A phase I clinical trial demonstrates that nfP2X7 -targeted antibodies provide a novel, safe and tolerable topical therapy for basal cell carcinoma.

BACKGROUND: Expression of P2X7 , an ATP-gated calcium channel, increases cancer cell proliferation and invasiveness. A variant of P2X7 (termed nfP2X7 ), in which a normally hidden epitope (E200) is exposed for antibody binding, is observed in a variety of different cancers. OBJECTIVES: To investigate the safety, tolerability and pharmacokinetics and assess indicative efficacy of a novel antibody ointment as a therapeutic for basal cell carcinoma (BCC). METHODS: An open-label, phase I clinical trial was undertaken at three dermatology clinics to evaluate the safety and tolerability of topical administration of an ointment containing 10% sheep polyclonal anti-nfP2X7 antibodies (BIL010t) to primary BCC lesions twice daily for 28 days. Twenty-one patients with primary BCC lesions at least 0·5 cm2 in area and less than 2·0 cm in diameter were enrolled. The primary end points were safety, tolerability and pharmacokinetics. Change in lesion size after treatment was determined and histology was performed on pretreatment and end-of-treatment (EOT) biopsies. RESULTS: Compliance was very high, with treatment being well tolerated. The most common adverse events were treatment site erythema, pruritus, dryness and pain. There was no evidence of systemic penetration of the sheep antibody. Lesions were measured prior to and after 28 days of treatment, with 65% of patients showing a reduction in lesion area, 20% showing no change and 15% showing an increase. Histopathology of post-treatment excision of lesion sites showed eight patients with stable disease, nine with partial response and three with complete response. CONCLUSIONS: Antibodies against nfP2X7 (BIL010t) provide a novel, safe and well-tolerated treatment for BCC.

Differentiating keratoacanthoma from squamous cell carcinoma by the use of apoptotic and cell adhesion markers.

AIMS: Keratoacanthomas (KA) are well-differentiated squamoproliferative skin lesions that grow rapidly and regress spontaneously. In contrast, squamous cell carcinomas (SCC) can have variable differentiation, inexorably progress and on occasion metastasize. Distinguishing between KA and SCC using haematoxylin and eosin-stained sections from an initial biopsy can often be difficult. There is also some debate as to whether KA is simply a variety of well-differentiated SCC or a distinct entity. METHODS AND RESULTS: Initial biopsy sections from 25 cases of SCC and 20 of KA were labelled with markers for both the initiation (the cytolytic receptor P2X7) and end-stage (terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling) of apoptosis, telomerase-associated protein (TP1) and the cell adhesion protein E-cadherin. As this was a retrospective study, the clinical outcome of each case was known. This resulted in a unique labelling pattern of each marker for SCC and KA, allowing a differential diagnosis between the two conditions. The simplest marker to use for this purpose was anti-P2X7. Sections from five cases that were initially very difficult to diagnose were correctly identified as SCC using this method. CONCLUSIONS: These results support the view that KA has a different pathogenesis and biochemistry from that of SCC, and is a distinct entity. Anti-P2X7 labelling, using routine immunohistochemical techniques, provides a method for differentially diagnosing these conditions.

Early prostate cancer detected using expression of non-functional cytolytic P2X7 receptors.

AIMS: To detect early prostate cancer reliably by monitoring the expression of non-functional P2X(7) cytolytic purinergic receptors. METHODS AND RESULTS: P2X(7) receptors were absent from normal prostate epithelium obtained from post mortem tissue and tissue from cases of transurethral resection collected from young men (n = 23) who were confirmed to be free of cancer at later procedures 5-10 years after collection of the original samples. However, P2X(7) was present in every case of 116 confirmed prostate cancers regardless of Gleason grade or patient age. P2X(7) was present in apparently normal epithelial cells in acini well outside the tumour margins, but appeared in a distinct stage-specific manner commencing with the nucleus, progressing to the cytoplasm and collecting finally on the apical membrane of the epithelial cells in morphologically distinct cancer. The pattern of P2X(7) receptor localization in the epithelial cells was recorded in earlier biopsies obtained from the same patient cohort. One hundred and fourteen of 116 prostates stained positively for P2X(7) at the earliest biopsy, though generally with a less advanced pattern of distribution. CONCLUSIONS: The appearance of P2X(7) receptors in normal prostate tissue adjacent to prostate tumours makes direct tumour biopsy less critical for positive cancer diagnosis and enables cancer progression to be monitored.

