Immunohistochemical localisation of the creatine transporter in the rat brain.

Creatine (Cr) is required to maintain ATP levels in the brain. The transport of Cr across the blood-brain barrier and into neurones requires a specific creatine transporter (CRT). Mutations in the CRT gene (SLC6A8) result in a novel form of X-linked mental retardation, characterised by developmental delays, seizures and a complete absence of Cr from the brain. To identify cell types and regions that depend on Cr for energy metabolism we have determined the regional and cellular localisation of CRT protein in the rat brain using immunohistochemical techniques with a highly specific, affinity-purified, CRT antibody. The results show high levels of CRT localisation is associated with specific brain regions and certain cell types. The CRT is predominantly found in neurones. CRT immunoreactivity is particularly abundant in the olfactory bulb, granule cells of the dentate gyrus of the hippocampus, pyramidal neurones of the cerebral cortex, Purkinje cells of the cerebellum, motor and sensory cranial nerve nuclei in the brainstem and the dorsal and ventral horns of the spinal cord. Low levels of CRT were seen in the basal ganglia and white matter. Overall, CRT was found to show high intensities of labelling in the major motor and sensory regions of the forebrain, brainstem and spinal cord and forebrain regions associated with learning, memory and limbic functions. It is hypothesised that regions with high CRT expression are likely to have high metabolic ATP requirements and that areas with low CRT levels are those regions which are particularly vulnerable in neurodegenerative diseases.

Cysteine 144 in the third transmembrane domain of the creatine transporter is located close to a substrate-binding site.

All creatine transporters contain a cysteine residue (Cys(144)) in the third transmembrane domain that is not present in other members of the Na+,Cl(-)-dependent family of neurotransmitter transporters. Site-directed mutagenesis and reaction with methane thiosulfonates were used to investigate the importance of Cys(144) for transporter function. Replacement of Cys(144) with Ser did not significantly affect the kinetics or activity of the transporter, whereas a C144A mutant had a higher K(m) (0.33 compared with 0.18 mm). Substitution of Cys(144) with Leu gave a mutant with a 5-fold higher K(m) and a reduced specificity for substrate. Low concentrations of 2-aminoethyl methanethiosulfonate (MTSEA) resulted in rapid inactivation of the creatine transporter. The C144S mutant was resistant to inactivation, indicating that modification of Cys(144) was responsible for the loss of transport activity. Creatine and analogues that function as substrates of the creatine transporter were able to protect from MTSEA inactivation. Na+ and Cl(-) ions were not necessary for MTSEA inactivation, but Na+ was found to be important for creatine protection from inactivation. Our results indicate that cysteine 144 is close to the binding site or part of a permeation channel for creatine.

Helicobacter pylori and Meckel's diverticula.

BACKGROUND: Helicobacter pylori is known to infect only gastric mucosa and is strongly associated with gastroduodenal ulceration. The authors studied whether H. pylori colonizes the gastric mucosa of Meckel's diverticula, and determined its relationship to "gastritis" and bleeding. METHODS: A 10-year retrospective review identified 45 children with Meckel's diverticulum. Hematoxylin-eosin and Diff-Quik stains were used to assess the presence and severity of gastritis, and to highlight organisms in the resected diverticula. Cases with organisms were then studied with antibodies specific for H. pylori using immunoperoxidase methods. RESULTS: Twenty-eight children, 7 months to 12.6 years of age, had lower gastrointestinal hemorrhage caused by Meckel's diverticulum and had positive radionuclide scans. All had acid-secreting mucosa in their diverticula, and ulceration. "Chronic gastritis" and eosinophilia were constant findings; "acute gastritis" was present in four patients. Twenty specimens exhibited lymphoid follicles in the gastric mucosa. Seventeen patients with Meckel's diverticula (age range, 1 month-14.7 years) who presented with acute abdominal pain associated with intussusception were used for comparison. Acid-secreting gastric mucosa was seen in four patients. H. pylori was identified in only one of the 45 patients; this patient had ulceration and moderate "acute gastritis." CONCLUSIONS: H. pylori does not colonize a substantial number of children who have ulcerated and bleeding Meckel's diverticulum in the presence of acid-secreting mucosa. Although H. pylori is a notable cause of ulceration, the authors confirm that ulceration is possible in its absence, and alternative mechanisms of ulceration are important. The presence of lymphoid follicles in Meckel's diverticula, unlike gastric biopsies, is not associated with H. pylori.

