Mechanisms of the inhibition of epithelial Na+ channels by CFTR and purinergic stimulation

The epithelial Na+ channel ENaC is inhibited when the cystic fibrosis transmembrane conductance regulator (CFTR) coexpressed in the same cell is activated by the cyclic adenosine monophosphate (cAMP)-dependent pathway. Regulation of ENaC by CFTR has been studied in detail in epithelial tissues from intestine and trachea and is also detected in renal cells. In the kidney, regulation of other membrane conductances might be the predominant function of CFTR. A similar inhibition of ENaC takes place when luminal purinergic receptors a re activated by 5 ' -adenosine triphosphate (ATP) or uridine triphosphate (UTP). Because both stimulation of purinergic receptors and activation of CFTR induce a Cl- conductance, it is likely that Cl- ions control ENaC activity.

Effects of purinergic stimulation, CFTR and osmotic stress on amiloride-sensitive Na+ transport in epithelia and Xenopus oocytes

Both stimulation of purinergic receptors by ATP and activation of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibit amiloride-sensitive Na+ transport and activate Cl-secretion. These changes in ion transport may well affect cell volume. We therefore examined whether cell shrinkage or cell swelling do affect amiloride-sensitive Na+ transport in epithelial tissues or Xenopus oocytes and whether osmotic stress interferes with regulation of Na+ transport by ATP or CFTR. Stimulation of purinergic receptors by ATP/UTP or activation of CFTR by IBMX and forskolin inhibited amiloride-sensitive transport in mouse trachea and colon, respectively, by a mechanism that was Cl- dependent. When exposed to a hypertonic but not hypotonic bath solution, amiloride-sensitive Na+ transport was inhibited in mouse trachea and colon, independent of the extracellular Cl- concentration. Both inhibition of Na+ transport by hypertonic bath solution and ATP were additive. When coexpressed in Xenopus oocytes, activation of CFTR by IBMX and forskolin inhibited the epithelial Na+ channel (ENaC) in a Cl(-)dependent fashion. However, both hypertonic and hypotonic bath solutions showed only minor effects on amiloride-sensitive conductance, independent of the bath Cl- concentration. Moreover, CFTR-induced inhibition of ENaC could be detected in chocytes even after exposure to hypertonic or bypotonic bath solutions. We conclude that amiloride-sensitive Na+ absorption in mouse airways and colon is inhibited by cell shrinkage by a mechanism that does not interfere with purinergic and CFTR-mediated inhibition of ENaC.

No Association Between MTHFR A1298C and MTRR A66G Polymorphisms, and MS in an Australian Cohort

Multiple sclerosis (MS) is a complex neurological disease that affects the central nervous system (CNS) resulting in debilitating neuropathology. Pathogenesis is primarily defined by CNS inflammation and demyelination of nerve axons. Methionine synthase reductase (MTRR) is an enzyme that catalyzes the remethylation of homocysteine (Hcy) to methionine via cobalamin and folate dependant reactions. Cobalamin acts as an intermediate methyl carrier between methylenetetrahydrofolate reductase (MTHFR) and Hcy. MTRR plays a critical role in maintaining cobalamin in an active form and is consequently an important determinant of total plasma Hcy (pHcy) concentrations. Elevated intracellular pHcy levels have been suggested to play a role in CNS dysfunction, neurodegenerative, and cerebrovascular diseases. Our investigation entailed the genotyping of a cohort of 140 cases and matched controls for MTRR and MTHFR, by restriction length polymorphism (RFLP) techniques. Two polymorphisms: MTRR A66G and MTHFR A1298C were investigated in an Australian age and gender matched case-control study. No significant allelic frequency difference was observed between cases and controls at the α = 0.05 level (MTRR χ^2 = 0.005, P = 0.95, MTHFR χ^2 = 1.15, P = 0.28). Our preliminary findings suggest no association between the MTRR A66G and MTHFR A1298C polymorphisms and MS.

