Search

Background: The ATP-gated channel (P2X2) receptor ectodomain is formed by β-strands from three subunits. Results: ATP-induced channel opening was prevented by disulfide locking between pairs of substituted cysteines in different subunits. Conclusion: The turret region, the lower body wall, and the outer ends of transmembrane domains move significantly in channel opening. Significance: Large ectodomain movements couple ATP binding to channel opening in P2X receptors., The ectodomain of the P2X receptor is formed mainly from two- or three-stranded β-sheets provided symmetrically by each of the three subunits. These enclose a central cavity that is closed off furthest from the plasma membrane (the turret) and that joins with the transmembrane helices to form the ion permeation pathway. Comparison of closed and open crystal structures indicates that ATP binds in a pocket positioned between strands provided by different subunits and that this flexes the β-sheets of the lower body and enlarges the central cavity: this pulls apart the outer ends of the transmembrane helices and thereby opens an aperture, or gate, where they intersect within the membrane bilayer. In the present work, we examined this opening model by introducing pairs of cysteines into the rat P2X2 receptor that might form disulfide bonds within or between subunits. Receptors were expressed in human embryonic kidney cells, and disulfide formation was assessed by observing the effect of dithiothreitol on currents evoked by ATP. Substitutions in the turret (P90C, P89C/S97C), body wall (S65C/S190C, S65C/D315C) and the transmembrane domains (V48C/I328C, V51C/I328C, S54C/I328C) strongly inhibited ATP-evoked currents prior to reduction with dithiothreitol. Western blotting showed that these channels also formed predominately as dimers and/or trimers rather than monomers. The results strongly support the channel opening mechanism proposed on the basis of available crystal structures.

Hans Walter Kosterlitz will be remembered as the winner of an international race to identify the first endogenous opioid, enkephalin. He came to the UK from Berlin in 1934, one of the many scientific émigrés of that period who later so enriched biochemistry, physiology and pharmacology. His entire career was spent at the University of Aberdeen, where he developed a reputation first in carbohydrate metabolism and later in the pharmacology of opiates. His strong experimental skills, and particularly the use of bioassays on tissues from laboratory animals, allowed him to predict the effectiveness and abuse potential of opiate drugs. Most notably, this led to the isolation of the peptide hormone enkephalin, the first of a group of such peptides now often known as endorphins. This work was done in his seventies, after his official retirement, with an energy and enthusiasm that could leave younger colleagues physically tired but intellectually stimulated.

Extracellular adenosine 5′ triphosphate (ATP) is a widespread cell-to-cell signaling molecule in the brain, where it activates cell surface P2X and P2Y receptors. P2X receptors define a protein family unlike other neurotransmitter-gated ion channels in terms of sequence, subunit topology, assembly, and architecture. Within milliseconds of binding ATP, they catalyze the opening of a cation-selective pore. However, recent data show that P2X receptors often underlie neuromodulatory responses on slower time scales of seconds or longer. Herein, we review these findings at molecular, cellular and systems levels. We propose that, while P2X receptors are fast ligand-gated cation channels, they are most adept at mediating slow neuromodulatory functions that are more widespread and more physiologically utilized than fast ATP synaptic transmission in the CNS.

P2X receptors are trimeric membrane proteins. When they bind extracellular ATP, a conformational change occurs that opens a transmembrane ion channel. The ATP-binding pocket is formed in a cleft between two subunits, and a critical amino acid residue for ATP contact is Lys⁶⁹ (P2X2 numbering). In the present work, we sought to determine whether the binding of fewer than three ATP molecules could open the ion channel. We expressed eight concatenated cDNAs in human embryonic kidney cells, which encoded three serially joined, epitope-tagged, subunits with either Lys or Ala at position 69 (denoted as KKK, KKA, KAK, AKK, KAA, AKA, AAK, and AAA). Western blotting of surface-biotinylated proteins indicated that breakdown of concatemers to individual subunits was minimal. Recording of membrane currents in response to ATP (whole cell and excised outside-out patch) showed that all formed functional channels except AAK, AKA, and AAA. There was no difference in the kinetics of activation and deactivation among KKK, KKA, KAK, and AKK channels, and amplitude of the unitary conductances was in all cases not different from that found after expression of a single wild-type subunit. Currents through KKA and KAK receptors were larger than those observed for AKK receptors. The results indicate that trimeric P2X receptors containing only two intact binding sites can be readily activated by ATP.

P2X(1) receptors belong to a family of cation channels gated by extracellular ATP; they are found inter alia in smooth muscle, platelets, and immune cells. Suramin has been widely used as an antagonist at P2X receptors, and its analog 4,4',4'',4'''-[carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino))] tetrakis-benzene-1,3-disulfonic acid (NF449) is selective for the P2X(1) subtype. Human and mouse P2X(1) receptors were expressed in human embryonic kidney cells, and membrane currents evoked by ATP were recorded. ATP (10 nm to 100 microm) was applied only once to each cell, to avoid the profound desensitization exhibited by P2X(1) receptors. Suramin (10 microm) and NF449 (3-300 nM) effectively blocked the human receptor. Suramin had little effect on the mouse receptor. Suramin and NF449 are polysulfonates, with six and eight negative charges, respectively. We hypothesized that species differences might result from differences in positive residues presented by the large receptor ectodomain. Four lysines in the human sequence (Lys(111), Lys(127), Lys(138), and Lys(148)) were changed individually and together to their counterparts in the mouse sequence. The substitution K138E, either alone or together with K111Q, K127Q, and K148N, reduced the sensitivity to block by both suramin and NF449. Conversely, when lysine was introduced into the mouse receptor, the sensitivity to block by suramin and NF449 was much increased for E138K, but not for Q111K, Q127K, or N148K. The results explain the marked species difference in antagonist sensitivity and identify an ectodomain lysine residue that plays a key role in the binding of both suramin and NF449 to P2X(1) receptors.

Transient currents occur at rest in cortical neurones that reflect the quantal release of transmitters such as glutamate and γ-aminobutyric acid (GABA). We found a bimodal amplitude distribution for spontaneously occurring inward currents recorded from mouse pyramidal neurones in situ, in acutely isolated brain slices superfused with picrotoxin. Larger events were blocked by glutamate receptor (AMPA, kainate) antagonists; smaller events were partially inhibited by P2X receptor antagonists suramin and PPADS. The decay of the larger events was selectively prolonged by cyclothiazide. Stimulation of single intracortical axons elicited quantal glutamate-mediated currents and also quantal currents with amplitudes corresponding to the smaller spontaneous inward currents. It is likely that the lower amplitude spontaneous events reflect packaged ATP release. This occurs with a lower probability than that of glutamate, and evokes unitary currents about half the amplitude of those mediated through AMPA receptors. Furthermore, the packets of ATP appear to be released from vesicle in a subset of glutamate-containing terminals.

Receptor desensitization can determine the time course of transmitter action and profoundly alter sensitivity to drugs. Among P2X receptors, ion currents through homomeric P2X4 receptors exhibit intermediate desensitization when compared with P2X1 and P2X3 (much faster) and P2X2 and P2X7 (slower). We recorded membrane currents in HEK293 cells transfected to express the human P2X4 receptor. The decline in current during a 4-s application of ATP (100 microm) was about 30%; this was not different during whole-cell or perforated patch recording. Alanine-scanning mutagenesis of the intracellular C terminus identified two positions with much accelerated desensitization kinetics (Lys373: 92% and Tyr374: 74%). At position 373, substitution of Arg or Cys also strongly accelerated desensitization: however, in the case of K373C the wild-type phenotype was fully restored by adding ethylammonium methanethiosulfonate. At position 374, phenylalanine could replace tyrosine. These results indicate that wild-type desensitization properties requires an aromatic moiety at position 374 and an amino rather than a guanidino group at position 373. These residues lie between previously identified motifs involved in membrane trafficking (YXXXK and YXXGL) and implicates the C-terminal also in rearrangements leading to channel closing during the presence of agonist.