Differential expression of the noradrenaline transporter in adrenergic chromaffin cells, ganglion cells and nerve fibres of the rat adrenal medulla.

Expression of the noradrenaline transporter (NAT) was identified in various cell and fibre populations of the rat adrenal medulla, examined with immunohistochemistry and confocal microscopy. Immunoreactivity for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), aromatic-L-amino-acid decarboxylase (AADC) and dopamine beta-hydroxylase (DBH) was present in all chromaffin cells, while phenylethanolamine N-methyltransferase (PNMT) was used to determine adrenergic chromaffin cell groups. Labelling with NAT antibody was predominantly cytoplasmic and colocalised with PNMT immunoreactivity. Noradrenergic chromaffin cells were not NAT immunoreactive. Additionally, NAT antibody labelling demonstrated clusters of ganglion cells (presumably Type I) and nerve fibres. Expression of TH, AADC, DBH, PNMT and NAT mRNA was examined using reverse transcription-polymerase chain reaction (RT-PCR) from adrenal medulla punches and single chromaffin cells, and results were consistent with those obtained with immunocytochemistry. Chromaffin cells and fibres labelled with antibodies against growth associated protein-43 (GAP-43) were not NAT immunoreactive, while ganglion cells were doubled labelled with the two antibodies. The presence of NAT in adrenergic chromaffin cells, and its absence from noradrenergic cells, suggests that the adrenergic cell type is primarily responsible for uptake of catecholamines in the adrenal medulla.

P2X2 receptor expression by interstitial cells of Cajal in vas deferens implicated in semen emission.

Male reproduction is dependent upon seminal emission mediated by vas deferens contraction. This drives spermatic fluid to the prostatic urethra during ejaculation. We localize interstitial cells of Cajal (ICC), which express P2X2 receptor, subunits of ATP-gated ion channels, to rat, mouse and guinea-pig vas deferens submucosa. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of rat vas deferens resolved two functional splice variant transcripts of the P2X2 receptor subunit. The P2X2 receptor mRNA was localized principally within the lamina propria (submucosal) region of the rat vas deferens using in situ hybridization (ISH) and in situ RT-PCR-ISH. Immunohistochemistry using rat, mouse and guinea-pig vas deferens tissues confirmed expression of P2X2 receptor protein within the lamina propria, particularly within a dense column of small spindle-shaped cells adjacent to the columnar epithelial cells which line the lumen. This immunoreactivity was co-localized with neurone-specific enolase (NSE) and c-Kit protein expression, gene markers for ICC. Mucosal mast cells were distinguished from ICC by toluidine blue staining. Choline acetyltransferase immunoreactivity, a marker for post-ganglionic parasympathetic innervation, occurred on the lateral margin of the lamina propria and extended into the inner longitudinal muscle layer. P2X1 receptor immunolabelling was associated with sympathetic innervation of the smooth muscle in the outer longitudinal and circular muscle layers, but not the inner longitudinal layer. The physiological significance of the vas deferens ICC which express P2X2 receptors remains to be established. Possible roles include regulation of smooth muscle activity or mucosal secretion utilizing local ATP signaling, both of which would affect semen transport.

Cloning of a mucin-desulfating sulfatase gene from Prevotella strain RS2 and its expression using a Bacteroides recombinant system.

