Biomedical subjects

R H Edwards

Publications and source records attributed to R H Edwards.

At least 73 records · Page 4Linked to original sources

PubMed1997

Charged residues in transmembrane domains II and XI of a vesicular monoamine transporter form a charge pair that promotes high affinity substrate recognition.

Vesicular monoamine transporters package monoamine neurotransmitters into secretory vesicles for regulated exocytotic release. Both vesicular monoamine transporter 1 and 2 contain several charged residues predicted to reside within transmembrane domains (TMDs), and conservation of these residues in multiple species and in other members of the gene family suggest important roles in transporter structure and function. To determine the role of these residues, we have used site-directed mutagenesis. Replacement of Asp-263 in TMD6 with Asn (D263N) had no effect on transport activity. However, replacement of Lys-139 in TMD2 with Ala (K139A), Asp-400 in TMD10 with Asn (D400N), or Asp-427 in TMD11 with Asn (D427N) eliminated transport activity despite normal levels of protein expression. Remarkably, the double mutant K139A/D427N showed substantial transport activity, suggesting that Lys-139 and Asp-427 interact to form an ion pair in the native protein and hence that TMD2 occurs next to TMD11. Nonetheless, the double mutant showed reduced apparent affinity for serotonin and reduced ability of serotonin to inhibit reserpine binding, suggesting that although not required for activity, the ion pair promotes high affinity interaction with the substrate. In addition, a double mutant in which the polarity of the charged residues was reversed (K139D/D427K) showed no active transport. Remarkably, however, this mutant displayed normal reserpine binding that remained coupled to DeltaH+, but serotonin failed to inhibit reserpine binding, suggesting that the charge reversal specifically disrupts substrate recognition.

Animals↗

PubMed1997

Vesicular monoamine transporter-2: immunogold localization in striatal axons and terminals.

The vesicular monoamine transporter-2 (VMAT2) mediates the reserpine-sensitive neuronal uptake of monoamines into vesicles and other intracellular organelles. Accordingly, this transporter is expressed at high levels in regions that contain a dense monoamine innervation, such as the rat dorsolateral striatum. We used ultrastructural immunocytochemistry in this region to show that immunogold labeling for VMAT2 is present in varicose axonal processes, many of which also contain the catecholamine-synthesizing enzyme tyrosine-hydroxylase. Within these mainly dopaminergic processes, VMAT2 was associated with small synaptic vesicles (SSVs) and more rarely with large dense-core vesicles or tubulovesicles. These findings suggest that SSVs are the major organelles involved in the storage and release of dopamine in the dorsolateral striatum.

Animals↗

PubMed1997

Chronic sympathetic innervation of islets in transgenic mice results in differential desensitization of alpha-adrenergic inhibition of insulin secretion.

The effects of chronic sympathetic hyperinnervation on pancreatic beta-cell insulin secretion were investigated utilizing the in vitro perfused pancreas from transgenic mice. These mice exhibit islet hyperinnervation of sympathetic neurons resulting from overexpression of nerve growth factor in their beta-cells (1). The goal was to determine whether sympathetic hyperinnervation increased classic alpha-adrenergic inhibition of beta-cell insulin secretion or, in contrast, down-regulated beta-cell sensitivity to adrenergic input resulting in enhanced insulin secretion. Both fasting and fed blood sugars and pancreatic insulin content were normal in the transgenics. Response of the transgenic perfused pancreas to low glucose (7 mM) was primarily first phase and normal whereas high glucose (22 mM) caused enhanced, rather than reduced, insulin secretion of both first and second phases. The alpha-antagonist, phentolamine, caused a six-fold increase in glucose-stimulated insulin secretion from the control pancreas, an effect that was blunted for the transgenic pancreas. A similarly blunted response to phentolamine occurred when this agent was superimposed on a combined glucose-forskolin stimulus. (The positive effect on insulin secretion by phentolamine in normal beta-cell preparations has arguably been ascribed to non-specific ionic effects.) Therefore, as a test of possible changes in the ATP regulated K+ channel or the linked Ca++ channels, glyburide was perfused during glucose stimulation. Insulin secretion in response to glyburide was increased two fold in the control pancreas. However, with the transgenic pancreas, in contrast to the enhanced response to glucose, the effect of glyburide was almost completely inhibited. It is concluded that: 1) chronic adrenergic hyperinnervation results in enhanced glucose-stimulated insulin secretion by desensitization of a major alpha-adrenergic inhibitory site(s); and 2) adrenergic hyperinnervation acts directly or indirectly on ion flux to partially inhibit insulin release, an effect which is not desensitized. Since down-regulation of a single alpha-adrenergic receptor would be expected to desensitize both phenomena the observed differential desensitization indicates that different post receptor events or more than one adrenergic receptor are involved.