ATP released from damaged or inflamed tissues can act at P2X receptors expressed on primary afferent neurones. The resulting depolarization can initiate action potentials that are interpreted centrally as pain. P2X3 subunits are found in a subset of small-diameter, primary afferent neurones, some of which are also sensitive to capsaicin. They can form homo-oligomeric channels, or they can assemble with P2X2 subunits into hetero-oligomers. Studies with antagonists selective for P2X3-containing receptors, experiments with antisense oligonucleotides to reduce P2X3 subunit levels, and behavioural testing of P2X3 knock-out mice, all suggest a role for the P2X2/3 receptor in the signalling of chronic inflammatory pain and some features of neuropathic pain. The availability of such tools and experimental approaches promises to accelerate our understanding of the other physiological roles for P2X receptors on primary afferent neurones.

1. Heteromeric P2X2/3 receptors are much more sensitive than homomeric P2X2 receptors to alphabeta-methylene-ATP, and this ATP analogue is widely used to discriminate the two receptors on sensory neurons and other cells. 2. We sought to determine the structural basis for this selectivity by synthesising ADP and ATP analogues in which the alphabeta and/or betagamma oxygen atoms were replaced by other moieties (including -CH2-, -CHF-, -CHCl-, -CHBr-, -CF2-, -CCl2-, -CBr2-, -CHSO3-, -CHPO3-, -CFPO3-, -CClPO3-, -CH2-CH2-, C triple bond C, -NH-, -CHCOOH-). 3. We tested their actions as agonists or antagonists by whole-cell recording from human embryonic kidney cells expressing P2X2 subunits alone (homomeric P2X2 receptors), or cells expressing both P2X2 and P2X3 subunits, in which the current through heteromeric P2X2/3 receptors was isolated. 4. ADP analogues had no agonist or antagonist effect at either P2X2 or P2X2/3 receptors. All the ATP analogues tested were without agonist or antagonist activity at homomeric P2X2 receptors, except betagamma-difluoromethylene-ATP, which was a weak agonist. 5. At P2X2/3 receptors, betagamma-imido-ATP, betagamma-methylene-ATP, and betagamma-acetylene-ATP were weak agonists, whereas alphabeta,betagamma- and betagamma,gammadelta-bismethylene-AP4 were potent full agonists. betagamma-Carboxymethylene-ATP and betagamma-chlorophosphonomethylene-ATP were weak antagonists at P2X2/3 receptors (IC50 about 10 microm). 6. The results indicate (a). that the homomeric P2X2 receptor presents very stringent structural requirements with respect to its activation by ATP; (b). that the heteromeric P2X2/3 receptor is much more tolerant of alphabeta and betagamma substitution; and (c). that a P2X2/3-selective antagonist can be obtained by introduction of additional negativity at the betagamma-methylene.

ATP receptors present on rat alveolar macrophages (NR8383 cells) were identified by recordings of membrane current, measurements of intracellular calcium, RT–PCR and immunocytochemistry. In whole-cell recordings with a sodium-based internal solution, ATP evoked an inward current at −60 mV. This reversed at 0 mV. The EC50 for ATP was 18 μM in normal external solution (calcium 2 mM, magnesium 1 mM). The currents evoked by 2′,3-O-(4-benzoyl)benzoyl-ATP were about five-fold smaller than those observed with ATP. ADP, UTP and αβ-methylene-ATP (αβmeATP) (up to 100 μM) had no effect. ATP-evoked currents were potentiated up to ten-fold by ivermectin and were unaffected by suramin (30–100 μM), pyridoxal-phosphate-6-azophenyl-(2,4-sulphonic acid) (30–100 μM), and brilliant blue G (1 μM). In whole-cell recordings with a potassium-based internal solution and low EGTA (0.01 mM), ATP evoked an inward current at −60 mV that was followed by larger outward current. ADP and UTP (1–100 μM) evoked only outward currents; these reversed polarity at the potassium equilibrium potential and were blocked by apamin (10 nM). Outward currents were also blocked by the phospholipase C inhibitor U73122 (1 μM), and they were not seen with higher intracellular EGTA (10 mM). Suramin (30 μM) blocked the outward currents evoked by ATP and UTP, but not that evoked by ADP. PPADS (10 μM) blocked the ADP-evoked outward current without altering the ATP or UTP currents. RT–PCR showed transcripts for P2X subunits 1, 4 and 7 (not 2, 3, 5, 6) and P2Y receptors 1, 2, 4 and 12 (not 6). Immunocytochemistry showed strong P2X4 receptor expression partly associated with the membrane, weak P2X7 staining that was not associated with the cell membrane, and no P2X1 receptor immunoreactivity. We conclude that rat alveolar macrophages express (probably homomeric) P2X4 receptors, but find no evidence for other functional P2X subtypes. The P2Y receptors are most likely P2Y1 and P2Y2 and these couple through phospholipase C to an increase in intracellular calcium and the opening of SK type potassium channels. British Journal of Pharmacology (2003) 140, 567–575. doi:10.1038/sj.bjp.0705459

A monoclonal antibody was developed to the extracellular domain of the rat P2X4 receptor. The antibody was highly selective among all rat P2X receptor subunits, and recognised only the oligomeric, non-denatured form of the P2X4 receptor. Immunohistochemistry showed an extensive pattern of distribution throughout the central and peripheral nervous systems, the epithelia of ducted glands and airways, smooth muscle of bladder, gastrointestinal tract, uterus, and arteries, uterine endometrium and fat cells. The protein was identified by Western blotting in membrane extracts of these tissues, and the ectodomain antibody immunoprecipitated a protein that was recognised with a P2X4 receptor C terminus antibody. The findings indicate that the P2X4 receptor subunit has a very extensive distribution among mammalian tissues, and this suggests possible new functional roles.

We constructed a full-length human P2X5 purinoceptor cDNA by incorporating a sequence corresponding to exon 10, which is missing in cDNAs cloned previously from human tissues. We studied the functional properties by patch-clamp recording and fluorescence imaging after expression in human embryonic kidney 293 cells. ATP (1-100 microM; half-maximal current at 4 microM) elicited inward currents at -60 mV; these persisted during brief (2 s) applications but declined during longer applications. The peak current was dependent on the holding potential and showed little rectification; however, both the desensitization during the application and the decline in the current when ATP was washed out were slower at +30 mV than at -60 mV. 2',3'-O-(4-Benzoyl)-benzoyl-ATP and alphabeta-methylene-ATP mimicked the action of ATP (half-maximal concentrations 6 and 161 microM, respectively). The currents were inhibited by suramin, pyridoxal-5-phosphate-6-azo-2',4'-disulfonic acid and Brilliant Blue G, with half-maximal inhibition at 3, 0.2, and 0.5 microM, respectively; 2',3'-O-(2',4',6'-trinitrophenol)-ATP (1 microM) was ineffective. Removing divalent cations did not significantly alter ATP concentration-response curves. Reversal potential measurements showed that the human P2X5 receptor was permeable to calcium (PCa/PNa = 1.5) and N-methyl-d-glucamine (NMDG) (PNMDG/PNa = 0.4); it was also permeable to chloride (PCl/PNa = 0.5) but not gluconate (Pgluc/PNa = 0.01) ions. The permeability to NMDG developed as quickly as the channel opened, in contrast to the P2X7 receptor where the NMDG permeability develops over several seconds. Cells expressing human P2X5 receptors also rapidly accumulated the propidium dye YO-PRO-1 in response to ATP.