A gene encoding the mucin-desulfating sulfatase in Prevotella strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl- and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into Escherichia coli expression vectors. However, cloning the gene into a Bacteroides expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.

Creatine accumulation and exchange by HEK293 cells stably expressing high levels of a creatine transporter.

We have generated a stable HEK293 cell line expressing high levels of a creatine transporter (CREAT). This cell line (HEK293-CREAT) was used to study the properties of CREAT in terms of the accumulation and release of creatine. HEK293-CREAT cells accumulated high steady state levels of creatine under saturating creatine levels (approx. 25-fold higher intracellular creatine levels than seen in control cells). The accumulation of high levels of creatine affected [3H]creatine uptake by decreasing the Vmax for transport. High intracellular creatine levels were maintained in the absence of extracellular creatine. External creatine stimulated the release of stored creatine by an exchange mechanism dependent on extracellular Na+. These studies have shown that cellular creatine levels can be affected by the amount of creatine transporter in the membrane and exchange through the creatine transporter. These findings highlight the importance of the creatine transporter in the maintenance of intracellular creatine levels.

Expression of the P2X(2) receptor subunit of the ATP-gated ion channel in the cochlea: implications for sound transduction and auditory neurotransmission.

Extracellular ATP has multimodal actions in the cochlea affecting hearing sensitivity. ATP-gated ion channels involved in this process were characterized in the guinea pig cochlea. Voltage-clamped hair cells exhibited a P2 receptor pharmacology compatible with the assembly of ATP-gated ion channels from P2X(2) receptor subunits. Reverse transcription-PCR experiments confirmed expression of the P2X(2-1) receptor subunit mRNA isoform in the sensory epithelium (organ of Corti); a splice variant that confers desensitization, P2X(2-2), was the predominant subunit isoform expressed by primary auditory neurons. Expression of the ATP-gated ion channel protein was localized using a P2X(2) receptor subunit-specific antiserum. The highest density of P2X(2) subunit-like immunoreactivity in the cochlea occurred on the hair cell stereocilia, which faces the endolymph. Tissues lining this compartment exhibited significant P2X(2) receptor subunit expression, with the exception of the stria vascularis. Expression of ATP-gated ion channels at these sites provides a pathway for the observed ATP-induced reduction in endocochlear potential and likely serves a protective role, decoupling the "cochlear amplifier" in response to stressors, such as noise and ischemia. Within the perilymphatic compartment, immunolabeling on Deiters' cells is compatible with purinergic modulation of cochlear micromechanics. P2X(2) receptor subunit expression was also detected in spiral ganglion primary afferent neurons, and immunoelectron microscopy localized these subunits to postsynaptic junctions at both inner and outer hair cells. The former supports a cotransmitter role for ATP in a subset of type I spiral ganglion neurons, and latter represents the first characterization of a receptor for a fast neurotransmitter associated with the type II spiral ganglion neurons.

Localization of the noradrenaline transporter in rat adrenal medulla and PC12 cells: evidence for its association with secretory granules in PC12 cells.

The noradrenaline transporter (NAT) is present in noradrenergic neurons and a few other specialized cells such as adrenal medullary chromaffin cells and the rat pheochromocytoma (PC12) cell line. We have raised antibodies to a 49-residue segment (NATM2) of the extracellular region (residues 184-232) of bovine NAT. Affinity-purified NATM2 antibodies specifically recognized an 80-kDa band in PC12 cell membranes by western blotting. Bands of a similar size were also detected in membranes from human neuroblastoma (SK-N-SH) cells expressing endogenous NAT and human embryonic kidney (HEK293) cells stably expressing bovine NAT. Immunocytochemistry of rat adrenal tissue showed that NAT staining was colocalized with tyrosine hydroxylase in medullary chromaffin cells. Most NAT immunoreactivity in rat adrenal chromaffin and PC12 cells was present in the cytoplasm and had a punctate appearance. Cell surface biotinylation experiments in PC12 cells confirmed that only a minor fraction of the NAT was present at the cell surface. Subcellular fractionation of PC12 cells showed that relatively little NAT colocalized with plasma membrane, synaptic-like microvesicles, recycling endosomes, or trans-Golgi vesicles. Most of the NAT was associated with [3H]noradrenaline-containing secretory granules. Following nerve growth factor treatment, NAT was localized to the growing tip of neurites. This distribution was similar to the secretory granule marker secretogranin I. We conclude that the majority of NAT is present intracellularly in secretory granules and suggest that NAT may undergo regulated trafficking in PC12 cells.

