Biomedical subjects

B R Ransom

Publications and source records attributed to B R Ransom.

At least 37 records · Page 2Linked to original sources

PubMed1997

Ischemic injury of optic nerve axons: the nuts and bolts.

Anterior ischemic optic neuropathy is the most common cause of persistent monocular visual loss in persons over the age of 50. At the heart of this form of optic neuropathy is a sequence of cytoplasmic and membrane events that culminate in axonal destruction. Early depletion of ATP is followed by membrane depolarization, influx of Na+ and Ca2+ via specific voltage-gated channels and reverse operation of the Na+/Ca2+ exchange protein. Toxic Ca2+ overload is the ultimate consequence of these events. Preventing or modulating any of these well-defined steps mitigates against the development of anoxic injury. Translating these molecular insights about how optic nerve axons are damaged by ischemia-like conditions into clinical gains remains the challenge for the future.

Adenosine Triphosphate↗

PubMed1997

Does astrocytic glycogen benefit axon function and survival in CNS white matter during glucose deprivation?

Axons, the functional elements in CNS white matter, are frequently injured by ischemia, especially in the context of stroke. The pathophysiology of axonal injury induced by energy deprivation has been analyzed in the rat optic nerve and involves excessive calcium influx by way of reverse Na+/Ca2+ exchange and Ca2+ channels. Evidence is presented that CNS axonal function can be supported in the absence of glucose by intrinsic energy reserves provided through the breakdown of astrocytic glycogen. It is argued that energy is transferred from astrocytes to axons in the form of lactate, which is able to maintain axonal function when substituted for glucose. These observations complement the increasingly convincing hypothesis that astrocytes and neurons interact metabolically, both in the course of normal activity and under pathological conditions such as ischemia. The emerging picture would be no surprise to Camillo Golgi, who predicted a close facsimile of this glial-neuronal interaction more than a century ago.

Animals↗

PubMed1996

Mechanisms of H+ and Na+ changes induced by glutamate, kainate, and D-aspartate in rat hippocampal astrocytes.

The excitatory transmitter glutamate (Glu), and its analogs kainate (KA), and D-aspartate (D-Asp) produce significant pH changes in glial cells. Transmitter-induced pH changes in glial cells, generating changes in extracellular pH, may represent a special form of neuronal-glial interaction. We investigated the mechanisms underlying these changes in intracellular H+ concentration ([H+]i) in cultured rat hippocampal astrocytes and studied their correlation with increases in intracellular Na+ concentration ([Na+]i), using fluorescence ratio imaging with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) or sodium-binding benzofuran isophthalate (SBFI). Glu, KA, or D-Asp evoked increases in [Na+]i; Glu or D-Asp produced parallel acidifications. KA, in contrast, evoked biphasic changes in [H+]i, alkaline followed by acid shifts, which were unaltered after Ca2+ removal and persisted in 0 CI(-)-saline, but were greatly reduced in CO2/HCO3(-)-free or Na(+)-free saline, or during 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) application. The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) blocked KA-evoked changes in [H+]i and [Na+]i, indicating that they were receptor-ionophore mediated. In contrast, CNQX increased the [H+]i change and decreased the [Na+]i change induced by Glu. D-Asp, which is transported but does not act at Glu receptors, induced [H+]i and [Na+]i changes that were virtually unaltered by CNQX. Our study indicates that [Na+]i increases are not primarily responsible for Glu- or KA-induced acidifications in astrocytes. Instead, intracellular acidifications evoked by Glu or D-Asp are mainly caused by transmembrane movement of acid equivalents associated with Glu/Asp-uptake into astrocytes. KA-evoked biphasic [H+]i changes, in contrast, are probably attributable to transmembrane ion movements mediated by inward, followed by outward, electrogenic Na+/HCO3- cotransport, reflecting KA-induced biphasic membrane potential changes.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1996

Characterization of Na+/H+ exchange activity in cultured rat hippocampal astrocytes.

Astrocytes actively maintain their intracellular pH (pHi) more alkaline than expected by passive distribution of H+. Acid extruding transporters such as the amiloride-sensitive Na+/H+ exchanger (NHE) are necessary for pH regulation. Currently, four mammalian NHEs (NHE1-NHE4) have been cloned, with a fifth (NHE5) partially cloned. We attempted to determine which isoform(s) of NHE was present in cultured hippocampal astrocytes using amiloride sensitivity and immunospecificity as criteria. In the absence of HCO3-, amiloride blocked pHi recovery after an acid load with an IC50 of approximately 3.18 microM, similar to values reported for the amiloride-sensitive isoforms NHE1 and NHE2. Immunoblotting with a highly specific antibody for NHE1 identified a 100 kDa protein, indicating the presence of NHE1 in whole brain, hippocampus, and cultured hippocampal astrocytes. Further probing for an additional amiloride-sensitive NHE failed to detect evidence of the presence of NHE4. Surprisingly, application of the potent analog of amiloride, ethylisopropylamiloride (EIPA), caused a reversible alkalinization of pHi, suggesting the presence of an additional acid/base transport mechanism that is EIPA-sensitive.