1. Currents through heteromeric P2X(2/3) receptors were evoked by applying alpha,beta-methylene-ATP to human embryonic kidney cells transfected with cDNAs encoding the P2X(2) and P2X(3) subunits. The concentration of alpha,beta-methylene-ATP were < or =30 microM because higher concentrations can activate homomeric P2X(2) receptors. The kinetics of action of three structurally unrelated antagonists were studied; these were 2', 3'-O-(2,4,6,trinitrophenyl)-ATP (TNP-ATP), pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonate (PPADS) and suramin. The association and dissociation rate constants were determined by pre-applying the antagonist for various periods prior to the co-application of agonist and antagonist, or by changing the solution from one containing only the agonist to one containing both agonist and antagonist. The high affinity of TNP-ATP for the P2X(2/3) receptor (K(D) approximately 2 nM) results from fast binding (k(+1) approximately 100 microM(-1) s(-1)) rather than slow unbinding (k(-1) approximately 0.3 s(-1)). For suramin (K(D) approximately 1 microM) the association rate constant ( approximately 1 microM(-1) s(-1)) was 100 times slower than that of TNP-ATP but the dissociation rate constant was similar (k(-1) approximately 1 s(-1)). PPADS (K(D) approximately 0.1 microM) associated and dissociated some 100 - 10,000 times more slowly than the other antagonists.

There are few antagonists selective for subtypes of the several P2X receptors, but these are needed to identify the receptors expressed on native cells and tissues. In particular, P2X(4) and P2X(7) receptor subunits are colocalized on immune, epithelial, and exocrine gland cells, but both are relatively insensitive to suramin and pyridoxal-5-phosphate-6-azo-2',4'-disulfonic acid derivative. In this article, we show that Coomassie Brilliant Blue G selectively inhibits P2X(7) receptors with nanomolar affinity. We measured currents in response to P2X receptor activation in HEK293 cells heterologously expressing human or rat P2X(1), P2X(2), P2X(3), P2X(2/3), P2X(4), P2X(1/5), and P2X(7) receptors. Brilliant Blue G produced a noncompetitive inhibition of rat and human P2X(7) receptors with IC(50) values of 10 and 200 nM, respectively. IC(50) values for inhibition of the other receptors ranged from 2 to >30 microM; the rat and human P2X(4) receptors showed IC(50) values of >10 and 3.2 microM. Coomassie Blue G also blocked YO-PRO1 uptake and membrane blebbing, which are uniquely associated with activation of P2X(7) receptors. Thus, Brilliant Blue G is at least 1000-fold more potent at rat P2X(7) receptors than at rat P2X(4) receptors.

There are seven P2X receptor cDNAs currently known. Six homomeric (P2X1, P2X2, P2X3, P2X4, P2X5, P2X7) and three heteromeric (P2X2/P2X3, P2X4/P2X6, P2X1/P2X5) P2X receptor channels have been characterized in heterologous expression systems. Homomeric P2X1 and P2X3 receptors are readily distinguishable by their rapid desensitization, the agonist action of αβmethyleneATP, and the block by 2′,3′-O-(2,4,6-trinitrophenyl)-ATP. P2X2 receptors are unique among homomeric forms in their potentiation by low pH. Homomeric P2X4 receptors are much less sensitive to antagonism by suramin and pyridoxal 5-phosphate-6-azo-2′,4′-disulfonic acid. Homomeric P2X7 receptors are the only form in which 2′,3′-O-(4-benzoylbenzoyl)-ATP is more potent than ATP. The heteromeric P2X2/P2X3 receptor resembles P2X2 in slow desensitization kinetics and potentiation by low pH and is similar to P2X3 with respect to agonism by αβmethyleneATP and block by 2′,3′-O-(2,4,6-trinitrophenyl)-ATP. Other agonists, antagonists, and ions that can be used to differentiate among the receptors are discussed.

The P2X3 receptor subunit, a member of the P2X family of ATP-gated ion channels, is almost exclusively localized in sensory neurons. In the present study, we sought to gain insight into the role of P2X3 and P2X3-containing neurons in sensory transmission, using immunohistochemical approaches. In rat dorsal root ganglia (DRG), P2X3-immunoreactivity (-ir) was observed in small- and medium-sized neurons. Approximately 40% of DRG neuronal profiles in normal rats contained P2X3-ir. In rats that had received neonatal capsaicin treatment, the number of P2X3-positive neurons was decreased by approximately 70%. Analysis of the colocalization of P2X3-ir with cytochemical markers of DRG neurons indicated that approximately 94% of the P2X3-positive neuronal profiles were labelled by isolectin B4 from Bandeiraea simplicifolia, while only 3% contained substance P-ir, and 7% contained somatostatin-ir. In dorsal horn of rat spinal cord, P2X3-ir was observed in the inner portion of lamina II and was reduced subsequent to dorsal rhizotomy, as well as subsequent to neonatal capsaicin treatment. Finally, P2X3-ir accumulated proximal to the site of sciatic nerve ligation, and was seen in nerve fibres in skin and corneal epithelium. In summary, our results suggest that P2X3 is expressed by a functionally heterogeneous population of BSI-B4-binding sensory neurons, and is transported into both central and peripheral processes of these neurons.

1. Intracellular recordings were made from neurones in slice of rat striatum in vitro. 2. The forty-nine neurones studied were immunoreactive for choline acetyltransferase and had the electrophysiological characteristics typical of large aspiny interneurones. 3. Focal stimulation of the slice elicited a hyperpolarizing inhibitory postsynaptic potential in thirty-five neurones. This IPSP lasted 0.5-1 s and reversed polarity at a membrane potential which was dependent on the logarithm of the extracellular potassium concentration. 4. The IPSP was reversibly blocked by scopolamine and methoctramine, which has some selectivity for M2 subtype of muscarinic receptor. It was unaffected by 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM), DL-2-amino-phosphonovaleric acid (30 microM) and bicuculline (30 microM). 5. Exogenous acetylcholine and muscarine also hyperpolarized the neurones, and this was blocked by methoctramine by not by pirenzepine, which is an M1 receptor-selective antagonist. 6. The findings demonstrate that muscarinic IPSPs occur in the central nervous system. The IPSP may mediate an 'autoinhibition' of striatal cholinergic neurone activity.

With a view to determining whether production of Th2 cytokines, IL-4 or IL-10, is responsible for the inability of mice to resolve infection with Mycobacterium tuberculosis, mice with targeted disruption of their IL-4 or IL-10 gene were compared with wild-type mice in terms of their ability to defend against an M. tuberculosis infection initiated via the respiratory route. The results show that mice that are unable to make either IL-4 or IL-10 are no more capable than wild-type mice at defending against tuberculosis (TB). Therefore, the results are inconsistent with the proposition that the inadequacy of Th1-mediated anti-tuberculosis immunity is due to its down-regulation by either of these Th2 cytokines.

