calcium-independent and sustained, calcium-dependent components. Amiloride, a drug which inhibits Na(+)-H+ countertransport, impaired the regulation of potassium permeability in sweat glands but not in the submandibular gland. It is suggested that the stimulus-permeability coupling process in the sweat gland may be sensitive to the lowering of internal pH.

Sr2+ can become incorporated into an agonist-sensitive, cytoplasmic Ca2+ store in a cell line derived from the equine sweat gland epithelium.

We have explored the properties of a Ca(2+)-dependent cell-signalling pathway that becomes active when cultured equine sweat gland cells are stimulated with ATP. The ATP-regulated, Ca(2+)-influx pathway allowed Sr2+ to enter the cytoplasm but permitted only a minimal influx of Ba2+. Experiments in which cells were repeatedly stimulated with ATP suggested that Sr2+, but not Ba2+, could become incorporated into the agonist-sensitive, cytoplasmic Ca2+ store. Further evidence for this was provided by experiments using ionomycin, a Ca2+ ionophore which has no affinity for Sr2+.

Potassium (86Rb+) efflux from the rat submandibular gland under sodium-free conditions in vitro.

1. Fragments of rat submandibular gland were pre-loaded with 86Rb+, an isotopic marker of potassium transport, and rate constants for 86Rb+ efflux were determined during superfusion with a physiological salt solution. 2. In sodium-containing solutions acetylcholine evoked a rapid and immediate increase in efflux rate. After reaching a peak value, the efflux rate initially declined rapidly, but a second, slowly declining phase to the response was also evident. The response could be resolved into Ca2(+)-independent and Ca2(+)-dependent phases. 3. The basal efflux rate was elevated during superfusion with solutions in which sodium had been replaced with either lithium or N-methyl-D-glucammonium (NMDG+). Although lithium had a greater effect, which was absent under calcium-free conditions, addition of calcium to initially calcium-free, lithium-containing solutions did not affect the rate of efflux. 4. In the presence of calcium the response to acetylcholine was augmented during exposure to lithium-containing, sodium-free solutions but, in contrast, slightly inhibited when NMDG+ was used as a sodium substituent. 5. The transient, calcium-independent component of the response to acetylcholine was unaffected by exposure to lithium, whereas the calcium-dependent phase of the response was inhibited. 6. Responsiveness to acetylcholine was reduced during superfusion with a calcium-free, NMDG+-containing solution. The response normally observed when extracellular Ca2+ was subsequently elevated, in the continued presence of acetylcholine, was also inhibited. Sensitivity to acetylcholine was retained, however, when the tissue was initially exposed to a solution containing approximately 20 mumol l-1 Ca2+. The response was smaller than that evoked in sodium-containing solutions. 7. The use of lithium as a sodium substituent presents special problems, possibly related to the effects of this ion on the metabolic cycling of phosphatidylinositol-4,5-bisphosphate metabolites.

The role of potassium and other ions in the control of aldosterone synthesis.

Fast and slow K+ efflux components, independently regulated by angiotensin II (AII), have been identified in bovine adrenocortical cells. We have further investigated the role of potassium in the control of aldosterone synthesis in two ways. Firstly, isotopic tracers, in conjunction with channel modulators, have been used to study the interrelationship of K+ and Ca2+ in the control of AII-stimulated aldosterone synthesis. Secondly, electron probe X-ray microanalysis (EPXMA) was used to quantify potassium, sodium, chlorine and phosphorous in control and AII-stimulated cells. The effects of verapamil on 43K efflux were measured at two stages during AII stimulation. During the first ten minutes of treatment, when efflux via the fast component predominates, AII and verapamil both slowed efflux and their effects were additive. If verapamil was added later, at the time when efflux by the fast component appeared exhausted and the stimulatory effect of AII on the slow efflux component was apparent, it again slowed efflux. These data suggest that verapamil prevents calcium-gated K+ channels from opening by blocking Ca2+ channels. However, verapamil had no effect on AII-stimulated calcium efflux. In addition to blocking Ca2+ channels, verapamil may directly inhibit potassium efflux. EPXMA showed a bimodal distribution of potassium concentrations in control cells. However, in cells stimulated with AII for five minutes, the mean potassium content was less than in controls and was not bimodally distributed. Sodium content was increased by AII-treatment, chlorine was lowered and phosphorus remained unchanged. The data confirm previous observations that AII inhibits Na+/K+ ATPase activity.