Amiloride↗

PubMed1996

Intracellular sodium homeostasis in rat hippocampal astrocytes.

1. We determined the intracellular Na+ concentration ([Na+]i) and mechanisms of its regulation in cultured rat hippocampal astrocytes using fluorescence ratio imaging of the Na+ indicator SBFI-AM (acetoxymethylester of sodium-binding benzofuran isophthalate, 10 microM). Dye signal calibration within the astrocytes showed that the ratiometric dye signal changed monotonically with changes in [Na+]i from 0 to 140 nM. The K+ sensitivity of the dye was negligible; intracellular pH changes, however, slightly affected the 'Na+' signal. 2. Baseline [Na+]i was 14.6 +/- 4.9 mM (mean +/- S.D.) in CO2/HCO3(-)-containing saline with 3 mM K+. Removal of extracellular Na+ decreased [Na+]i in two phases: a rapid phase of [Na+]i reduction (0.58 +/- 0.32 mM min-1) followed by a slower phase (0.15 +/- 0.09 mM min-1). 3. Changing from CO2/HCO3(-)-free to CO2/HCO3(-)-buffered saline resulted in a transient increase in [Na+]i of approximately 5 mM, suggesting activation of inward Na(+)-HCO3- cotransport by CO2/HCO3-. During furosemide (frusemide, 1 mM) or bumetanide (50 microM) application, a slow decrease in [Na+]i of approximately 2 mM was observed, indicating a steady inward transport of Na+ via Na(+)-K(+)-2Cl- cotransport under control conditions. Tetrodotoxin (100 microM) did not influence [Na+]i in the majority of cells (85%), suggesting that influx of Na+ through voltage-gated Na+ channels contributed to baseline [Na+]i in only a small subpopulation of hippocampal astrocytes. 4. Blocking Na+, K(+)-ATPase activity with cardiac glycosides (ouabain or strophanthidin, 1 mM) or removal of extracellular K+ led to an increase in [Na+]i of about 2 and 4 mM min-1, respectively. This indicated that Na+, K(+)-ATPase activity was critical in maintaining low [Na+]i in the face of a steep electrochemical gradient, which would favour a much higher [Na+]i. 5. Elevation of extracellular K+ concentration ([K+]o) by as little as 1 mM (from 3 to 4 mM) resulted in a rapid and reversible decrease in [Na+]i. Both the slope and the amplitude of the [K+]o-induced reductions in [Na+]i were sensitive to bumetanide. A reduction of [K+]o by 1 mM increased [Na+]i by 3.0 +/- 2.3 mM. In contrast, changing extracellular Na+ concentration by 20 mM resulted in changes in [Na+]i of less than 3 mM. 6. These results implied that in hippocampal astrocytes low baseline [Na+]i is determined by the action of Na(+)-HCO3- cotransport, Na(+)-K(+)-2Cl- cotransport and Na+, K(+)-ATPase, and that both Na+, K(+)-ATPase and inward Na(+)-K(+)-2Cl cotransport are activated by small, physiologically relevant increases in [K+]o. These mechanisms are well suited to help buffer increases in [K+]o associated with neural activity.

Animals↗

PubMed1996

Effects of CO2 on excitatory transmission apparently caused by changes in intracellular pH in the rat hippocampal slice.

It is generally known that hyperventilation produces an increase in neuronal excitability. However, the mechanism whereby a change in CO2 partial pressure (PCO2) leads to changes in neural excitability is not known. We have studied this phenomenon in rat hippocampal slices using double-barrelled microelectrodes for simultaneous recording of field excitatory postsynaptic potentials (EPSPs) and extracellular pH in stratum radiatum of area CA1. A drop in PCO2 from the control level, 36 mmHg to 7 mmHg, produced an increase in extracellular pH of 0.4-0.6 pH units and a transient increase in EPSP slope by about 20-30%. Despite the stable extracellular alkalosis, the EPSP reverted back to its original level within 10 min. Switching back to 36 mmHg PCO2 restored the original extracellular pH and caused a transient decrease in the EPSP slope. Pharmacological blockade of NMDA receptor and/or GABAA receptor had no influence on the effects of CO2. An increase in PCO2 to 145 mmHg led to a stable fall in extracellular pH by 0.6 units and to a transient 30-50% decrease in EPSP slope. The above results indicate that the CO2-induced changes in neuronal excitability were not caused by changes in extracellular pH but they might have been mediated by changes in intracellular pH. Indeed, exposing the slices to the permeant weak base, trimethylamine (20 mM), which is known to produce a rise in intracellular pH, increased the EPSP slope by 50-70%. Application of 20 mM propionate (a permeant weak acid) decreased the EPSP slope by 40-60%. We conclude that the transient changes in the EPSP seen in response to changes in PCO2 are mediated by in intracellular pH.