P2X receptors are trimeric membrane ion channels activated by extracellular ATP. Elucidation of their physiological roles has been handicapped by a lack of specific tools. In the present work, the P2X2 receptor was engineered to open and close with different wavelengths of light. We synthesized a photoswitchable crosslinker, bis(maleimido)azobenzene, of molecular dimensions appropriate to bridge cysteines introduced at different subunits at the outer pore (P329C). This produced a P2X2 receptor that was opened within milliseconds at 440 nm light and closed rapidly at 360 nm light, as measured by the ionic currents. When the P329C mutation was combined with the ATP-binding site mutation, K69A, ATP had no effect while light-induced currents were still present. The light-gated receptor displayed similar unitary currents, inward rectification and calcium permeability (PCa/PNa = 2.6), as the P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) could be controlled with light, displaying typical rapid desensitization. P2X3[P320C] subunits also co-assembled with native P2X2 subunits in pheochromocytoma 12 cells to form light-gated heteromeric P2X2/3 receptors. We extended this approach to acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The structurally equivalent mutation in human ASIC1 (G430C) was readily opened and closed by brief applications of light following modification with bis(maleimido)azobenzene. This provides functional evidence that P2X receptors and ASICs can open by a similar mechanism at the level of the pore. The generation of these engineered receptors should facilitate investigation of the functional roles of P2X receptors and ASICs in the cell, tissue and intact organism.

A cDNA was isolated from a human monocyte library that encodes the P2X7 receptor; the predicted protein is 80% identical to the rat receptor. Whole cell recordings were made from human embryonic kidney cells transfected with the human cDNA and from human macrophages. Brief applications (1-3 s) of ATP and 2′,3′-(4-benzoyl)-benzoyl-ATP elicited cation-selective currents. When compared with the rat P2X7 receptor, these effects required higher concentrations of agonists, were more potentiated by removal of extracellular magnesium ions, and reversed more rapidly on agonist removal. Longer applications of agonists permeabilized the cells, as evidenced by uptake of the propidium dye YO-PRO1, but this was less marked than for cells expressing the rat P2X7 receptor. Expression of chimeric molecules indicated that some of the differences between the rat and human receptor could be reversed by exchanging the intracellular C-terminal domain of the proteins.

Purinergic P2X receptors are widely distributed in the nervous system and are known to play roles in primary afferent transmission and central respiratory regulation. They are trimeric membrane proteins, with the extracellular domain that provides three intersubunit ATP binding sites. We expressed the rat P2X7 receptor in human embryonic kidney cells and measured membrane currents before and after photo-affinity labeling with the agonist 2′(3′)-O-(4-benzoylbenzoyl)-ATP (BzATP). After tethering BzATP with ultraviolet light, a persistent current remained after washing out the agonist. Additional current could now be elicited by other nucleotides (CTP and ADP) that are not normally effective as P2X receptor agonists. Similar results were obtained at P2X2 receptors even without previous agonist tethering: exposure to low concentrations of ATP caused the receptor to become sensitive to activation by CTP and ADP. The results show that ATP binding to the first of the three binding sites causes a conformational change to an intermediate closed state that shows increased effectiveness of pyrimidine and diphosphate nucleotide analogs.

The study of P2X receptors has long been handicapped by a poverty of small-molecule tools that serve as selective agonists and antagonists. There has been progress, particularly in the past 10 years, as cell-based high-throughput screening methods were applied, together with large chemical libraries. This has delivered some drug-like molecules in several chemical classes that selectively target P2X1, P2X3, or P2X7 receptors. Some of these are, or have been, in clinical trials for rheumatoid arthritis, pain, and cough. Current preclinical research programs are studying P2X receptor involvement in pain, inflammation, osteoporosis, multiple sclerosis, spinal cord injury, and bladder dysfunction. The determination of the atomic structure of P2X receptors in closed and open (ATP-bound) states by X-ray crystallography is now allowing new approaches by molecular modeling. This is supported by a large body of previous work using mutagenesis and functional expression, and is now being supplemented by molecular dynamic simulations and in silico ligand docking. These approaches should lead to P2X receptors soon taking their place alongside other ion channel proteins as therapeutically important drug targets.

ATP-gated ion channels (P2X receptors) are abundantly expressed in both neuronal and nonneuronal tissues, where they can serve as postsynaptic receptors. The response to ATP shows marked desensitization in some tissues but not others. Currents induced by ATP in Xenopus oocytes expressing cloned P2X 1 (or P2X 3 ) receptors had strong desensitization, whereas currents in cells expressing P2X 2 receptors desensitized relatively little (90% vs. 14% decline of current in a 10-s application). In chimeric receptors, substitution into the P2X 1 receptor of either one of two 34-residue segments from the P2X 2 receptor removed the desensitization; these segments included the first or the second hydrophobic domain. In contrast, desensitization was introduced into the P2X 2 receptor only by providing both these segments of the P2X 1 (or P2X 3 ) receptor. This suggests that desensitization requires interaction between the two hydrophobic domains of the receptor, and supports the view that these are membrane-spanning segments.

1. The effects of multivalent cations on membrane currents recorded from Xenopus oocytes were studied. 2. The hyperpolarization-activated chloride current was reversibly blocked by lanthanum; half-maximal block occurred at a concentration of 8 microM. Zinc, cadmium, cobalt and nickel were less potent than lanthanum, and gadolinium, manganese, barium and strontium had no effect at a concentration of 100 microM. 3. The calcium-activated chloride current was blocked by gadolinium (50 microM), and lanthanum, cadmium, cobalt, nickel and manganese were equally effective. The actions of gadolinium and lanthanum were almost irreversible, while partial (30-80%) recovery was observed with the other cations. Zinc (100 microM) had no effect. 4. In lanthanum (100 microM), membrane depolarizations from -70 mV activated an outward potassium current that was partially blocked by barium (0.1-2 mM). The barium-sensitive current was confined to potentials less negative than -70 mV. The current consisted of a time-independent as well as a time-dependent component, the latter of which had voltage dependence similar to the M-current. 5. It is proposed that lanthanum, gadolinium and barium can usefully separate these endogenous membrane currents in Xenopus oocytes.

New exciting developments on the occurrence and functional role of purinoceptors in mammalian brain were presented at the session Purinoceptors in the central nervous system chaired by Flaminio Cattabeni and Tom Dunwiddie at the Purines '96 international conference. The focus of the session were topics of recent interest, including the sources and mechanisms involved in ATP and adenosine release during physiological neurotransmission in hippocampus, the brain expression of the recently cloned P2 receptors, and the role of the various adenosine receptor subtypes in brain protection from neurodegeneration associated with trauma-, ischemia- and excessive excitatory amino acid neurotransmission. New important insights into the mechanisms responsible for the formation and release of adenosine into the extracellular space were provided by data obtained by Dunwiddie and coworkers in hippocampal pyramidal neurons. These data may have functional implications for the role of purines in modulation of synaptic plasticity and long-term potentiation in this brain area, and hence in cognitive functions. Buell provided an updated overview on the cloning, molecular characteristics and brain expression of various ligand-gated P2X purinoceptors; although the functional role of these receptors in mammalian brain still awaits elucidation, their widespread distribution in the nervous system strongly suggests that ATP-mediated events are more prevalent and important in brain than expected. Pedata presented data on the functional interrelationships between adenosine and glutamate in the brain, and also provided evidence for alterations of the reciprocal regulation between these two systems in aged brain, which may have important implications for both ischemia- and trauma-associated neurodegenerative events and senescence-associated cognitive impairment. Finally, von Lubitz provided novel data on the molecular mechanisms likely to be at the basis of the brain protective effects associated with the chronic stimulation of the adenosine A 3 receptor, further confirming that this receptor represents a crucial target for the development of new antiischemic and antineurodegenerative therapeutic agents.