Adrenergic alpha-Antagonists↗

PubMed1997

In vivo model of muscle pain: quantification of intramuscular chemical, electrical, and pressure changes associated with saline-induced muscle pain in humans.

Intramuscular injection of hypertonic saline is a good model to study human muscle pain (Kellgren 1938). The present study concerns the intramuscular (i.m.) pain mediators in saline-induced muscle pain. In experiment 1, the diffusion of infused hypertonic and isotonic saline (0.5 ml) in m. tibialis anterior was illustrated by magnetic resonance imaging (MRI) in one subject. In experiment 2, six volunteers received four sequential infusions (0.5 ml given at 5 min intervals) of isotonic saline and thereafter four sequential infusions (0.5 ml given at 5 min intervals) of hypertonic saline into m. tibialis anterior. The isotonic and hypertonic saline infusions were computer-controlled and separated by 20 min. The muscle pain intensity was assessed by continuous recordings on a visual analogue scale (VAS). One microdialysis probe was inserted 1 cm from the infusion needle in m. tibialis anterior and another probe in the other m. tibialis anterior. Concentrations of the i.m. sodium, potassium, magnesium, and prostaglandin E2 (PGE2) were assessed from the dialysates. Intramuscular electromyography (EMG) and pressure were assessed in the area of the infused saline. In experiment 1, the infusion of hypertonic and isotonic saline created a visible saline-pool on the MRI scans. These saline-pool volumes were stable and not correlated to the pain scores. In experiment 2, infusion of isotonic saline produced little pain compared to infusion of hypertonic saline. Maximal pain was reported after the first infusion of hypertonic saline and thereafter the pain gradually decreased with subsequent infusions of hypertonic saline. During infusion of hypertonic saline the i.m. sodium and potassium concentrations increased significantly, i.m. magnesium concentration tended to be increased, and the i.m. PGE2 concentration tended to be decreased although these changes were not significant. The i.m. EMG was smaller during and after infusions of hypertonic saline compared with isotonic saline. The i.m. pressure was not different during the infusions of hypertonic and isotonic saline but was increased between the infusions of hypertonic saline. This study has shown that i.m. infusion of hypertonic saline produced a saline-pool, causing the i.m. pressure to increase. Possibly, pain activation and cessation are related to increased intramuscular sodium and potassium content respectively.

Adult↗

PubMed1997

Expression of a putative vesicular acetylcholine transporter facilitates quantal transmitter packaging.

A putative vesicular acetylcholine transporter (VAChT) was overexpressed in developing Xenopus spinal neurons by injection of rat VAChT cDNA or synthetic mRNA into Xenopus embryos. This resulted in a marked increase in the amplitude and frequency of miniature excitatory postsynaptic currents at neuromuscular synapses, reflecting an over 10-fold increase in the vesicular packaging of acetylcholine (ACh). The effect appeared in developing neurons even before synaptogenesis and was blocked by L-vesamicol, a specific blocker of ACh uptake into synaptic vesicles. Mutational studies showed that two highly conserved aspartate residues within putative transmembrane domains 4 and 10 are essential for the transport activity. These results provide direct evidence for the physiological function of a putative VAChT and demonstrate that quantal size can be regulated by changes in vesicular transporter activity.

Acetylcholine↗

PubMed1997

Vesicular transport regulates monoamine storage and release but is not essential for amphetamine action.