Autoimmune Neurological Disease

This book provides a comprehensive and critical overview of the immunological aspects of autoimmune neurological disease. These diseases include common conditions such as multiple sclerosis, the Guillain–Barré syndrome and myasthenia gravis. The introductory chapters on antigen recognition and self–non-self discrimination, and on neuroimmunology, are followed by chapters on specific diseases. These are presented in a standardized format with sections on clinical features, genetics, neuropathology, pathophysiology, immunology and therapy. Each chapter has a concluding section which summarizes key points and suggests directions for future research. Animal models of autoimmune neurological disease are also covered in detail because of their importance in understanding the human diseases. The book is suitable for clinicians and neurologists managing patients with these diseases, and for immunologists, neuroscientists and neurologists investigating the pathogenesis and pathophysiology of these disorders.

Are there functional P2X receptors on cell bodies in intact dorsal root ganglia of rats?

P2X purinoceptors have been suggested to participate in transduction of painful stimuli in nociceptive neurons. In the current experiments, ATP (1-10 mM), alpha,beta-methylene-ATP (10-30 mu M) and capsaicin (10 nM-1 mu M) were applied to neurons impaled with high resistance microelectrodes in rat dorsal root ganglia (L4 and L5) isolated in vitro together with the sciatic nerve and dorsal roots. The agonists were either bath applied or focally applied using a picospritzer. GABA (100 mu M) and 40-80 mM K+ solutions gave brisk responses when applied by either technique. Only three of 22 neurons with slowly conducting axons (C cells) showed evidence of P2X-purinoceptor-mediated responses. Only two of 13 cells which responded to capsaicin (putative nociceptors), and none of 29 cells with rapidly conducting axons (A cells), responded to the purinergic agonists. When acutely dissociated dorsal root ganglion cells were studied using patch-clamp techniques, all but four of 30 cells of all sizes responded with an inward current to either ATP or alpha,beta-methylene-ATP (both 100 mu M). Our data suggest that few sensory cell bodies in intact dorsal root ganglia express functional purinoceptors. (C) 1998 IBRO. Published by Elsevier Science Ltd.

Pharmacotherapy of the ion transport defect in cystic fibrosis

1. More than 1300 different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF), a disease characterized by deficient epithelial Cl- secretion and enhanced Na+ absorption. The clinical course of the disease is determined by the progressive lung disease. Thus, novel approaches in pharmacotherapy are based primarily on correction of the ion transport defect in the airways. 2. The current therapeutic strategies try to counteract the deficiency in Cl- secretion and the enhanced Na+ absorption. A number of compounds have been identified, such as genistein and xanthine derivatives, which directly activate mutant CFTR. Other compounds may activate alternative Ca2+-activated Cl- channels or basolateral K+ channels, which supply the driving force for Cl- secretion. Apart from that, Na+ channel blockers, such as phenamil and benzamil, are being explored, which counteract the hyperabsorption of NaCl in CF airways. 3. Clinical trials are under way using purinergic compounds such as the P2Y(2) receptor agonist INS365. Activation of P2Y(2) receptors has been found to both activate Cl- secretion and inhibit Na+ absorption. 4. The ultimate goal is to recover Cl- channel activity of mutant CFTR by either enhancing synthesis and expression of the protein or by activating silent CFTR Cl- channels. Strategies combining these drugs with compounds facilitating Cl- secretion and inhibiting Na+ absorption in vivo may have the best chance to counteract the ion transport defect in cystic fibrosis.

A. W. Campbells Australian career: 1905-1937

The Australian Alfred Walter Campbell (1868-1937) is remembered as one of the two chief pioneers of the study of the cytoarchitectonics of the primate cerebral cortex. He had worked in Britain carrying out neuroanatomical and neuropathological research for almost two decades before his famous monograph on Histological Studies on the Localisation of Cerebral Function appeared in 1905. In that year he returned to his native Australia and practiced for over 30 years in Sydney as a neurologist rather than a neuropathologist, publishing mainly clinical material though he was involved in the investigation of the epidemic of Australian X disease, a viral encephalitis. His abrupt change in both the nature and the location of his career at a time when he was well established in Britain appears to have been a consequence of his marriage and the need to provide for a family. His simultaneous apparent abandonment of research seems not to have really been the case. As judged from the contents of a paper presented to a local medical congress in Sydney in 1911, it appears that, in Australia, Campbell did carry out a major comparative anatomical and histological investigation of the possibility of localization of function in the cerebellar cortex. He never published this work in detail. His investigation let him to conclude that no such localization of function existed, a view contrary to the then topical interpretation of Bolk (1906), but one in accordance with Gordon Holmes' views a decade later. Campbell's circumstances in Sydney, his extremely reticent nature and the essentially negative outcome of his investigation probably explain his failure to make his study more widely known.