Calcium-dependent regulation of membrane ion permeability in a cell line derived from the equine sweat gland epithelium.

We measured the rates of 125I- and 86Rb+ efflux from preloaded, cultured equine sweat gland cells. The calcium ionophore ionomycin increased the efflux of both isotopes. Anion efflux was unaffected by Ba2+, but this cation inhibited 86Rb(+)-efflux, suggesting that [Ca2+]i-activated potassium channels were present. Activation of these channels was not, however, important for the efflux of anions. We measured 125I- efflux from valinomycin-depolarised cells in which anion cotransport was inhibited. Changes in 125I- efflux reflect changes in anion permeability under these conditions, and ionomycin caused a clear permeability increase that was abolished by the anion channel blocker diphenylamine-2-carboxylate. ATP and UTP increased the efflux of both isotopes, suggesting that type P2U purine receptors allow these nucleotides to regulate membrane permeability.

Localisation of calcium ions and calcium-ATPase activity within myelinated nerve fibres of the adult guinea-pig optic nerve.

There is no published description of the distribution of free Ca2+, nor of the distribution of Ca(2+)-ATPase activity associated with the maintenance of low axoplasmic Ca2+ concentrations, in normal central myelinated nerve fibres. We have used the oxalate-pyroantimonate technique to localise free Ca2+, together with the lead-citrate technique to localise Ca(2+)-ATPase activity within myelinated fibres from the adult guinea-pig optic nerve. Pyroantimonate precipitate occurred within the axoplasm at nodes of Ranvier and the internode, at areas of myelin disruption, within Schmidt-Lanterman incisures (SLI) and glial paranodal loops. But precipitate was absent from the axoplasm beneath SLI and at the paranode. Ca(2+)-ATPase activity was localised in axonal smooth endoplasmic reticulum (SER), the outer membrane of mitochondria, the nodal axolemma, the glial membranes of the paranodal loops, the SLI and the external aspect of the myelin sheath. We have demonstrated large domains within the axons of CNS fibres where calcium is present or absent. Moreover, we have shown that, where calcium is absent, there is localisation of Ca(2+)-ATPase activity, which would serve to remove calcium from the adjacent axoplasm. Our results are compared with information obtained from PNS fibres and some differences of distribution discussed.

Single-cell recombinant pharmacology: bovine alpha(1a)-adrenoceptors in rat-1 fibroblasts release intracellular ca(2+), display subtype-characteristic agonism and antagonism, and exhibit an antagonist-reversible inverse concentration-response phase.

Phe-activated Ca(2+) signals recorded from single rat-1 fibroblasts stably expressing the bovine alpha(1a)-adrenoceptor (AR) were characterized and used to analyze functional agonist-antagonist interactions. The response to Phe was initiated by the mobilization of stored Ca(2+) and subsequently sustained by receptor-regulated Ca(2+) influx. The selective alpha(1A)-AR agonist (R)-A-61603 was 141-fold more potent as an agonist than Phe. This potency ratio was consistent with the pharmacology of the native alpha(1A)-ARs. Functional responses evoked by concentrations of Phe of more than 0. 3 microM displayed fade, which could be explained by agonist-dependent depletion of Ca(2+) stores. The antagonists tested did not conform to the predictions of the Schild equation for competitive antagonism as expected from the nonequilibrium nature of the response. The antagonist potency series WB4101 > or = prazosin >> BMY7378, however, was consistent with alpha(1A)-ARs. Antagonism exhibited by WB4101 and prazosin was compatible with a model in which antagonists dissociate so slowly from the receptor that this is a major factor in their inhibition of the transient agonist-mediated response, leading to the appearance of insurmountable antagonism. A consequence of this phenomenon was that an inverse concentration-response relationship at high agonist concentrations was abolished by low concentrations of antagonists. Overall, the results indicate that quantitative pharmacology can be studied successfully in single cells even though equilibrium could not be achieved in the agonist-antagonist-response relationship in this particular cell phenotype. The study also showed a form of fade that could be readily explained.