Several P2X receptor subunits were recently cloned; of these, one was cloned from the rat vas deferens (P2X1) and another from pheochromocytoma (PC12) cells differentiated with nerve growth factor (P2X2). Peptides corresponding to the C-terminal portions of the predicted receptor proteins (P2X1 391-399 and P2X2 460-472) were used to generate antisera in rabbits. The specificities of antisera were determined by staining human embryonic kidney cells stably transfected with either P2X1 or P2X2 receptors and by absorption controls with the cognate peptides. In the vas deferens and the ileal submucosa, P2X1 immunoreactivity (ir) was restricted to smooth muscle, whereas P2X2-ir was restricted to neurons and their processes. Chromaffin cells of the adrenal medulla and PC12 cells contained both P2X1- and P2X2-ir. P2X1-ir was also found in smooth muscle cells of the bladder, cardiac myocytes, and nerve fibers and terminals in the superficial dorsal horn of the spinal cord. In contrast, P2X2-ir was observed in scattered cells of the anterior pituitary, neurons in the hypothalamic arcuate and paraventricular nuclei, and catecholaminergic neurons in the olfactory bulb, the substantia nigra, ventral tegmental area, and locus coeruleus. A plexus of nerve fibers and terminals in the nucleus of the solitary tract contained P2X2-ir. This staining disappeared after nodose ganglionectomy, consistent with a presynaptic function. The location of the P2X1 subunit in smooth muscle is consistent with its role as a postjunctional receptor in autonomic transmission, while in neurons, these receptors appear in both postsynaptic and presynaptic locations.

Cation-selective P2X receptor channels were first described in sensory neurons where they are important for primary afferent neurotransmission and nociception. Here we report the cloning of a complementary DNA (P2X3) from rat dorsal root ganglia that had properties dissimilar to those of sensory neurons. We also found RNA for (P2X1)(ref. 7), (P2X2)(ref. 8) and P2X4 (ref. 9) in sensory neurons; channels expressed from individual cDNAs did not reproduce those of sensory ganglia. Coexpression of P2X3 with P2X2, but not other combinations, yielded ATP-activated currents that closely resembled those in sensory neurons. These properties could not be accounted for by addition of the two sets of channels, indicating that a new channel had formed by subunit heteropolymerization. Although in some tissues responses to ATP can be accounted for by homomeric channels, our results indicate that ATP-gated channels of sensory neurons may form by a specific heteropolymerization of P2X receptor subunits.

P2X receptors are cation-selective ion channels that open on binding to extracellular ATP; they play a role in fast synaptic transmission between neurones, and from autonomic nerves to smooth muscles. Isolation of cDNAs that encode P2X receptors in the smooth muscle of vas deferens and in phaeochromocytoma cells indicates that the receptors are not related to other ligand-gated ion channels. Their overall structure resembles more closely that of epithelial Na+ channels and the proteins that are thought to form mechanosensitive channels in Caenorhabditis elegans. The type of P2X RNA that is found in vas deferens is expressed preferentially by apoptotic thymocytes, and the type of P2X RNA that is found in PC12 cells is abundant in the pituitary gland, suggesting hitherto unsuspected roles for ATP-gated channels in endocrine and immune function.

Intracellular recordings were made with conventional microelectrodes and with whole-cell patch-clamp electrodes from neurons of the rat ventral tegmental area and substantia nigra zona compacta in vitro. Neurons were distinguished as principal cells and secondary cells; it is known from previous work that most principal cells contain dopamine whereas secondary cells do not. 5-Hydroxytryptamine (5-HT; 3–100 μM) depolarized (or evoked an inward current at −60 mV) 46% of 153 principal cells; a small proportion (11%) of cells were hyperpolarized (or showed outward current at −60 mV). Secondary cells were equally likely to be depolarized (or inward current at −60 mV, 30% of 80 cells) or hyperpolarized (or outward current at −60 mV, 28%). ∼40% of each type of cell were unaffected by 5-HT. Depolarizing responses of 5-HT were mimicked by (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and blocked by ketanserin. Hyperpolarizing responses were mimicked by dipropyl-5-carboxamidotryptamine and reversed polarity at the K + equilibrium potential. Inhibitory postsynaptic potentials (or currents) mediated at GABA A receptors occured spontaneously in some principal cells; they were reversibly blocked by tetrodotoxin and bicuculline. 5-HT either increased or decreased the frequency of these synaptic potentials but did not change their mean amplitude or decay time. The results support the interpretation that 5-HT, released onto dopamine neurons by inputs from the dorsal raphe, acts on 5-HT 2 receptors to increase firing in a large proportion of dopamine-containing cells in the ventral tegmental area; however, this would be complemented by indirect changes in dopamine cell-firing resulting from 5-HT exciting or inhibiting local GABA-containing interneurons.

5-Hydroxytryptamine type 3 receptors were expressed in Xenopus oocytes from a cloned cDNA. The peak inward current evoked by 5-hydroxytryptamine (30 microM) was linearly related to the holding potential (-100 to +20 mV) and reversed near 0 mV. The inward current (at -60 mV) declined during the continued presence of 5-hydroxytryptamine with a half-time of about 2 s; this desensitization was 20 times slower in calcium-free solution. Desensitization was markedly different in channels in which Leu286 was changed by site-directed mutagenesis; this residue is thought to lie near the middle of the M2 segment. Desensitization was faster with Phe, Tyr, or Ala in this position and slower with Thr. Phe and Thr substitutions in the equivalent position of the nicotinic acetylcholine receptor have similar effects on desensitization, suggesting that the underlying conformational change might be common to ligand-gated channels.

Intracellular recordings were made from neurons in the lateral nucleus of the amygdala, in a slice of rat brain that was superfused in vitro. [Met5]enkephalin (3-30 microM) and the mu receptor selective agonist DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol; 0.3-3 microM) hyperpolarized about 50% of cells; this was blocked by naloxone and by the mu receptor antagonist CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2). The pA2s for naloxone and CTOP were 8.3 and 7.7, respectively. DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen: delta receptor selective) and U50488 (trans-(+-)-3,4-dichloro-N-methyl-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide methane sulfonate; kappa receptor selective) had no effect. Synaptic potentials mediated by gamma-aminobutyric acid (GABA) acting at GABAA receptors were elicited by focal stimulation of the slice in a combination of 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (10 microM) and 4-aminophosphonovaleric acid (30 microM). They were inhibited by up to 60% by DAMGO and by DPDPE. The action of DAMGO was blocked by CTOP but not by the delta-selective antagonist ICI174864 (N,N-bisallyl-Tyr-Aib-Aib-Phe-Leu-OH, Aib = aminoisobutyrate). The action of DPDPE was blocked by ICI174864 but not by CTOP. Depolarizations elicited by addition of GABA to the superfusing solution were not affected by opioids. It is concluded that activation of mu opioid receptors hyperpolarizes about 50% of lateral amygdala neurons. Activation of either mu or delta receptors also inhibits presynaptically the release of GABA.