To assess the role of exocytotic release in signaling by monoamines, we have disrupted the neuronal vesicular monoamine transporter 2 (VMAT2) gene. VMAT2-/- mice move little, feed poorly, and die within a few days after birth. Monoamine cell groups and their projections are indistinguishable from those of wild-type littermates, but the brains of mutant mice show a drastic reduction in monoamines. Using midbrain cultures from the mutant animals, amphetamine but not depolarization induces dopamine release. In vivo, amphetamine increases movement, promotes feeding, and prolongs the survival of VMAT2-/- animals, indicating that precise, temporally regulated exocytotic release of monoamine is not required for certain complex behaviors. In addition, the brains of VMAT2 heterozygotes contain substantially lower monoamine levels than those of wild-type littermates, and depolarization induces less dopamine release from heterozygous than from wild-type cultures, suggesting that VMAT2 expression regulates monoamine storage and release.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1997

Quality of life, body composition and muscle strength in adult growth hormone deficiency: the influence of growth hormone replacement therapy for up to 3 years.

OBJECTIVE: Adults with GH deficiency complain frequently of low energy levels, emotional lability and mental fatigue resulting in a low perceived quality of life (QOL). Body composition is altered with increased fat mass and decreased lean body mass and muscle strength is reduced. The aims of this study were to determine the effects of replacement GH treatment on: (a) body composition and muscle strength and (b) QOL, using specifically selected and adapted measures. DESIGN: A 12-month study (double-blind placebo-controlled for the first 6 months and open for the second 6 months) of GH replacement injections (0.125 iu/kg/week for the first month and 0.25 iu/kg/week for the following 5 months of each study period) in GH deficient adults on QOL, body composition and muscle strength. This was followed by an open study of a further 12 months' GH treatment assessing QOL and muscle strength. Finally, QOL was assessed after up to 3 years of GH replacement treatment. PATIENTS: Thirty of the 32 adult patients with GH deficiency enrolled completed the initial 12-month study (10 male, mean age 33.5 years, mean (SD) stimulated serum GH response 3.0 mU/l (2.86)). Nineteen patients then opted to continue GH treatment. Of these, 13 patients were available for assessment after a further 12 months' and 24 months' treatment. MEASUREMENTS: Health-related QOL was assessed using 2 specifically adapted scales for adults with GH deficiency: the Life Fulfillment Scale and the Impact Scale. In addition 4 other self-rating questionnaires were used: Nottingham Health Profile, Hospital Anxiety and Depression Scale, Self Esteem Scale and Mental Fatigue Scale. Body composition was assessed by DEXA and quadriceps muscle strength by measuring maximum voluntary contractions. RESULTS: In the initial 12 months' placebo-controlled study perceived energy levels increased after 6 and 12 months of GH treatment (P < 0.01 compared with baseline) in the patients receiving GH for the full 12-month period. There were no changes in energy levels throughout the study in the group receiving placebo for the first 6 months. Also small improvements in impact scores were found after 6 months of GH treatment (P < 0.05) but this was not sustained at 12 months. In both GH and placebo groups life fulfillment worsened after 6 months, but then improved to baseline values after 12 months. In the patients who persisted with GH replacement, energy levels continued to improve (at 2 years, P < 0.01 compared with baseline) but then fell (at 3 years, P = NS compared with baseline). A similar pattern was observed in emotional reaction scores. However, improvements in self-esteem were maintained (at 3 years, P < 0.05 compared with baseline). Body composition altered favourably over the initial 12-month study period with a significant increase in lean mass and decrease in fat mass in both groups after 6-12 months of GH. There were no changes in muscle strength in either group during the initial 12-month study. However, in the patients who were available for assessment after a further 12 months of GH treatment, muscle strength increased significantly (P < 0.02 compared with baseline). CONCLUSION: GH replacement treatment for 6-12 months leads to significant improvements in body composition (DEXA) but longer-term treatment may be needed to increase muscle strength. Self-esteem scores improve and are maintained after 3 years of treatment. Energy levels and emotional reaction improve during treatment for up to 2 years but decline thereafter.

Adult↗

PubMed1997

Epstein-Barr virus and human herpesvirus 8 prevalence in human immunodeficiency virus-associated oral mucosal lesions.