2-Amino-5-phosphonovalerate↗

PubMed1996

Octanol, a gap junction uncoupling agent, changes intracellular [H+] in rat astrocytes.

Octanol rapidly closes gap junction channels but its mechanism of action is not known. Because intracellular [H+], pHi, also affects the conductance of gap junctions, we studied octanol's effects on pHi in cultured rat astrocytes, which are highly coupled cells. Octanol (1 mM) caused an acid shift in the pHi of 90% of rat hippocampal astrocytes which averaged -0.19 +/- 0.09 pH units in magnitude. In 58% of the cells tested, a biphasic change in pHi was seen; octanol produced an initial acidification lasting approximately 10 min that was followed by a persistent alkalinization. The related gap junction uncoupling agent, heptanol, had similar effects on pHi. Octanol-induced changes in pHi were similar in nominally HCO(3-)-free and HCO(3-)-containing solutions, although the rate of initial acidification was significantly greater in the presence of HCO3-. The initial acidification was inhibited in the presence of the stilbene DIDS, an inhibitor of Na+/HCO3- cotransport, indicating that octanol caused acidification by blocking this powerful acid extruder. The alkalinization was inhibited by amiloride which blocks the Na+/H+ exchanger (NHE), an acid extruder, suggesting that the alkaline shift induced by octanol was caused by stimulation of NHE. As expected, octanol's effects on astrocytic pHi were prevented by removal of external Na+, which blocks both Na+/HCO3- cotransport and NHE. Octanol had only small effects on intracellular Ca2+ (Ca2+i) in astrocytes. Hepatocytes which, like astrocytes, are strongly coupled to one another, showed no change in pHi with octanol application. Fluorescence recovery after photobleaching (FRAP) was used to study the effect of changes in astrocyte pHi on degree of coupling in hippocampal astrocytes. Coupling was decreased by intracellular acid shifts approximately -0.2 pH units in size. Octanol's effects on astrocyte pHi were complex but a prompt initial acidification was nearly always seen and could contribute to the uncoupling action of this drug in astrocytes. Because octanol uncouples hepatocytes without changing their pHi, this compound clearly can influence gap junctional conductance independent of changes in pHi.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1996

Effects of osmotically driven cell volume changes on diffusion-weighted imaging of the rat optic nerve.

The apparent diffusion coefficient (ADC) of the rat optic nerve was measured in vitro, using magnetic resonance imaging, to determine the effects of changes in cellular volume fraction on the diffusion of tissue water. Nerve ADC was determined under conditions of cell membrane depolarization and (i) increased intracellular volume, (ii) decreased intracellular volume, and (iii) negligible volume change. Depolarization alone had little affect on ADC, whereas volume changes produced strong, reversible effects. Increased cell volume decreased ADC and vice versa. These results are consistent with the view that changes in the extracellular space are the major source of ADC changes in brain tissue.

Animals↗

PubMed1996

Autoprotective mechanisms in the CNS: some new lessons from white matter.

Anoxia/ischemia in the CNS is a common and devastating phenomenon. It is possible that the best hopes for protection against anoxic/ischemic injury may involve recruiting and/or augmenting any autoprotective systems that evolution has provided for the CNS. We describe here the existence of such an autoprotective system present in CNS white matter. White matter is both well suited to studying extrasynaptic systems, such as the system we describe here, and is a highly appropriate target for research into anoxic-ischemic injury in its own right. We show that white matter contains functional GABAB and adenosine receptors that respond to an anoxic efflux of GABA and adenosine by recruiting a convergent intracellular mechanism involving protein kinase C (PKC). The net result of this receptor-mediated cascade is an increase in resistance to anoxia, which presumably allows CNS white matter to tolerate better a common class of ischemic events that are located solely in white matter and that comprises approximately 25% of all strokes seen clinically.

Animals↗

PubMed1996

Glial-neuronal interactions in non-synaptic areas of the brain: studies in the optic nerve.