Localisation of P2X2 receptor subunit immunoreactivity on nitric oxide synthase expressing neurones in the brain stem and hypothalamus of the rat: a fluorescence immunohistochemical study.

A large body of evidence suggests that nitric oxide (NO) and ATP act as neurotransmitters in the regulatory mechanisms concerning several autonomic functions at the level of both the hypothalamus and the brain stem. In the present study, we investigated whether neuronal NO synthase containing neurones also express P2X(2) receptor subunit of the ATP-gated ion channel via double-labelling fluorescence immunohistochemistry. Our data demonstrate that a high percentage of neuronal NO synthase-immunoreactive neurones are also P2X(2)-immunoreactive in the rostral ventrolateral medulla (98%) and supraoptic nucleus of the hypothalamus (92%). Significant numbers of neuronal NO synthase-immunoreactive neurones are also P2X(2)-immunoreactive in the subpostremal (48%) and commissural (65%) subdivisions of the nucleus tractus solitarius. In the caudal ventrolateral medulla and raphe obscurus, 96% and 89%, respectively, of neuronal NO synthase containing neurones also express P2X(2) receptor subunit. In contrast to the supraoptic nucleus, there was a lower percentage of co-localisation between NO synthase and P2X(2) receptor subunit in the paraventricular nucleus of the hypothalamus. In summary, this study demonstrates for the first time that there is a widespread co-localisation of neuronal NO synthase and P2X(2) receptor subunit in the hypothalamus and brain stem of the rat. Further studies are required to elucidate whether NO and ATP functionally interact within the hypothalamus and the brain stem.

Effects of pathophysiological concentrations of albumin on NHE3 activity and cell proliferation in primary cultures of human proximal tubule cells.

The progression of renal disease correlates strongly with hypertension and the degree of proteinuria, suggesting a link between excessive Na+ reabsorption and exposure of the proximal tubule to protein. The present study investigated the effects of albumin on cell growth and Na+ uptake in primary cultures of human proximal tubule cells (PTC). Albumin (1.0 mg/ml) increased cell proliferation to 134.1 +/- 11.8% (P < 0.001) of control levels with no change in levels of apoptosis. Exposure to 0.1 and 1.0 mg/ml albumin increased total 22Na+ uptake to 119.1 +/- 6.3% (P = 0.005) and 115.6 +/- 5.3% (P < 0.006) of control levels, respectively, because of an increase in Na+/H+ exchanger isoform 3 (NHE3) activity. This was associated with an increase in NHE3 mRNA to 161.1 +/- 15.1% (P < 0.005) of control levels in response to 0.1 mg/ml albumin. Using confocal microscopy with a novel antibody raised against the predicted extracellular NH2 terminus of human NHE3, we observed in nonpermeabilized cells that exposure of PTC to albumin (0.1 and 1.0 mg/ml) increased NHE3 at the cell surface to 115.4 +/- 2.7% (P < 0.0005) and 122.4 +/- 3.7% (P < 0.0001) of control levels, respectively. This effect was paralleled by significant increases in NHE3 in the subplasmalemmal region as measured in permeabilized cells. These albumin-induced increases in expression and activity of NHE3 in PTC suggest a possible mechanism for Na+ retention in response to proteinuria.

Specific detection of non-functional human P2X(7) receptors in HEK293 cells and B-lymphocytes.

P2X(7) receptor/channels mediate ATP-induced apoptosis in a range of cells including lymphocytes. HEK293 cells were transfected with wild-type human P2X(7) receptor or site-directed mutant constructs (K193A, K311A and E496A) known to be non-functional from measurements of barium/ethidium influx in the presence of ATP or 2',3'-O-(4-benzoylbenzoyl)-ATP. An antibody was designed against an epitope from a loop adjacent to the extracellular ATP site. The epitope was unavailable in cells expressing normal functional surface receptors. Non-functional surface receptors as well as intracellular receptors selectively bound the antibody. So did B-lymphocytes from chronic lymphocytic leukemia patients expressing non-functional (E496A) mutant receptor.