The prevalence of Epstein-Barr virus (EBV) and the recently identified Kaposi's sarcoma (KS)-associated herpesvirus (also designated human herpesvirus 8 [HHV-8]) was determined in oral lesions and oral neoplasms common to persons with human immunodeficiency virus (HIV) infection. Oral lesions were examined by polymerase chain reaction (PCR) for EBV and HHV-8 DNA and by Southern blot analysis for EBV clonality. EBV was detected by Southern blot in hairy leukoplakia lesions, in a subset of AIDS-related lymphomas, and in saliva from HIV-positive persons but not in pseudohairy leukoplakia lesions, oral aphthous ulcers, or oral KS lesions. EBV was detected, however, by PCR in most of the lesions, while HHV-8 was detected only in oral KSs. The absence of HHV-8 DNA in both the EBV-associated hairy leukoplakia lesions and in the EBV-associated AIDS-related lymphomas strengthens the etiologic relationship of EBV to these pathologies and the etiologic role of HHV-8 in KS.

AIDS-Related Opportunistic Infections↗

PubMed1997

Physiological abnormalities of skeletal muscle in dialysis patients.

BACKGROUND: Muscle weakness is a common but unexplained feature of dialysis patients. This study investigated the prevalence and causes of muscle weakness in dialysis patients by examining the quadriceps muscle force and contractile properties. METHODS: The quadriceps femoris was studied in terms of force, force-frequency curve, and speed of muscle relaxation in 49 dialysis patients and 27 healthy subjects. In addition nutritional, haematological, biochemical, and histological assessments were performed, and steps of force generation were analysed to reach the possible mechanisms leading to the observed weakness. RESULTS: Muscle weakness, though invariable as a symptom, was subtle or absent on clinical examination. Quadriceps force measurements, however, revealed unequivocal weakness in most of the patients (71%). The quadriceps muscle was weaker (317 +/- 115 versus 460 +/- 159 N, P < 0.01) compared to healthy individuals, but there was no evidence of impaired excitation-contraction coupling (0.79 +/- 0.05 versus 0.76 +/- 0.07, P = 0.1). Among dialysis patients the older and the malnourished (n = 23) were the weaker but there was no relationship to the type or duration of dialysis. The serum albumin was the only biochemical parameter related to the muscle force (r = 0.6, P = 0.01). The other most prominent abnormality of quadriceps muscle function observed in this study was slowing of relaxation (patients versus controls; 8.7 +/- 1.8% versus 10.8 +/- 1.1% force loss/10 ms, P < 0.0001) particularly in the malnourished group (malnourished versus well nourished; 8.3 +/- 2.1 versus 9.4 +/- 0.95, P = 0.03). Muscle histology was investigated (n = 12) and revealed that type II fibres were mildly atrophic in 40% of the biopsies in most areas, but predominantly type IIB. Although type IIB fibre areas are slightly smaller in the dialysis patients compared to the controls, this was not statistically significant (3025 +/- 578 versus 4406 +/- 1582, P = 0.1) except in the malnourished group compared to the well-nourished dialysis patients (2092 +/- 304 versus 4346 +/- 1496, P = 0.04), and in the malnourished dialysis patients type IIB fibre area was significantly correlated to the strength (r = 0.6, P = 0.02). CONCLUSIONS: The only significant predictor of loss of muscle strength and abnormality of relaxation in this study was the nutritional state. A regular assessment of the nutritional state is required to ensure adequate nutrition to prevent the observed abnormalities of the skeletal muscles.

Adult↗

PubMed1997

Detection of gene fusions in rhabdomyosarcoma by reverse transcriptase-polymerase chain reaction assay of archival samples.

Alveolar rhabdomyosarcoma is a pediatric soft-tissue tumor that is often difficult to distinguish from other small round-cell tumors. The PAX3-FKHR and PAX7-FKHR gene fusions that result from chromosomal translocations in this tumor provide potential molecular diagnostic markers. To apply these molecular markers to commonly available archival material, we used reverse transcriptase-polymerase chain reaction and oligonucleotide hybridization methodology to develop an assay capable of identifying PAX3-FKHR and PAX7-FKHR fusion transcripts in formalin-fixed, paraffin-embedded tissue. Use of a control assay for wild-type FKHR mRNA indicated that RNA was successfully isolated, reverse-transcribed, and amplified in 15 of 16 archival cases. Comparison of assay results for the PAX3-FKHR and PAX7-FKHR fusions with standard molecular assays of paired frozen material revealed that all eight cases of known fusion-positive rhabdomyosarcoma were correctly identified and distinguished as PAX3-FKHR or PAX7-FKHR. The seven cases of known fusion-negative rhabdomyosarcoma showed no evidence of either product. These results indicate that we have developed a molecular assay that accurately identifies the fusion transcripts characteristic of alveolar rhabdomyosarcoma in archival samples. This assay will be useful for diagnosis and for retrospective clinicopathologic correlative studies.