1. Intracellular recordings were made from layer V pyramidal neurons in slices of rat cingulate cortex. Electrodes contained potassium methylsulphate and biocytin for subsequent histology. 2. Synaptic potentials were separated pharmacologically with DL-2-amino-5-phosphonovaleric acid (APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), bicuculline, and 2-hydroxysaclofen into components mediated, respectively, by N-methyl-D-aspartate (NMDA) type excitatory amino acid, non-NMDA type excitatory amino acid, gamma-aminobutyric acid (GABAA), and GABAB receptors. Components mediated at excitatory amino acid, GABAA and GABAB receptors reversed polarity at -11, -76, and -108 mV, respectively. 3. When synaptic potentials were evoked by stimulation to the subcortical white matter, 5-hydroxytryptamine (5-HT; 1-100 microM) reversibly reduced the amplitude of NMDA, non-NMDA, GABAA, and GABAB components. Selective agonists and antagonists were used to show that this resulted from activation of 5-HT1B receptors. 4. When synaptic potentials were evoked by stimulation within layer V, 5-HT reduced the amplitude only of the NMDA and non-NMDA components but did not affect the GABAA and GABAB components. 5-HT did not change the amplitude of depolarizations evoked by direct application of glutamate. 5. It is concluded that 5-HT presynaptically inhibits the release of excitatory amino acids at synapses onto prefrontal pyramidal neurons and at synapses onto local feed-forward inhibitory interneurons. 6. 5-HT also hyperpolarized, depolarized, or did not change the membrane potential. The hyperpolarization involved 5-HT1A receptors and resulted from potassium conductance increase. The depolarization involved 5-HT2 receptors and resulted from potassium conductance decrease.

1. Intracellular recordings were made from the lateral nucleus of the amygdala in tissue slices cut from rat brain and maintained in vitro. 2. Three types of neurones were distinguished according to the after-potential that followed an action potential. Type 1 cells (44%, n = 225) had depolarizing after-potentials, resulting from a calcium-dependent chloride conductance. Type 2 cells (48%) had long-lasting (> 250 ms) hyperpolarizing after-potentials and type 3 cells (8%) had shorter hyperpolarizing after-potentials. The average resting potentials of the three cell types were -78, -69 and -62 mV respectively. Intracellular labelling with biocytin showed that type 1 cells were pyramidal neurones; type 2 and type 3 cells were non-pyramidal. 3. Experiments with receptor antagonists identified synaptic potentials mediated by excitatory amino acids and by GABA (acting at GABAA receptors) in all three cell types. A longer duration inhibitory synaptic potential resulting from activation of GABAB receptors was present in type 1 (pyramidal) and type 2 cells. 4. Cholecystokinin (100 nM to 1 microM) depolarized type 2 and type 3 cells but had no effect on type 1 (pyramidal) cells. Baclofen (1-3 microM) hyperpolarized type 1 and type 2, but not type 3 cells. [Met5]enkephalin (1-10 microM) hyperpolarized only type 2 cells. 5. It is concluded that the lateral nucleus of the amygdala contains pyramidal neurones and two types of non-pyramidal neurone; these can be differentiated by membrane properties, synaptic inputs and sensitivities to transmitters.

The widely expressed Na(+)-independent transporter for basic amino acids (system y+) is the cell surface receptor (ecoR) for ecotropic host-range mouse retroviruses (murine leukemia viruses (MuLVs)), a class of retroviruses that naturally infects only mice or rats. Accordingly, expression of mouse ecoR cDNA in mink CCL64 fibroblasts yields cells (CEN cells) that have y+ transporter activity above the endogenous background and that bind and are infected by ecotropic MuLVs. The effect of ecotropic MuLV infection on expression of y+ transporter was analyzed in mouse and in mink CEN fibroblasts. Chronic infection with ecotropic MuLVs caused 50-70% loss (down-modulation) of mouse y+ transporter in plasma membranes, detected as a reduced Vmax for uptake and outflow of L-[3H]arginine with no effect on Km values. Down-modulation was specific for mouse y+ and did not affect other transporters or the endogenous mink y+, suggesting that it results from specific interaction between mouse y+ and the viral envelope glycoprotein gp70 in the infected cells. Because this partial loss of mouse y+ from cell surfaces is insufficient to explain the complete interference to superinfection that occurs in cells chronically infected with ecotropic MuLVs, alternative explanations for interference are proposed. In contrast to the y+ down-modulation caused by chronic infection, binding of extracellular envelope glycoprotein gp70 at 37 degrees C resulted in noncompetitive inhibition of amino acid import by mouse y+ but had no effect on export through this same transporter or on any transporter properties of mink y+. The effects of gp70 on transport kinetics suggest that it slows the rate-limiting step of the amino acid import cycle, a conformational transition of the empty transporter in which the binding site moves from the inside back to the outside of the cell, and that gp70 has no effect on the rate-limiting step of the amino acid export cycle. Infected cells retain substantial y+ activity. Moreover, the virus binding site on ecoR is in a mobile region that changes conformation during the amino acid transport cycle.

The tetradecapeptide somatostatin (SRIF) is a hormone release-inhibiting substance that mediates diverse effects in brain and peripheral organs via specific receptors. A cDNA encoding a rat SRIF receptor was identified by use of degenerate oligonucleotide primers and polymerase chain reaction amplification of cDNA prepared from transcripts expressed in rat brain. The complete cDNA encodes a protein of 391 amino acids with seven potential transmembrane domains. Expression of the cDNA product in transfected COS-7 cell lines provides the same high affinity of binding to [125I-Tyr11]SRIF-14 as that of rat cerebral cortex tissues. However, the binding of [125I-Tyr11]SRIF-14 to cloned rat SRIF receptor is not displaced by MK678, a SRIF analog that partially displaces [125I-Tyr11]SRIF-14 binding sites in membranes of rat cerebral cortex. Northern analysis and in situ hybridization indicate that mRNA (4.0 kilobases) for cloned rat SRIF receptor is preferentially expressed in rat brain regions such as cerebral cortex and hippocampus with no detectable expression in most peripheral organs. This pattern contrasts with the exclusive peripheral expression of a recently cloned human SRIF receptor. The cDNA probe of rat receptor detects mRNA from mouse brain but not from human cerebral cortex and cerebellum.

Cryoinjury in ram sperm was investigated by direct observation, using cryomicroscopy, to validate model hypotheses of freezing injury in such a specialized cell. Fluorescein diacetate was used to determine when during the freeze-thaw cycle the sperm membrane became permeable. In noncryoprotected sperm plasma membrane, integrity was maintained throughout the cooling and freezing process, but fluorescein leakage occurred during rewarming. The temperature of post-thaw permeabilization varied in relation to the minimum temperature reached during freezing; cells cooled to -10 degrees C retained fluorescence into the post-thaw temperature range of 9-24 degrees C (mean +/- SEM; 13.25 +/- 0.91 degrees C), whereas cells cooled to -20 degrees C lost fluorescence shortly after thawing (mean +/- SEM; 2.62 +/- 0.91 degrees C). Sperm cooled to 5 degrees C, but not frozen, retained fluorescence during rewarming up to 20-30 degrees C. The inclusion of glycerol and egg yolk in the freezing medium significantly and independently increased the post-thaw permeabilization temperature. Maintenance of fluorescence was also correlated with ability to resume motility after thawing. Sperm reactivation experiments were undertaken to examine deleterious effects of freezing upon the flagellar microtubular assembly. No direct evidence for such effects was obtained. Instead, a highly significant correlation between minimum freezing temperature and post-thaw temperature of initial reactivation was detected.