Distribution of the P2X2 receptor subunit of the ATP-gated ion channels in the rat central nervous system.

The distribution of the P2X2 receptor subunit of the adenosine 5'-triphosphate (ATP)-gated ion channels was examined in the adult rat central nervous system (CNS) by using P2X2 receptor-specific antisera and riboprobe-based in situ hybridisation. P2X2 receptor mRNA expression matched the P2X2 receptor protein localisation. An extensive expression pattern was observed, including: olfactory bulb, cerebral cortex, hippocampus, habenula, thalamic and subthalamic nuclei, caudate putamen, posteromedial amygdalo-hippocampal and amygdalo-cortical nuclei, substantia nigra pars compacta, ventromedial and arcuate hypothalamic nuclei, supraoptic nucleus, tuberomammillary nucleus, mesencephalic trigeminal nucleus, dorsal raphe, locus coeruleus, medial parabrachial nucleus, tegmental areas, pontine nuclei, red nucleus, lateral superior olive, cochlear nuclei, spinal trigeminal nuclei, cranial motor nuclei, ventrolateral medulla, area postrema, nucleus of solitary tract, and cerebellar cortex. In the spinal cord, P2X2 receptor expression was highest in the dorsal horn, with significant neuronal labeling in the ventral horn and intermediolateral cell column. The identification of extensive P2X2 receptor immunoreactivity and mRNA distribution within the CNS demonstrated here provides a basis for the P2X receptor antagonist pharmacology reported in electrophysiological studies. These data support the role for extracellular ATP acting as a fast neurotransmitter at pre- and postsynaptic sites in processes such as sensory transmission, sensory-motor integration, motor and autonomic control, and in neuronal phenomena such as long-term potentiation (LTP) and depression (LTD). Additionally, labelling of neuroglia and fibre tracts supports a diverse role for extracellular ATP in CNS homeostasis.

Noradrenaline transporter expression in the pons and medulla oblongata of the rat: localisation to noradrenergic and some C1 adrenergic neurones.

Catecholaminergic neurotransmission is normally terminated by rapid re-uptake of the neurotransmitter by a high-affinity Na+/Cl--dependent plasma membrane transporter. Specific transporters have been cloned for both dopamine (DAT) and noradrenaline (NAT) in the rat. While DAT has been studied extensively, NAT expression has received less attention, particularly at the protein level. We used an antibody generated against a 49 residue segment of an extracellular loop region of NAT to study expression of the transporter protein throughout the rat pons and medulla oblongata. NAT was expressed in over 95% of noradrenergic neurones in the A1, A2/area postrema, A5, A6/locus subcoeruleus, and A7 noradrenergic groups. Approximately 10% of C1 adrenergic neurones located in the rostral ventrolateral medulla (RVL) also expressed NAT. Expression of NAT mRNA in bulbospinal C1 cells was confirmed using single-cell reverse transcription polymerase chain reaction (RT-PCR) of acutely isolated RVL neurones. Spinally projecting neurones were identified by retrograde labelling with rhodamine beads, and C1 neurones were identified by RT-PCR using primers specific for tyrosine hydroxylase (TH) or phenylethanolamine N-methyltransferase (PNMT) mRNAs. Thirteen percent of adrenergic bulbospinal neurones tested expressed NAT mRNA. C1 neurones are potentially important in cardiovascular control and blood pressure regulation, and the identification of NAT expression in a sub-population of these neurones provides further evidence for the heterogeneity of this neuronal population.