Base Sequence↗

PubMed1997

Improving outpatient clinic efficiency using computer simulation.

To assess and plan alterations in outpatient clinic structure, produces a computer simulation of an outpatient clinic based on detailed time and role measurements from the authors' clinic. The stimulation which used an object-oriented design method is able to indicate the impact of changes in clinic structure using patient and doctor waiting times in clinic as endpoint measures. The effects of changes in clinic size, consultation time, patient mix, appointment scheduling and non-attendance were examined. We found that patient waiting time could be shortened considerably by using an optimizing appointment scheduler to determine appointment intervals. Clinic mix influences patient waiting time, which was shorter with a 1 in 4 ratio of new to follow-up patients. In mixed clinics, new patients appointments are optimally spread throughout the clinic to reduce patient waiting time. In all new or all follow-up clinics, waiting time is improved if the appointment interval reflects the consultation time. Computer modelling can help in optimizing clinic management so improving the delivery of care in outpatient services.

Appointments and Schedules↗

PubMed1997

Dissociation between metabolic and contractile responses during intermittent isometric exercise in man.

This study examines the temporal changes in high-energy phosphate and metabolic levels, and in force-generating capacity, during and after voluntary submaximal repetitive isometric exercise (RIE). Eight male subjects performed one-legged RIE with the knee extensors at 40% maximum voluntary contraction (MVC) target force (duty cycle: 6 s contraction, 4 s rest) in a 48 cm bore whole body 1.5 T superconducting magnet. Phosphocreatine (PCr), inorganic phosphate (P(i)), ATP and pH were measured every 9 s. Force-generating capacity was repeatedly measured using MVC force and electrically stimulated contractions (sequential train of impulses of 1-100 Hz). During RIE, MVC declined gradually by 56 +/- 5% (mean +/- S.E.M.). Electrically stimulated force also declined, with a disproportionally large drop in low-frequency force, seen as a decline from 0.76 +/- 0.02 to 0.33 +/- 0.02 in 20:50 Hz force ratio. The PCr decline during RIE was 65 +/- 9%, in most subjects seen as a rapid initial drop followed by less or no further decline to exhaustion. pH declined in parallel by 0.18 +/- 0.04 units, whilst ATP levels remained unchanged throughout the exercise. PCr, P(i) and pH recovered to near control values within 5 min of exhaustion. Force, however, was not fully restored after 30 min recovery. The results support the hypothesis that fatigue from submaximal RIE is unrelated to changes in P(i) and H+ levels. The decline in 20:50 Hz force ratio implies that fatigue may be associated with excitation-contraction coupling impairment. No sudden changes were observed in mechanical or metabolic factors at exhaustion. Exhaustion was probably not caused by lack of substrates for ATP resynthesis, since pH had decreased only marginally.

Adenosine Triphosphate↗

PubMed1997

The role of vesicular transport proteins in synaptic transmission and neural degeneration.

Classical neurotransmitters are synthesized in the cytoplasm, so they require transport into secretory vesicles for regulated exocytotic release. Previous work has identified distinct vesicular transport activities for the different classical transmitters, and all depend on the H+-electrochemical gradient across the vesicle membrane but differ in the extent to which they rely on the chemical and electrical components of this gradient. Drugs that interfere with vesicular amine transport have implicated this activity in psychiatric disease. Selection for a cDNA encoding vesicular amine transport in the neurotoxin MPP+ also implicates the activity in Parkinson's disease. Molecular cloning of vesicular monoamine transporters shows sequence similarity to bacterial antibiotic resistance proteins, supporting a role for transport in detoxification and defining a novel mammalian gene family that now also includes a transporter for acetylcholine. Current work focuses on the mechanism of transport and the role that regulation of activity and its subcellular localization have in transmitter release, behavior, and neural degeneration.