Intracellular recordings were made from presumed dopamine-containing neurons in slices cut from the midbrain of the rat. Focal electrical stimulation produced a hyperpolarizing synaptic potential that was reduced by 75–95% by the GABAB-receptor antagonist 2-hydroxysaclofen (300 microM). 5-HT (3–100 microM) reduced the amplitude of the GABAB synaptic potential by 20–74%, with a 50% reduction at 10 microM, but did not reduce the amplitude of synaptic potentials mediated by GABAA receptors. 5-HT acted presynaptically because hyperpolarizations produced by exogenously administered GABA (1 mM) in picrotoxin (100 microM) were not affected by 5-HT (30 microM). (+/-)-Cyanopindolol (100 nM), a 5-HT1B antagonist, blocked the effect of 5-HT (10 microM); spiperone (1 microM), which is an antagonist at 5-HT1A and 5-HT2 receptors, had no effect. The amplitude of the GABAB synaptic potential was reduced by the 5-HT1B receptor agonists 1-[3-(trifluoromethyl)- phenyl]-piperazine (300 nM) and 7-trifluoromethyl-4-(4-methyl-1- piperazinyl)-pyrrolo[1,2-a]quinoxaline (1 microM), but not by the 5- HT1A agonist N,N-dipropyl-5-carboxamidotryptamine (1 microM) or the 5- HT2 agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (10 microM). We conclude that 5-HT activates presynaptic 5-HT1B receptors that inhibit the release of GABA onto GABAB but not GABAA receptors.

1. Unitary potassium currents were recorded in outside-out patches of membrane from guinea-pig submucosal neurones. The actions of alpha 2-adrenoceptor agonists, somatostatin and [Met5]enkephalin were studied. 2. Three main groups of background potassium channels were active. At -70 mV with 160 mM-potassium on both sides of the membrane, they had conductances of 30-65 (small), 120-160 (intermediate) and 220-260 pS (large). 3. The open channel current-voltage relation showed only constant-field rectification. Extracellular barium (2 mM) and caesium (2 mM) decreased inward but not outward currents. Tetraethylammonium (10 mM) had no effect. 4. Noradrenaline, somatostatin and [Met5]enkephalin each increased the open probability of all three classes of channel when two or more unitary amplitude channels were active in the membrane patch. Agonists were ineffective when no channel, or a single channel, was discernible in the patch. Agonists did not cause the appearance of unitary currents distinct from those seen prior to their application. 5. The effect of the agonists required intracellular guanosine 5'-triphosphate. 6. The results show that the hyperpolarization of submucosal plexus neurones by noradrenaline, somatostatin and [Met5]enkephalin results from the increased opening of at least three types of background potassium channel, and that the coupling from the receptors to the channels is maintained in excised membrane patches.

The physiological role of opioid peptides in the rat striatum was sought by intracellular recording in vitro. Excitatory synaptic potentials (mediated by glutamate or aspartate) and inhibitory synaptic potentials (mediated by GABA) were isolated pharmacologically and/or by positioning the stimulation electrode over the external capsule. Opioid agonists and antagonists selective for mu-, delta-, and kappa-receptors were applied by superfusion. Two main actions of opioids were observed. First, mu- and delta-selective opioids presynaptically inhibited the excitatory postsynaptic potential, whereas only delta-selective opioids decreased the inhibitory synaptic potential. Second, a small subpopulation of cells (not medium spiny neurons) were hyperpolarized by delta-selective agonists. The results indicate that the main action of opioids on striatal neurons is presynaptic inhibition of the corticostriate excitatory synaptic input.

gamma-Aminobutyric acid (GABA) inhibits neurons by acting at GABAA and GABAB receptors but it is not known whether the two receptors are associated with discretely separate afferent inputs or whether GABA released from a single presynaptic neuron activates both receptors. Intracellular recordings were used to show that, in the lateral amygdala and ventral tegmental area of the rat, distinct sets of GABA-containing neurons provide the synaptic input to GABAA and GABAB receptors. Synaptic potentials resulting from GABAA receptor activation (blocked by bicuculline) and from GABAB receptor activation (blocked by 2-hydroxysaclofen) occurred spontaneously but as unrelated events. Furthermore, the two components of evoked synaptic potentials were differentially inhibited by agonists acting presynaptically (muscarine and 5-hydroxytryptamine). The finding that GABA acting at GABAA and GABAB receptors originates from distinct sets of presynaptic fibers suggests that two groups of GABA-containing neurons might be generally distinguishable in the mammalian nervous system.

The excitability of the second order neurons within the trigeminal subnucleus caudalis underlies pain perception and processing in migraine and trigeminal neuralgia. These neurons were studied with whole-cell patch-clamp technique in slices from mouse brain stem. Electrical and morphological characteristics of 56 neurons were determined. Four categories were distinguished from electrophysiological properties: tonic (39%), phasic (34%), delayed (16%) and single spiking (11%). These categories did not show distinct morphological properties. Neurons had tetrodotoxin-sensitive sodium currents that activated and inactivated within milliseconds. They also showed a high voltage-activated, slowly inactivating calcium current: up to half of this current was blocked by omega-conotoxin GVIA (1microM) and omega-agatoxin IVA (100-300 nM), but it was not affected by nifedipine (10microM). Exogenously applied capsaicin (1microM) and alphabetamethylene-5'-adenosine triphosphate (100microM) elicited large amplitude, spontaneous excitatory postsynaptic currents that were blocked by capsazepine (10microM) and 5-[(3-phenoxybenzyl)-(1,2,3,4-tetrahydro-naphthalen-1-yl)-carbamoyl]-benzene-1,2,4-tricarboxylic acid (A-317491: 10microM), respectively. Thus, neurons of the mouse trigeminal subnucleus caudalis substantia gelatinosa exhibit N-type and P/Q-type voltage-gated calcium channels, and receive presynaptic afferents that express TRPV1 and P2X(2/3) receptors. These results suggest possible therapeutic interventions, and serve as a basis for the characterization of cellular changes that may underlie trigeminal neuropathic pain.

Intracellular recordings were made from neurons of rat lateral amygdala, nucleus accumbens, and striatum in vitro. Synaptic potentials mediated by gamma-aminobutyric acid and by excitatory amino acids were isolated pharmacologically by using receptor antagonists, and their amplitudes were used as a measure of transmitter release. Muscarine and acetylcholine inhibited the release of both gamma-aminobutyric acid and excitatory amino acids, but measurements of the dissociation equilibrium constants for the antagonists pirenzepine, 11-(2-[(diethylamino)methyl]-1-piperidinyl)acetyl-5,11-dihydro-6H-pyrido [2,3-b][1,4]benzodiazepine-6-one, methoctramine, and hexahydrosiladifenidol indicated clearly that different muscarinic receptors were involved (M1 and probably M3, respectively). The differential localization of distinct muscarinic receptor subtypes on terminals releasing the major inhibitory and excitatory transmitters of the brain could be exploited therapeutically in some movement disorders and Alzheimer disease.