A variant of the bovine noradrenaline transporter reveals the importance of the C-terminal region for correct targeting to the membrane and functional expression.

We have characterized a cDNA clone which encodes a variant (bNAT2) of the bovine noradrenaline transporter. This cDNA differs from the previously identified bovine noradrenaline transporter (bNAT1) in the sequence encoding part of the cytoplasmic-facing C-terminus and the 3'-untranslated region. The bNAT1 and bNAT2 cDNA clones are encoded by a 5.8 and 3.6 kb mRNA species respectively. The bNAT1 and bNAT2 proteins, which are identical apart from their C-terminal 31 and 18 residues, were stably expressed in HEK293 cells. Cells expressing bNAT1 showed a high level of desipramine-sensitive [3H]noradrenaline uptake activity, whereas no activity was present in bNAT2 cells. The bNAT1 and bNAT2 proteins were present as major 80 and 50 kDa species respectively. Cells expressing bNAT1 showed strong immunostaining of the plasma membrane, whereas bNAT2 was present in the endoplasmic reticulum/Golgi region. Treatment of membrane samples from bNAT1 cells with peptide N-glycosidase F resulted in the formation of a predominantly 50 kDa species, but little effect was observed after similar treatment of bNAT2 cell membranes. These results indicate that bNAT2 is retained in the endoplasmic reticulum and that the glycosylation of this variant differs from that of bNAT1. The characterization of bNAT2 and its comparison with bNAT1 highlight the importance of the cytoplasmic-facing C-terminus for the intracellular trafficking of neurotransmitter transporters.

Predictors of hemolytic uremic syndrome in children during a large outbreak of Escherichia coli O157:H7 infections.

OBJECTIVE: To evaluate risk factors for progression of Escherichia coli O157:H7 infection to the hemolytic uremic syndrome (HUS). STUDY DESIGN: We conducted a retrospective cohort study among 278 Washington State children <16 years old who developed symptomatic culture-confirmed E coli O157:H7 infection during a large 1993 outbreak. The purpose of the study was to determine the relative risk (RR) of developing HUS according to demographic characteristics, symptoms, laboratory test results, and medication use in the first 3 days of illness. RESULTS: Thirty-seven (14%) children developed HUS. In univariate analysis, no associations were observed between HUS risk and any demographic characteristic, the presence of bloody diarrhea or of fever, or medication use. In multivariate analysis, HUS risk was associated with, in the first 3 days of illness, use of antimotility agents (odds ratio [OR] = 2.9; 95% confidence interval [CI] 1.2-7.5) and, among children <5.5 years old, vomiting (OR = 4. 2; 95% CI 1.4-12.7). Among the 128 children tested, those whose white blood cell (WBC) count was >/=13 000/microL in the first 3 days of illness had a 7-fold increased risk of developing HUS (RR 7. 2; 95% CI 2.8-18.5). Thirteen (38%) of the 34 patients with a WBC count >/=13 000/microL developed HUS, but only 5 (5%) of the 94 children whose initial WBC count was <13 000/microL progressed to HUS. Among children who did not develop HUS, use of antimotility agents in the first 3 days of illness was associated with longer duration of bloody diarrhea. CONCLUSIONS: Prospective studies are needed to further evaluate measures to prevent the progression of E coli O157:H7 infection to HUS and to assess further clinical and laboratory risk factors. These data argue against the use of antimotility agents in acute childhood diarrhea. Our finding that no intervention decreased HUS risk underscores the importance of preventing E coli O157:H7 infections.

Localization of ATP-gated ion channels in cerebellum using P2x2R subunit-specific antisera.