Tenascin, E-cadherin and P2X calcium channel receptor expression is increased during rat blastocyst implantation.

The calcium-activated cell-adhesion proteins tenascin, E-cadherin and the purinergic (P2X) calcium channel receptors are expressed in an identical spatial and temporal pattern in uterine epithelium in the rat during implantation. On Day 1 of pregnancy (estrous), a diffuse cytoplasmic and specific basement membrane label for each of the proteins was observed throughout the uterine epithelium. On Day 3 of pregnancy, a specific and prominent lateral plasma membrane label for each protein was seen. At the time of implantation on Day 6, an additional and significant increase in the label for each was observed on the apical epithelium. At this time, the label for tenascin in the apical epithelium was increased 2.1-fold (p < 0.0004), that of E-cadherin was increased 2.5-fold (p < 0.0001) and the P2X receptor label was increased 2.0-fold (p < 0.0001). These observations suggest a major role for the calcium-activated adhesion proteins tenascin and E-cadherin in attachment and implantation, with ionic calcium for protein activation possibly provided by the P2X calcium channels. These events occur along the entire length of the uterine epithelium in preparation for blastocyst adhesion.

Antisense co-suppression of G(alpha)(q) and G(alpha)(11) demonstrates that both isoforms mediate M(3)-receptor-activated Ca(2+) signalling in intact epithelial cells.

We used replication-deficient adenoviruses overexpressing antisense against G(q) class alpha-subunits to determine the roles of G(q) and G(11) in mediating M(3)-receptor-coupled Ca(2+) mobilization in intact HT29 human colonic carcinoma epithelial cells. Western blot analysis and confocal microscopy showed that the viruses expressing antisense directed against the alpha-subunits of G(q) or G(11) produced isoform-specific reductions in the levels of these alpha-subunits. Fura-2 was used to measure changes in the Ca(2+) response following activation of the M(3) receptors by carbachol. The G(alpha)(q) antisense virus suppressed the peak Ca(2+) response by 70%, whereas the G(alpha)(11) antisense virus reduced it by 34%. We then used co-infection with both viruses to determine the effect of concomitant suppression of both G(alpha)(q) and G(alpha)(11). Overexpression of antisense to both alpha-subunits reduced by approximately 50% the levels of both G(alpha)(q) and G(alpha)(11). It also almost completely inhibited the Ca(2+) response to carbachol. These data show that both G(q) and G(11) are involved in mediating the action of the M(3) receptor on cytosolic Ca(2+) in HT29 cells. Furthermore, they suggest that the coupling of the M(3) receptor to these G proteins is specific, in that G(alpha)(q) cannot substitute for G(alpha)(11), and vice versa.

Purinergic receptor expression in the apical plasma membrane of rat uterine epithelial cells during implantation.

We examined the expression of the metabotropic P2Y(1), P2Y(2), P2Y(4), and ionotropic P2X(7) purinergic receptor subtypes in the uterine epithelium during early pregnancy in the rat. On Day 1 of pregnancy, there was no expression of P2X(7), P2Y(2), or P2Y(4) in the uterine epithelium. P2Y(1) was detected only as a diffuse label. On Day 3, P2X(7) and P2Y(2) receptor distribution was confined to the lateral plasma membranes in the epithelium. There was no expression of P2Y(4) while P2Y(1) was again detected only as a diffuse label throughout the epithelium. At the time of implantation on Day 6, a strong, continuous and area-specific P2X(7) and P2Y(2) label was noted along the entire surface of the apical epithelium suggesting a major role in calcium-modified events preceding and facilitating attachment and implantation of the blastocyst. P2Y(1) and P2Y(4) were present as a ubiquitous and nonspecific label, although the latter exhibited a minor apical deposition. These and earlier experiments with P2X subtype-specific antibodies indicate that both P2X and P2Y purinergic receptors play a role in conditioning the entire uterine epithelium for blastocyst implantation regardless of the site of attachment.

Point mutations confer loss of ATP-induced human P2X(7) receptor function.