Optic nerves, like other CNS tracts, consist of axons closely apposed across narrow extracellular clefts to the cell bodies and processes of glial cells. Despite the anatomical simplicity of these pathways and the absence of synapses, a surprising range of interactions occurs between axons and glial cells mediated by changes in the chemical composition of the extracellular fluid produced by glial or neuronal stimulation. Some of the interactions are relatively brief, resulting from alterations in extracellular ions such as K+ or H+, or alterations of small molecules like glutamate or ATP. Other interactions involve much longer time periods and presumably larger signaling molecules, like peptides or proteins. These play a role not only in the development of axonal pathways but also in the processes of degeneration and regeneration that follow brain injury or disease.

Animals↗

PubMed1996

Cd(2+)-induced injury in CNS white matter.

1. The affect of extracellular Cd2+ on CNS white matter was studied using an isolated rat optic nerve preparation. A 100-min exposure to 200 microM Cd2+ reduced the area of the compound action potential (CAP) recorded from the optic nerve to 32.6 +/- 3.8% (mean +/- SE) of the preexposure area, compared with a reduction to 74.9 +/- 2.9% after 100 min in control conditions (P > 0.001). This CAP reduction was not reversed after 120 min of reperfusion with Cd(2+)-free solution, or by perfusion with Cd2+ chelators. 2. Cd(2+)-induced CAP loss occurred in the absence of extracellular Ca2+. Increasing extracellular Ca2+ concentration to 16 mM, however, prevented Cd(2+)-induced CAP loss. Once evident, Cd(2+)-induced CAP reduction could not subsequently be reversed by addition of 16 mM Ca2+. 3. Low concentrations of Cd2+ (60 microM) did not significantly reduce CAP area. This concentration of Cd2+ combined with high extracellular K+ (30 mM) caused CAP loss that was blocked by 10 microM nifedipine, an antagonist of L-type voltage-gated Ca2+ channels. 4. Treatment with pharmacological inhibitors of membrane proteins known to be inhibited by Cd2+ did not affect the CAP. These included inhibitors of voltage-gated Ca2+ channels, Ca(2+)-activated K+ channels, Ca(2+)-ATPase and the Na+/Ca2+ exchanger. 5. Treatment with pharmacological agents that inhibit calmodulin or disrupt tubulin, two intracellular proteins affected by Cd2+, did not affect CAP area. 6. The effect of Cd2+ was not prevented by pretreatment with (+)-cyanidanol-3, an agent that prevents Cd(2+)-induced lipid peroxidation. 7. Treatment with antimycin A, a inhibitor of mitochondrial respiration, resulted in irreversible CAP reduction with a time course and extent similar to that produced by 200 microM Cd2+. Cd(2+)-induced CAP reduction was prevented by 1 mM cysteine, which prevents Cd(2+)-induced disruption of mitochondrial respiration. 8. The ultrastructure of optic nerves exposed to 200 microM Cd2+ for 100 min was characterized by swollen mitochondria with disrupted cristae and dissolution of microtubules, which were replaced by flocculent debris. Occasional regions of axonal swelling and empty spaces beneath the myelin also were found. Qualitatively similar changes in mitochondria and cytoskeletal elements were found in optic nerves exposed to antimycin A for 100 min. Astrocytes also displayed disrupted mitochondria and had an electron-lucent appearance under both conditions. 9. The neurological injury produced by exposure to Cd2+ is characterized by lesions of CNS white matter. Our results indicate that Cd(2+)-induced white matter injury in vitro results largely from disruption of mitochondrial respiration after Cd2+ influx through routes that include voltage-gated Ca2+ channels.

Animals↗

PubMed1995

Isoform-specific expression of sodium channels in astrocytes in vitro: immunocytochemical observations.