The distribution of the P2x2 purinoceptor subunit protein, which forms ATP-gated ion channels by homo- and hetero-multimeric assembly, was examined in the adult rat and guinea-pig cerebellum using two novel antisera generated against separate 18 amino acid sequences located in the predicted extracellular domain of this subunit. These antisera, the first available for labelling the P2x2R subunit protein, were validated by selective labelling of a fusion protein containing the target amino acid sequences, and in cerebellum, by peptide specific block of immunoreactivity and by comparison with the distribution of P2x2R mRNA. P2x2R-like immunoreactivity was seen in Purkinje cells, specifically the soma and dendrites, neurons in the granular and molecular layers and deep cerebellar nuclei. The identification of P2x2R-like immunoreactivity within the cerebellar neural circuitry is consistent with a role for extracellular ATP acting as a fast neurotransmitter in motor learning and coordination of movement. Additionally, labelling of neuroglia and fibre tracts supports a diverse role for extracellular ATP in CNS homeostasis.

Biliary atresia, cytomegalovirus, and age at referral.

OBJECTIVE: To determine the frequency with which patients with extrahepatic biliary atresia (EHBA) are infected with cytomegalovirus (CMV) and to ascertain the age at referral to a specialty center for surgical correction of EHBA. METHODS: The charts of all patients discharged from the Children's Hospital and Medical Center between July 1, 1989 and December 31, 1993 with a new diagnosis of EHBA were reviewed to determine the frequency with which EHBA was accompanied by CMV infection. Data analyzed included age at referral and sex of patients, histopathologic evidence of CMV infection and size of bile ducts in the resected liver, and serologic (IgM) or culture diagnosis of CMV infection. RESULTS: Twenty-three patients with EHBA were evaluated at Children's Hospital and Medical Center in the study period. Twenty-one of the patients with EHBA were appropriately evaluated for infection with CMV and infection was documented in 5 (24%) patients. The median age of referral for all patients was 61 days (range 10 to 124 days). Infected patients were referred later (82.4 +/- 28.7 days) than noninfected patients (48.8 +/- 21.8 days) (P = .01) and were more likely to be girls, bu the medians of the diameters of the bile ducts in the resected porta hepatis were similar. Viral inclusions were not identified in any of the liver specimens. CONCLUSIONS: CMV infection is present in an unexpectedly large proportion of patients with EHBA at the time of referral. The establishment of CMV infection in infants with cholestasis should not deter the search for EHBA. Physicians should strive to reduce the age of referral of patients with EHBA to pediatric surgical centers by evaluating infants who remained jaundiced at 4 weeks of age.

Comparison of the molecular forms of the Kex2/subtilisin-like serine proteases SPC2, SPC3, and furin in neuroendocrine secretory vesicles reveals differences in carboxyl-terminus truncation and membrane association.

The molecular forms and membrane association of SPC2, SPC3, and furin were investigated in neuroendocrine secretory vesicles from the anterior, intermediate, and neural lobes of bovine pituitary and bovine adrenal medulla. The major immunoreactive form of SPC2 was the full-length enzyme with a molecular mass of 64 kDa. The major immunoreactive form of SPC3 was truncated at the carboxyl terminus and had a molecular mass of 64 kDa. Full-length 86-kDa SPC3 with an intact carboxyl terminus was found only in bovine chromaffin granules. Immunoreactive furin was also detected in secretory vesicles. The molecular masses of 80 and 76 kDa were consistent with carboxyl-terminal truncation of furin to remove the transmembrane domain. All three enzymes were distributed between the soluble and membrane fractions of secretory vesicles although the degree of membrane association was tissue specific and, in the case of SPC3, dependent on the molecular form of the enzyme. Significant amounts of membrane-associated and soluble forms of SPC2, SPC3, and furin were found in pituitary secretory vesicles, whereas the majority of the immunoreactivity in chromaffin granules was membrane associated. More detailed analyses of chromaffin granule membranes revealed that 86-kDa SPC3 was more tightly associated with the membrane fraction than the carboxyl terminus-truncated 64-kDa form.