The effects of controlled stress, i.e. cooling, upon the distribution of actin in ram spermatozoa were examined to investigate the hypothesis that cytoskeletal proteins are involved in the maintenance of sperm plasma membrane integrity. The normal distribution of actin on the spermatozoon was initially determined. A monoclonal antibody (IgM) interacted exclusively with the post-acrosomal region and the principal piece of the flagellum. By the use of a polyclonal antibody, actin was detected on the acrosome (excluding the equatorial segment), the post-acrosomal region and the whole of the flagellum. The actin was present in its non-filamentous form. Spermatozoa fixed at 39 degrees C and then treated for the immunofluorescent detection of actin with the monoclonal antibody were mostly unstained (proportion stained = 4.4% (+/- 1.6; s.e.m.); n = 8 ejaculates). Provided spermatozoa were permeabilized by greater than 0.025% Triton X-100 before immunofluorescence, actin was localized in the postacrosomal region of all sperm heads, and to a minor extent on the principal piece of the flagellum. Use of the polyclonal antibody confirmed that the post-acrosomal antigen was unmasked by detergent treatment. Slow cooling, over 2-h periods to various temperatures between 5 and 15 degrees C, also induced an increase in the proportion of cells showing post-acrosomal actin immunoreactivity. Cooling through the temperature range 15 to 10 degrees C markedly increased the proportion of immunoreactive cells (mean +/- s.e.m.; 12 +/- 4.5% at 15 degrees C; 27 +/- 4.5% at 10 degrees C; n = 4 ejaculates). Further cooling to 5 degrees C failed to elicit increased staining. Ultrastructural examination of cooled spermatozoa confirmed that a subpopulation of spermatozoa exhibited post-acrosomal actin immunoreactivity after cooling. These results are compatible with the suggestion that actin fulfills a stabilizing function in spermatozoa.

Intracellular microelectrode and whole cell patch-clamp recordings were made from guinea pig enteric neurons in vitro. In most myenteric AH neurons (but not S neurons), step hyperpolarizations from the resting potential evoked an inward current (IH). IH peaked in approximately 400 ms (at 36 degrees C), was fully activated at -100 mV, and did not inactivate during 10 s. IH was associated with a conductance increase and reversed polarity at about -40 mV (by extrapolation). IH was reduced in low-sodium concentrations and increased when the concentration of extracellular potassium ions was increased. Cesium (2 mM) blocked IH in a voltage-dependent manner; this led to an increase in the amplitude of the spike after-hyperpolarization. Cobalt (2-4 mM) or barium (0.01-1 mM) did not alter IH. Only 12% of submucous plexus neurons showed IH and this subgroup included both S and AH neurons. In myenteric AH neurons, IH would act in opposition to the persistent calcium-activated potassium current and thus stabilize the resting potential.

1. Whole-cell recordings were made from submucosal neurones acutely dissociated from guinea-pigs. The actions of noradrenaline, somatostatin and [Met5]enkephalin on currents carried by calcium ions were studied. 2. On depolarization from a holding potential of -70 mV, an inward current activated at -40 mV, reached its peak amplitude at 10 mV and reversed to outward at 72 mV (with external calcium of 5 mM and internal caesium of 160 mM). 3. Cadmium, nickel and cobalt reversibly blocked the calcium current; concentrations causing 50% block were 2.5, 500 and 2000 microM respectively. The calcium current (holding at -70 or -30 mV) was reversibly blocked by omega-conotoxin (100 nM), and unaffected by Bay K 8644 (0.1-10 microM) and nifedipine (1 microM). Cadmium caused an outward shift in holding current at -30 mV, implying that there was a persistent inward calcium current at this potential. 4. Noradrenaline, somatostatin and [Met5]enkephalin decreased the calcium current. The maximal inhibition observed with any one agonist, or with a combination of two agonists, did not exceed 50%; concentrations giving half-maximal inhibition were 5.5 microM for noradrenaline, 4 nM for somatostatin and 1 microM for [Met5]enkephalin. The inhibition was independent of membrane potential. All three agonists also reduced the persistent calcium current at -30 mV. 5. Inhibition of the calcium current by noradrenaline occurred with a latency of not less than 175 ms; cadmium applied by the same method depressed the current within 5-45 ms. 6. Experiments with selective agonists and antagonists indicated that the receptor types involved in calcium current inhibition were alpha 2-adrenoceptors and delta-opioid receptors. Somatostatin acted at a distinct receptor. 7. Calcium currents were also inhibited by intracellular dialysis with guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S). Agonists were ineffective in cells pre-treated with pertussis toxin, but their action was restored when purified GTP-binding proteins (Go or Gi) were included in the intracellular recording solution. 8. It is concluded that noradrenaline, somatostatin and [Met5]enkephalin act at their respective receptors on guinea-pig submucosal neurones to inhibit a voltage-dependent calcium current. Activation of the same receptors also increases a potassium conductance in these cells: in both cases a pertussis-sensitive G protein is involved.

1. Intracellular recordings were made from neurons in slices of rat nucleus accumbens in vitro. Membrane currents were measured in the potential range -60 to -120 mV with the use of a single-electrode, voltage-clamp amplifier. 2. A minority of neurons (28/285) was identified that had resting membrane potentials almost 20 mV less negative than the majority of the cells. These cells, but not the majority, had an inward current that activated slowly when the cells were hyperpolarized from -60 to -120 mV. The time constant of activation was approximately 3 s at -70 mV and 100 ms at -120 mV. 3. This inward current was completely blocked by external cesium (2 mM) but unaffected by barium. The current was reduced in solutions containing low-sodium concentration and increased in solutions with high-potassium concentration; its reversal potential was estimated to be -36 mV. 4. It is concluded that two types of neuron can be distinguished in the rat nucleus accumbens on the basis of the presence or absence of a cation current activated by hyperpolarization. This current (IH, also called If and IQ) causes the neurons to have less-polarized resting potentials than the majority of nucleus accumbens neurons.

Routine radionuclide bone scans have not been considered cost-effective for the routine follow-up after treatment of primary breast cancer. However subgroups of patients exist in whom early relapse in the skeleton is likely and this study examines again the role of the bone scan in routine follow-up. Serial radionuclide bone scans were performed every 6 months during the first 2 years of follow-up of 560 patients with breast cancer. Tumor characteristics which predict early relapse in bone were identified and the scan conversion rate from negative to positive determined for each prognostic group. A total of 199 (28%) of patients have relapsed, 50 (9%) with first recurrence in bone within two years of diagnosis. All were identified on the bone scan with a median lead time of 4 months over radiological evidence of bone involvement. The overall scan conversion rate was 2.8%. This was significantly higher in poor prognosis patients with T4 tumours (6.3%), more than four involved axillary lymph nodes (6.1%) and inoperable tumours (6.5%), than in good prognosis patients with T1 tumours (1.1%), negative axillary lymph node involvement (1.2%) or well-differentiated ductal grade tumours (1.1%). We do not recommend routine bone scans in the follow-up of all patients with breast cancer. In patients with a good prognosis after primary treatment they cannot be considered cost-effective. However, in those with features which predict early recurrence in the skeleton the frequency of scan conversion is sufficient to justify serial bone scanning during the first two years of follow-up.

Agonists at alpha 2-adrenoceptors, delta-opioid receptors, and somatostatin receptors were applied to dissociated guinea pig submucous plexus neurons; whole-cell recordings of membrane current showed that they increased the membrane potassium conductance. The conductance affected showed inward rectification, being described by Gag(max)/[1 + exp((V - V0.5)/k)] where V0.5 was about -65 mV and Gag(max) was about 10 nS. The agonists were ineffective when the potassium conductance of the neurons had first been increased by intracellular dialysis with purified guanosine 5-triphosphate (GTP)-binding proteins (Gi or Go). Agonist actions were prevented by pertussis toxin, applied intracellularly (10–100 ng/ml for several minutes) or extracellularly 1– 10 micrograms/ml for 1 hr); in the latter case, the agonist responses were reconstituted by intracellular dialysis with GTP-binding proteins.