Animals↗

PubMed1997

Implications of socio-cultural contexts for the ethics of clinical trials.

BACKGROUND: Health technology assesment (HTA) requires scientifically rigorous experimentation involving patients as subjects. HTA itself is required so that treatment given to patients will be both effective and efficient; this requirement is itself ethical in nature. At the same time it is essential that the methods used in HTA are ethically sound. Most healthcare researchers agree that the most effective and soundest method for assessing treatments is the randomised controlled trial (RCT). However, some researchers believe that the RCT is unethical, either in essence, or for use in some forms of medical research and HTA. Furthermore, many patients seem unable to understand the principles and purposes of the RCT, a factor which is highly detrimental for the validity of informed consent. Informed consent is the key to the ethics of medical research, both in most theories and in all codes of research conduct. Many RCTs therefore risk being unethical in practice, even if ethical in principle. AIM OF REPORT: To survey the main objections to the RCT and its alternatives. To assess the philosophical and methodological basis of these objections, and of the methods recommended for addressing them. To identify areas where objections are founded in social or cultural factors normally overlooked in ethical argument about the RCT methodology. To identify alternative arguments or methods which might resolve ethical conflicts in this area. HOW THE RESEARCH WAS CONDUCTED: The methods used were adapted from systematic reviews in medicine. Systematic searches of Medline, Psychlit and Sociofile CD-ROM databases; hand-searches of the major journals in general medicine and surgery, medical ethics and philosophy; and searches of books were carried out. The literature survey was restricted to articles published or abstracted in English. A database of the most relevant and useful materials was compiled, and is accessible on the Internet (http://www.liv.ac.uk/sdthomps/page1.html). RESEARCH FINDINGS: UNDERSTANDING RCTS AND THEIR ALTERNATIVES: There is some evidence of difficulty in understanding the aims and methods of RCTs, and some disquiet about elements of the RCT methodologies. These objections are well known and much discussed, and concern the use of placebo, the continuation of trials after significant differentials in benefit or harm are apparent, and randomisation. CULTURAL OR RELIGIOUS OBJECTIONS: There was an absence of evidence of cultural or religious objections to randomisation, placebo or other kinds of controlled prospective trials. This most likely reflects an absence of research rather than absence of objections. (ABSTRACT TRUNCATED)

Cultural Diversity↗

PubMed1996

Differential distribution of the putative vesicular transporter for acetylcholine in the rat central nervous system.

The organization and distribution of the mRNA for the putative vesicular transporter for acetylcholine (VAChT) was studied in the rat brain by use of digoxigenin-labeled riboprobes and in situ hybridization technology. Signal was observed in all neural regions deduced to contain cholinergic somata on the basis of previous histochemical investigations employing choline acetyltransferase riboprobes and prior immunocytochemical studies with antibodies against choline acetyltransferase. It was absent in areas believed to contain no cholinergic neurons. Anti-sense riboprobes hybridized to the mRNA for the putative VAChT: (a) the projection neurons of the various nuclei of the basal nuclear complex, (b) the local circuit cells of the dorsal and ventral striata, (c) the projection neurons of the mesopontine complex, (d) perikarya in the ventral 2/3 of the medial habenula, (e) the somatic motor and autonomic cells of cranial nerves 3-7 and 9-12, as well as perikarya in the dorsal and ventral cochlear nuclei presumably giving rise to efferent fibers of cranial nerve 8, and (f) the alpha-motor and gamma-efferent motor neurons of the spinal cord. In addition, the mRNA for the VAChT was found in a few somata, probably ectopically located cells of the basal nuclear complex, in the internal capsule, central nucleus of the amygdala, entopeduncular nucleus, and zona incerta. It was also detected in some cell bodies in the reticular part of the substantia nigra, probably the rostral extension of the mesopontine complex, in the parabigeminal nucleus, and around the central canal in the spinal cord but not in cortical, hippocampal, and cerebellar perikarya. It is concluded that, like choline acetyltransferase, the mRNA for the putative acetylcholine vesicular transporter is another specific marker for neurons utilizing acetylcholine as a neurotransmitter. Further investigations of that transporter could have important implications for various diseases involving cholinergic systems, such as Alzheimer's disease.