Residues considered essential for ATP binding to the human P2X(7) receptor (hP2X(7)R) were investigated. HEK293 cells or Xenopus oocytes were transfected with wild-type or site-directed mutants of hP2X(7)R constructs and channel/pore activity measured in the presence of ATP or 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP). Barium uptake and ethidium influx into HEK293 cells were abolished in cells expressing K193A and K311A mutants, and were partially reduced in cells expressing mutant P210A. K193A and K311A mutations also completely abolished responses to ATP and BzATP in Xenopus oocytes as measured by electrophysiology. These results indicate that K193 and K311 are essential residues in ATP binding in the hP2X(7)R.

On the immunohistochemical distribution of ionotropic P2X receptors in the nucleus tractus solitarius of the rat.

ATP has been shown to excite neurones of the nucleus tractus solitarius (NTS) via the activation of P2X receptors. In the present study, the distribution of six P2X receptors (P2X(1)-P2X(6)) within the rat NTS was investigated by peroxidase immunohistochemistry. Immunopositive neurones for P2X receptor subtypes were detected in all divisions of the NTS, although the staining densities differed according to receptor subtype and sub-nuclei. P2X(1)-immunopositive cells were distributed throughout the rostro-caudal extent of the NTS, while varicose fibres were mainly located along the postremal border. P2X(2) immunoreactivity was present in neurones and fibres located throughout the NTS. In the commissural NTS intense staining was observed medial of the solitary tract while in the sub-postremal NTS neurones were observed along the postremal border. A high density of P2X(3)-positive neurones and fibres was observed in the sub-postremal NTS along the border of the area postrema and in the rostral NTS in the medial subdivision. In comparison to the staining observed with the other receptor antibodies, there was considerably reduced P2X(4) receptor immunoreactivity. P2X(4)-positive neurones tended to be more sparsely distributed, and found mainly in the intermediate portion of the commissural NTS, and along the postremal border. In contrast, we observed dense staining for the P2X(5) receptor subtype in a majority of regions within the NTS. The most striking staining was observed in the intermediate subdivision at the level of the sub-postremal NTS and the medial portion of the rostral NTS. P2X(6) immunoreactive neurones were observed in the medial commissural NTS, along the postremal border and in the dorso-medial and medial subdivisions of the rostral NTS.Taken together, our findings confirm the presence of six P2X receptor subtypes within the NTS of the rat, consistent with a neurotransmitter role for ATP in the rat NTS. These results indicate the need for more extensive functional studies to elucidate the roles of the individual and heterodimeric assemblies of P2X receptor subtypes within the NTS.

Loss of purinergic P2X(3) and P2X(5) receptor innervation in human detrusor from adults with urge incontinence.

Activation of purinergic P2X receptors associated with the parasympathetic nerves that supply the human bladder smooth muscle (detrusor) is implicated in control of detrusor contractility. The relative abundance of all seven subtypes colocalized with synaptic vesicles on parasympathetic nerves was examined in specimens from normal adult bladder, infants, and in adults with overactive detrusor contractility and a diagnosis of idiopathic detrusor instability (IDI) to determine whether receptor distribution varied with age or in patients with incontinence. Alteration in control of detrusor innervation was examined with P2X subtype-specific antibodies and an antibody against synaptic vesicles, using immunofluorescence and confocal microscopy. Detrusor samples were taken from: controls, at cystectomy for cancer or cystoscopic biopsy for hematuria (n = 22; age 33-88), child bladder, at surgical correction of vesico-ureteric reflux (n = 21; age 4 months to 2 years), and adults with detrusor instability at cystoscopy-cystodistension (n = 18; age 30-81). Adult specimens contained muscle with large varicosities (1.2 microm) along parasympathetic nerves with colocalized patches of all P2X(1-7) subtypes. Infant bladder revealed little evidence of P2X at age <9 months but approached adult levels at 2 years. Detrusor from IDI patients revealed selective absence of P2X(3) and P2X(5) beneath all the varicosities. This specific lack of P2X(3) and P2X(5) may impair control of detrusor contractility and contribute to the pathophysiology of urge incontinence.

Distribution of P2X receptors on astrocytes in juvenile rat hippocampus.