Importance of agonists in alpha-adrenoceptor classification and localisation of alpha1-adrenoceptors in human prostate.

alpha-Adrenoceptor blocker drugs are commonly used in the clinical (non-surgical) treatment of BPH. alpha1-adrenoceptors were originally sub-divided using agonists but, subsequently, were sub-divided using only antagonists in ligand-ligand interactions, which did not require agonists at all. Ultimately, proof that adrenoceptors are functional receptors for the natural ligands, noradrenaline and adrenaline, requires that agonists be used. The earlier excitement engendered by finding varying agonist potency series in different tissues has not been revisited to place it in the context of current concepts of alpha1-adrenoceptor subtypes. This review will consider the advantages and limitations of different agonists for the study of alpha1-adrenoceptor subtypes including 'extreme' examples where the archetypal alpha1-adrenoceptor agonist phenylephrine activates alpha2-adrenoceptors and others where UK14304, often the alpha2-adrenoceptor agonist of choice, activates alpha1-adrenoceptors. New work will also be presented showing the interaction between agonists and the fluorescent alpha1-adrenoceptor antagonist QAPB. This introduces the novel point of view of studying the displacement of antagonists by agonists. Possible errors in antagonist classification arising from complexity in the actions of agonists and the recently developed method of fluorescent ligand binding on isolated living human prostatic smooth muscle cells will be discussed.

The effects of removing external sodium upon the control of potassium (86Rb+) permeability in the isolated human sweat gland.

The changes in cytoplasmic free calcium ([Ca2+]i) which occur in isolated human sweat glands during cholinergic stimulation have been studied indirectly by monitoring potassium permeability. The acetylcholine-evoked permeability increase normally consists of transient and sustained phases which are attributed to the mobilization of intracellular calcium stores and to calcium influx respectively. Such consistent responses to acetylcholine could not be obtained during superfusion with bicarbonate-free, HEPES-buffered solutions. The human sweat gland in vitro therefore appears to have a strict requirement for bicarbonate. The sustained component of the response was not affected by total removal of external sodium, suggesting that calcium influx does not occur via a sodium-dependent system. The transient component, however, was abolished when external sodium was replaced by N-methyl-D-glucammonium (NMDG+). It therefore appears that secretagogue-evoked mobilization of cytoplasmic calcium is dependent, in some way, upon external sodium. This dependence is not, however, absolute as the response was essentially normal when sodium was replaced by lithium.

The regulation of membrane 125I- and 86Rb+ permeability in a virally transformed cell line (NCL-SG3) derived from the human sweat gland epithelium.

We have explored the factors that may regulate membrane permeability in a cell line (NCL-SG3) derived from the human sweat gland epithelium. Ionomycin increased the rate of 125I-efflux from preloaded cells and this action appeared to be due to an increase in intracellular free calcium ([Ca2+]i). The ionomycin-evoked increase in 125I- efflux was reduced in cells that were exposed either to barium or to valinomycin in the presence of a high concentration of external potassium. It thus appears that a fraction of the ionomycin-evoked increase in 125I- efflux is due to the activation of potassium channels and experiments using 86Rb+ also suggested that ionomycin increased the rate of potassium efflux, an effect which was totally abolished by barium. Blockade of Na(+)-K(+)-2Cl- cotransport and of Cl- -HCO3- exchange reduced the basal rate of 125I- efflux and the ionomycin-evoked increase in 125I-efflux from control cells and from cells depolarized by valinomycin. These transport systems thus contribute to anion efflux, although [Ca2+]i-dependent chloride channels also appear to be present. Acetylcholine increases [Ca2+]i in the secretory cells of human sweat glands, but this neurotransmitter did not increase [Ca2+]i in NCL-SG3 cells and so membrane permeability was not under cholinergic control. Adrenaline did not increase [Ca2+]i, but this hormone did evoke cyclic-3',5'-adenosine monophosphate (cyclic AMP) production. However, membrane permeability was not under adrenergic control, as the cells did not appear to express functional, cyclic AMP-dependent anion channels. This may be because they were not fully differentiated under the culture conditions. ATP consistently evoked a dose-dependent increase in anion efflux that appeared to be mediated by [Ca2+]i. The increase in [Ca2+]i was initiated by the release of calcium from a limited internal store and was subsequently sustained by calcium influx. UTP and ADP also increased [Ca2+]i, whereas adenosine, AMP and alpha,beta-methylene ATP were without effect. These data thus suggest that a subclass of type 2 purine receptor, which is functionally coupled to phosphoinositidase C, is present in these cells.