Spatial distribution and developmental appearance of postjunctional P2X(1) receptors on smooth muscle cells of the mouse vas deferens

P2X(1)-type purinoceptors, have been shown to mediate fast transmission between sympathetic varicosities and smooth muscle cells in the mouse vas deferens but the spatial organization of these receptors on the smooth muscle cells remains inconclusive. Voltage clamp techniques were used to estimate the amplitudes of spontaneous excitatory junction currents (SEJCs) in cells of the vas deferens longitudinal smooth muscle layer. These currents involved the activation of about 6% of the P2X-type channels present on the cell, as compared to whole cell currents produced when isolated smooth muscle cells were exposed to maximal concentrations of either ATP or alpha,beta -MeATP. Immunofluorescence staining of the vas deferens with antibodies against P2X(1) receptor showed a diffuse, grainy distribution over the entire membrane of each smooth muscle cell. Anti-P2X(1) staining was not markedly clustered beneath anti-SV2-stained sympathetic varicosities. Similar results were obtained for cells in the urinary bladder. During development, P2X(1) mRNA was detected as early as embryonic day 15 (E15). Increasing intensities of diffuse immunostaining for P2X(1) were observed in the walls of the bladder, tail artery, and aorta from E15 until 6 weeks postnatal. The vas deferens showed increasing intensities of diffuse staining of its smooth muscle layers between 2 and 6 weeks postnatal, consistent with the time-course of development of fast purinergic transmission described previously. Together, the results suggest that the response of smooth muscle of the vas deferens to ATP released from sympathetic varicosities relies on rapidly desensitizing P2X(1) receptors, distributed diffusely across the smooth muscle cell surface. Synapse 42:1-11, 2001. (C) 2001 Wiley-Liss, Inc.

Pre- and postsynaptic actions of ATP on neurotransmission in rat submandibular ganglia

The pre- and postsynaptic actions of exogenously applied ATP were investigated in intact and dissociated parasympathetic neurotics of rat submandibular ganglia. Nerve-evoked excitatory postsynaptic potentials (EPSPs) were not inhibited by the purinergic receptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2 ' ,4 ' -disulphonic acid (PPADS), or the desensitising agonist, alpha,beta -methylene ATP. In contrast. EPSPs were abolished by the nicotinic acetylcholine receptor antagonists, hexamethonium and mecamylamine. Focal application of ATP (100 muM) had no effect on membrane potential of the postsynaptic neurone or on the amplitude of spontaneous EPSPs. Taken together, these results suggest the absence of functional purinergic (P2) receptors on the postganglionic neurone in situ. In contrast, focally applied ATP (100 muM) reversibly inhibited nerve-evoked EPSPs. Similarly, bath application of the non-hydrolysable analogue of ATP, ATP gammaS, reversibly depressed EPSPs amplitude, The inhibitory effects of ATP and ATP gammaS on nerve-evoked transmitter release were antagonised by bath application of either PPADS or suramin, suggesting ATP activates a presynaptic P2 purinoceptor to inhibit acetylcholine release from preganglionic nerves in the submandibular ganglia. In acutely dissociated postganglionic neurotics from rat submandibular ganglia. focal application of ATP (100 LM) evoked an inward current and subsequent excitatory response and action potential firing, which was reversibly inhibited by PPADS (10 muM). The expression of P2X purinoceptors in wholemount and dissociated submandibular ganglion neurones was examined using polyclonal antibodies raised against the extracellular domain of six P2X purinoceptor subtypes (P2X(1-6)). In intact wholemount preparations, only the P2X(5) purinoceptor subtype was found to be expressed in the submandibular ganglion neurones and no P2X immunoreactivity was detected in the nerve fibres innervating the ganglion. Surprisingly, in dissociated submandibular ganglion neurones, high levels of P2X(2) and P2X(4) purinoceptors immunoreactivity were found on the cell surface. This increase in expression of P2X(2) and P2X(4) purinoceptors in dissociated submandibular neurones could explain the increased responsiveness of the neurotics to exogenous ATP. We conclude that disruption of ganglionic transmission in vivo by either nerve damage or synaptic blockade may up-regulate P2X expression or availability and alter neuronal excitability. (C) 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Patterns of gene expression are altered in the frontal and motor cortices of human alcoholics