Recent evidence suggested that ATP acting via ionotropic (P2X) and metabotropic (P2Y) purinergic receptors might be involved in signaling between glial cells and within glial-neuronal networks. In contrast to their neuronal counterpart, the identity of P2X receptors in CNS glial cells is largely unknown. In the present study, antibodies recognizing the subunits P2X1-P2X7 were applied together with the astroglial marker S100beta and nuclear labeling with Hoechst 33342 to investigate semiquantitatively the distribution of the whole set of P2X receptors in astrocytes of the juvenile rat hippocampus. Expression of P2X1-P2X4, P2X6, and P2X7 subunits was observed in astrocytes of various hippocampal subregions, but the cells were completely devoid of P2X5 protein. S100beta-positive cells expressing subunits P2X3-P2X7 occurred evenly in the different subfields, while P2X1- and P2X2-positive astrocytes were distributed more heterogeneously. The staining pattern of P2X subunits also differed at the subcellular level. Antibodies against P2X2 and P2X4 labeled both astroglial cell bodies and processes. Immunoreactivity for P2X1 and P2X6 was mainly confined to somatic areas of S100beta-positive cells, whereas the subunit P2X3 was primarily localized along astroglial processes. Knowledge of the distribution of P2X receptors might provide a basis for a better understanding of their specific role in cell-cell signaling.

Detection of P2X purinergic receptors on human B lymphocytes.

B lymphocytes are known to synthesise the P2X7 subtype of the P2X purinergic receptor family; however, the identification of the other six P2X subtypes on these cells has been limited by the absence of specific antibodies. In this study, we used a panel of anti-P2X polyclonal antibodies and confocal microscopy to examine the presence of each P2X receptor on human B lymphocytes. We observed that P2X1, P2X2, P2X4 and P2X7 subtypes, but not P2X3, P2X5 and P2X6 subtypes, are present on B lymphocytes.

A Glu-496 to Ala polymorphism leads to loss of function of the human P2X7 receptor.

The P2X(7) receptor is a ligand-gated cation-selective channel that mediates ATP-induced apoptosis of cells of the immune system. We and others have shown that P2X(7) is nonfunctional both in lymphocytes and monocytes from some subjects. To study a possible genetic basis we sequenced DNA coding for the carboxyl-terminal tail of P2X(7). In 9 of 45 normal subjects a heterozygous nucleotide substitution (1513A-->C) was found, whereas 1 subject carried the homozygous substitution that codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X(7) on lymphocytes was not affected by this E496A polymorphism, demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the E496A homozygote subject expressed nonfunctional receptor, whereas heterozygotes showed P2X(7) function that was half that of germline P2X(7). Results of transfection experiments showed that the mutant P2X(7) receptor was nonfunctional when expressed at low receptor density but regained function at a high receptor density. This density dependence of mutant P2X(7) function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma, which up-regulated mutant P2X(7) and partially restored its function. P2X(7)-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X(7) compared with germline (8.6 versus 35.2%). The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X(7) receptor.

Detection of preneoplasia in histologically normal prostate biopsies.

P2X immunolabeling of prostate detected preneoplastic changes in apparently normal tissue. Labeling occurred in two well-defined stages before the diagnostic histological markers of cancer were visible. As cancer progressed, the location of P2X expression changed from confinement within individual nuclei in the acini (stage 1) to a cytoplasmic punctate label in the acinal epithelium, with an associated removal of nuclear stain (stage 2). Finally, in advanced cases, where clear morphological evidence of cancer was apparent, the P2X label condensed exclusively on the apical epithelium (stage 3). BPH/normal tissue was entirely devoid of P2X label. Biopsy samples (77) were tested in three categories. One group (35) were diagnosed as normal benign prostatic hyperplasia (BPH) on the basis of haematoxylin and eosin (H&E) stain, although underlying disease was suspected. Of these, 14 (40%) were clearly normal and appeared entirely devoid of label, 13 (37%) exhibited the first stage of P2X receptor labeling and the remaining eight (23%) exhibited second stage labeling. The accompanying H&E-stained sections of all these cases had a normal appearance. Low grade cancer biopsy samples with Gleason scores G4-7 (25) all revealed widespread second stage receptor labeling in areas of both normal and cancerous morphology, while 17 high grade cancer biopsy samples (Gleason G8-10) all showed third stage labeling along with some residual second stage labeling. The features of each P2X labeling stage occupied the entire histological area affected, offering more opportunity to diagnose the tissue than was supplied by the more-localised diagnostic features identified by H&E-stain. Besides detecting cases of preneoplasia in biopsies with a normal H&E appearance, this technique was also able to rule out the presence of neoplasia in purely hyperplasic prostates by the absence of any P2X labeling.Prostate Cancer and Prostatic Diseases (2001) 4, 92-96

The purinergic calcium channels P2X1,2,5,7 are down-regulated while P2X3,4,6 are up-regulated during apoptosis in the ageing rat prostate.