The cholinergic regulation of potassium (86Rb+) permeability in sweat glands isolated from patients with cystic fibrosis.

Sweat glands isolated from skin obtained from normal subjects and patients with cystic fibrosis (CF) were pre-loaded with 86Rb+ and superfused with a physiological salt solution and the rate of 86Rb+ efflux was measured as an indicator of cellular potassium permeability. Acetylcholine always evoked a permeability increase in the glands from control subjects and this response could be resolved into calcium-dependent and calcium-independent components. Sweat glands from CF patients did not show such consistent responses. In three individuals the glands were abnormally insensitive to acetylcholine but normal responsiveness was seen in a fourth case. It is proposed that CF can induce dysfunction of calcium-dependent control processes in sweat glands.

Activation of apical P2U purine receptors permits inhibition of adrenaline-evoked cyclic AMP accumulation in cultured equine sweat gland epithelial cells.

Experiments were undertaken using cultured equine sweat gland epithelial cells that express purine receptors belonging to the P2U subclass which allow the selective agonist uridine triphosphate (UTP) to increase the concentration of intracellular free Ca2+ ([Ca2+]i). Experiments using pertussis toxin (Ptx), which inactivates certain guanine-nucleotide-binding proteins (G-proteins), showed that this response consisted of Ptx-sensitive and Ptx-resistant components, and immunochemical analyses of the G-protein alpha subunits present in the cells showed that both Ptx-sensitive (alpha i1-3) and Ptx-resistant (alpha q/11) G-proteins were expressed. P2U receptors may, therefore, normally activate both of these G-protein families. Ptx-sensitive, alpha i2/3 subunits permit inhibitory control of adenylate cyclase, and UTP was shown to cause Ptx-sensitive inhibition of adrenaline-evoked cyclic AMP accumulation, suggesting that the receptors activate Gi2/3. Experiments using cells grown on permeable supports suggested that P2U receptors became essentially confined to the apical membrane in post-confluent cultures. Polarised epithelia may, therefore, express apical P2U receptors which influence two centrally important signal transduction pathways. It is highly improbable that these receptors could be activated by nucleotides released from purinergic nerves, but they may be involved in the autocrine regulation of epithelial function.

Extracellular ATP can activate autonomic signal transduction pathways in cultured equine sweat gland epithelial cells.

Changes in intracellular free calcium concentration ([Ca2+]i) were monitored in a cell line that was derived from the equine sweat gland epithelium. ATP and closely related compounds could increase [Ca2+]i with a rank order of potency of UTP > or = ATP > ADP >> AMP = adenosine = alpha,beta-methylene-ATP. The responses to ATP and to UTP were initiated by the release of calcium from an internal store and subsequently sustained by calcium influx. The rise in [Ca2+]i thus seems to be mediated by P2U receptors that are coupled to phosphoinositidase C. Some desensitisation of this response developed during repeated stimulation with ATP and this was blocked by staurosporine, an inhibitor of protein kinase C, and augmented by a phorbol ester which acts as an exogenous activator of this enzyme. A protein-kinase-C-dependent inhibitory pathway thus seems to become active during repeated stimulation with ATP. ATP and related compounds could also raise cellular cyclic AMP content. The order of potency was ATP > ADP = AMP = adenosine >> UTP, suggesting that this response is mediated via a separate subclass of P2 receptor. The present results demonstrate that ATP can activate autonomic signal-transduction pathways in cultured equine sweat gland cells and suggest that there may be a purinergic component to the control of secretory activity in the equine sweat gland.