Alcoholism is a major health problem in Western countries, yet relatively little is known about the mechanisms by which chronic alcohol abuse causes the pathologic changes associated with the disease. It is likely that chronic alcoholism affects a number of signaling cascades and transcription factors, which in turn result in distinct gene expression patterns. These patterns are difficult to detect by traditional experiments measuring a few mRNAs at a time, but are well suited to microarray analyses. We used cDNA microarrays to analyze expression of approximately 10 000 genes in the frontal and motor cortices of three groups of chronic alcoholic and matched control cases. A functional hierarchy was devised for classification of brain genes and the resulting groups were compared based on differential expression. Comparison of gene expression patterns in these brain regions revealed a selective reprogramming of gene expression in distinct functional groups. The most pronounced differences were found in myelin-related genes and genes involved in protein trafficking. Significant changes in the expression of known alcohol-responsive genes, and genes involved in calcium, cAMP, and thyroid signaling pathways were also identified. These results suggest that multiple pathways may be important for neuropathology and altered neuronal function observed in alcoholism.

Consensus neuropathological diagnosis of common dementia syndromes: testing and standardising the use of multiple diagnostic criteria

The aim of this study was to assess the variation between neuropathologists in the diagnosis of common dementia syndromes when multiple published protocols are applied. Fourteen out of 18 Australian neuropathologists participated in diagnosing 20 cases (16 cases of dementia, 4 age-matched controls) using consensus diagnostic methods. Diagnostic criteria, clinical synopses and slides from multiple brain regions were sent to participants who were asked for case diagnoses. Diagnostic sensitivity, specificity, predictive value, accuracy and variability were determined using percentage agreement and kappa statistics. Using CERAD criteria, there was a high inter-rater agreement for cases with probable and definite Alzheimer's disease but low agreement for cases with possible Alzheimer's disease. Braak staging and the application of criteria for dementia with Lewy bodies also resulted in high inter-rater agreement. There was poor agreement for the diagnosis of frontotemporal dementia and for identifying small vessel disease. Participants rarely diagnosed more than one disease in any case. To improve efficiency when applying multiple diagnostic criteria, several simplifications were proposed and tested on 5 of the original 210 cases. Inter-rater reliability for the diagnosis of Alzheimer's disease and dementia with Lewy bodies significantly improved. Further development of simple and accurate methods to identify small vessel lesions and diagnose frontotemporal dementia is warranted.

Neuronal calcium-binding proteins and schizophrenia

Calcium-binding proteins (CBPs) such as calbindin, parvalbumin and calretinin are used as immunohistochemical markers for discrete neuronal subpopulations. They are particularly useful in identifying the various subpopulations of GABAergic interneurons that control output from prefrontal and cingulate cortices as well as from the hippocampus. The strategic role these interneurons play in regulating output from these three crucial brain regions has made them a focus for neuropathological investigation in schizophrenia. The number of pathological reports detailing subtle changes in these CBP-containing interneurons in patients with schizophrenia is rapidly growing. These proteins however are more than convenient neuronal markers. They confer survival advantages to neurons and can increase the neuron's ability to sustain firing. These properties may be important in the subtle pathophysiology of nondegenerative phenomena such as schizophrenia. The aim of this review is to introduce the reader to the functional properties of CBPs and to examine the emerging literature reporting alterations in these proteins in schizophrenia as well as draw some conclusions about the significance of these findings. (C) 2002 Elsevier Science B.V. All rights reserved.