Subtype-specific antibodies were used to measure purinergic (P2X) receptor expression in the rat prostate. In mature Wistar rats, apoptosis and expression of P2X1, P2X2, P2X5 and P2X7 subtypes were all significantly decreased compared with the levels found in immature rat prostates. Accompanying this age-related reduction in purinergic calcium channel expression was a reduction in epithelial and stromal calcium as well as the calcium-regulating hormone stanniocalcin. In contrast, expression of P2X3, P2X4 and P2X6 increased with age. These results suggest that distinct changes in P2X subtype expression accompany apoptosis in the rat prostate.

Changes in the distribution of different subtypes of P2X receptor clusters on smooth muscle cells in relation to nerve varicosities in the pregnant rat urinary bladder.

Clusters of purinergic receptor subunits, about 1 microm diameter, are found on the smooth muscle cell membrane beneath junctional varicosities in the detrusor muscle of the rat urinary bladder. We have examined the extent of redistribution of the six different subunit clusters, P2X(1) to P2X(6), with respect to junctional varicosities during pregnancy, as it is known that the detrusor muscle undergoes changes in purinergic innervation during this period. Before pregnancy, clusters at junctional varicosities are principally composed of the subtypes P2X(1), P2X(2), P2X(3) and P2X(5). However this subtype distribution changes dramatically during pregnancy, such that by day 14 of pregnancy, the extent of P2X(1), P2X(2), P2X(3) and P2X(5) junctional clusters has decreased by more than 80% whereas the extent of P2X(4) and P2X(6) junctional clusters has increased by more than 80%. These changes were confirmed with Western blots for different subtypes. It is suggested that the changes in the purinergic innervation of the detrusor muscle during pregnancy reflect changes in the P2X subtypes found on the smooth muscle membrane beneath junctional varicosities.

Comparative study on the distribution patterns of P2X(1)-P2X(6) receptor immunoreactivity in the brainstem of the rat and the common marmoset (Callithrix jacchus): association with catecholamine cell groups.

The present study investigated the topographical distribution of P2X(1)-P2X(6) receptor subtypes in the rat and common marmoset hindbrain by immunohistochemistry. In addition, double-labeling immunofluorescence was used to determine the extent of colocalization between catecholamine cell groups and the various P2X receptors. The data demonstrate a widespread distribution pattern for all six P2X receptors throughout both the rat hindbrain and the marmoset hindbrain, although distinctions between species, brain nuclei, and P2X receptor subtypes exist. In rat, dense staining for the P2X receptors was found in the nucleus of the solitary tract (NTS), medial vestibular nucleus, and medial and lateral parabrachial nuclei. Moderate staining was observed in the hypoglossal nucleus, cuneate nucleus, inferior olive, prepositus hypoglossi, rostral ventrolateral medulla (RVLM), and locus coeruleus. Staining was also observed in the gracile nucleus, the mesencephalic trigeminal nucleus, and the central pontine gray. In marmoset, prominent P2X receptor-like immunoreactivity occurred in the NTS, medial cuneate nucleus, prepositus hypoglossi, and medial vestibular nucleus. Moderate staining was observed in the area postrema, dorsal motor nucleus of the vagus, lateral cuneate, lateral reticular, spinal trigeminal nucleus, RVLM, and inferior olive. Immunofluorescent double labeling of tyrosine hydroxylase (TH)-containing cells revealed that all subtypes of P2X receptors show some degree of colocalization with TH. The highest proportion of TH and P2X receptor double labeling was in the A5 region (with the P2X(2) subunit), whereas the lowest proportion of double-labeled cells occurred in the C2 region of the NTS for the P2X(5) subunit. These findings support a role for extracellular adenosine 5'-triphosphate in fast synaptic neurotransmission within the brainstem.