Acetylcholine↗

PubMed1996

Vesicular monoamine transporter 2 expression in enteric neurons and enterochromaffin-like cells of the rat.

The cellular localization of the vesicular monoamine transporter 2 (VMAT2) in the rat digestive tract was investigated with immunohistochemistry. VMAT2-immunoreactivity (IR) was localized to neurons and fibers of enteric and pancreatic ganglia, to processes supplying the gut wall, the pancreas and blood vessels, and to enterochromaffin-like (ECL) cells in the gastric corpus, which contained calbindin-IR. Few VMAT2-IR cells were also found in the gastric antrum, but they did not contain gastrin-IR. VMAT2-IR was expressed in extrinsic sympathetic fibers as demonstrated by the elimination of a portion of VMAT2-IR processes by sympathectomy. The VMAT2-IR pattern is consistent with the overall distribution of biogenic amine cell groups in the digestive tract. Our results provide further evidence that VMAT2 is the vesicular amine transporter responsible for accumulation of monoamines into secretory vesicles of monoaminergic neurons and ECL cells.

Animals↗

PubMed1996

Measurement of the turnover of glycogen phosphorylase by GC/MS using stable isotope derivatives of pyridoxine (vitamin B6).

The majority of vitamin B6 in the body is in skeletal muscle, bound as the cofactor pyridoxal 5'-phosphate to one abundant protein, glycogen phosphorylase. Previous work has established that radiolabelled vitamin B6 can be used as a turnover label for glycogen phosphorylase. In this study, a stable isotope derivative of pyridoxine {dideuterated pyridoxine; 3-hydroxy-4-(hydroxymethyl) -5-[hydroxymethyl-2H2]-2-methylpyridine} ([2H2]PN) has been used as a metabolic tracer to study the kinetics of labelling of the body pools of vitamin B6 in mice. A non-invasive method was developed in which the isotope abundance of the urinary excretory product of vitamin B6 metabolism, 4-pyridoxic acid, was analysed by GC/MS. The change in isotope abundance of urinary 4-pyridoxic acid following administration of [2H2]PN reflects the kinetics of labelling of the body pools of vitamin B6, and yields, non-invasively, the rate of degradation of glycogen phosphorylase.

Animals↗

PubMed1996

Ultrastructural localization of the vesicular monoamine transporter-2 in midbrain dopaminergic neurons: potential sites for somatodendritic storage and release of dopamine.

Midbrain dopaminergic neurons are known to release dopamine from somata and/or dendrites located in the substantia nigra (SN) and the ventral tegmental area (VTA). There is considerable controversy, however, about the subcellular sites for somatodendritic dopamine storage in these regions. In the present study, we used dual-labeling electron microscopic immunocytochemistry to localize the vesicular monoamine transporter-2 (VMAT2), a novel marker for sites of intracellular monoamine storage, within identified dopaminergic (tyrosine hydroxylase-containing) neurons in the rat SN and VTA. In dopaminergic perikarya, immunogold labeling for VMAT2 was localized to the Golgi apparatus, tubulovesicles that resembled smooth endoplasmic reticulum (SER), and the limiting membranes of multivesicular bodies. In dopaminergic dendrites, VMAT2 was extensively localized to tubulovesicles that resembled saccules of SER, and less frequently localized to isolated small synaptic vesicles (SSVs) or large dense-core vesicles (DCVs). In rare cases, VMAT2-immunoreactive SSVs were clustered within the cytoplasm of an SN or a VTA dendrite. Dopaminergic dendrites in the VTA contained a significantly higher number of immunogold particles for VMAT2 per unit than those in the SN. Together, these observations support the proposal that dopamine is stored in and may be released from dendritic SSVs and DCVs, but suggest that the SER is the major site of dopamine storage within midbrain dopaminergic neurons. In addition, they provide new evidence that dopaminergic dendrites in the VTA may have greater potential for reserpine-sensitive storage and release of dopamine than those in the SN.

Animals↗