The expression of sodium channel alpha-subunit isoforms in astrocytes cultured from P-0 rat spinal cord and P-7 rat optic nerve was examined utilizing immunocytochemical methods with antibodies generated against conserved and isoform-specific amino acid sequences of the rat brain sodium channel. In spinal cord cultures at 5 days in vitro (DIV), both stellate and flat astrocytes were immunostained with antibody SP20, which recognizes a conserved sequence common to sodium channel types I, II/IIA, and III. Antibody SP11-I, which is directed against a subtype-specific sequence in sodium channel I, did not yield detectable staining in spinal cord astrocytes. Antibody SP11-II, which is directed against a subtype-specific sequence in sodium channel II, immunostained both stellate and flat spinal cord astrocytes, although with less intensity than SP20. Antibody SP32-III, which is directed against a subtype-sequence in sodium channel III, immunostained stellate but not flat spinal cord astrocytes. SP20, SP11-II, and SP32-III staining persisted in stellate spinal cord astrocytes through 14-21 DIV, while SP20 and SP11-II immunostaining in flat spinal cord astrocytes was attenuated with time in culture. In optic nerve cultures at 5 DIV, SP20 staining was present in both stellate and flat astrocytes, but at reduced levels compared to spinal cord astrocytes. With increased time in culture SP20 staining was maintained in stellate optic nerve astrocytes but was gradually lost in flat optic nerve astrocytes. Stellate optic nerve astrocytes exhibited low levels of staining with SP11-I, SP11-II, and SP32-III. Flat optic nerve astrocytes lacked or displayed very low SP11-II staining, and SP11-I and SP32-III staining was not detectable. These observations demonstrate that cultures astrocytes are immunoreactive to antibodies generated against conserved and isotype-specific peptide sequences of rat brain sodium channels, and further suggest that there are different patterns of sodium channel expression between flat vs. stellate astrocytes and in astrocytes derived from different regions of the CNS.

Animals↗

PubMed1995

Voltage-gated calcium channels in CNS white matter: role in anoxic injury.

1. The effect of Ca2+ channel antagonists on the extent of anoxia-induced white matter injury was studied in the rat optic nerve, a white matter tract. Compound action potentials (CAPs) were recorded before and after a standard 60-min anoxic period to assess the extent of anoxic injury. 2. The L-type Ca2+ channel antagonists verapamil (90 microM), diltiazem (50 microM), and nifedepine (2.5 microM) significantly protected the rat optic nerve from anoxic injury. Mean recovery of CAP area was 51.3 +/- 3.0% (mean +/- SE, n = 8, P < 0.01), 65.6 +/- 5.3% (n = 8, P < 0.01), and 54.3 +/- 6.1% (n = 8, P < 0.01), respectively. Mean CAP recovery under control conditions was 35.2 +/- 0.3 (n = 33). 3. Simultaneous block of L-type and N-type Ca2+ channels by coapplication of 50 microM diltiazem and 1 microM SNX-124 [synthetic omega-conotoxin (CgTx) GVIA], resulted in postanoxic CAP recovery of 73.6 +/- 6.0% (n = 12), significantly larger than CAP recovery in diltiazem alone (P < 0.001). Block of CgTx MVIIC-sensitive channels in addition to L-type and N-type channels by coapplication of 50 microM diltiazem + 1 microM SNX-230 + 1 microM SNX-124 failed to produce any additional increase in CAP recovery (71.3 +/- 5.6%, n = 8). Application of 1 microM SNX-124 alone did not significantly protect against anoxic injury (CAP recovery, 36.3 +/- 2.9%, n = 10).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Endogenous GABA attenuates CNS white matter dysfunction following anoxia.

We studied the effects of GABA on anoxia-induced injury in CNS white matter using optic nerves exposed to 60 min of anoxia. Injury was assessed by recording pre- and postanoxic compound action potentials (CAPs). GABA (1 microM) significantly increased postanoxic CAP recovery when applied 60 min prior to anoxia. This effect was bicuculline (100 microM) insensitive, mimicked by baclofen (1 microM), blocked by GABA-B antagonists, and not mimicked by selective GABA-A agonists. GABA therefore acted at GABA-B receptors. High concentrations of GABA and baclofen did not influence recovery, possibly indicating GABA-B receptor desensitization at high agonist concentrations. Pertussis toxin (PTX) treatment reduced postanoxic CAP recovery in the presence of 1 microM GABA to control levels, indicating the recruitment of a G-protein-linked intracellular pathway. Protein kinase C (PKC) activation with 12-myristate 13-acetate (PMA) mimicked the effects of GABA. Inhibition of PKC with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) or staurosporine reduced postanoxic recovery in the presence of GABA to lower levels than under control conditions, confirming the involvement of PKC in the protective effect of GABA and indicating that this GABA-B receptor/G-protein/PKC protective pathway might be active under control conditions. This was confirmed by the observation that GABA-B receptor blockade, in the absence of exogenous GABA, significantly reduced postanoxia recovery. Thus, activation of the protective mechanism under control conditions is due to endogenous GABA release. Increasing the level of endogenous extracellular GABA by blocking GABA uptake with 1 mM nipecotic acid also protected against anoxia. We propose a model where release of GABA in white matter helps to limit nerve fiber injury during anoxia via recruitment of a G-protein/PKC pathway with subsequent phosphorylation of an unknown target protein.

Animals↗

PubMed1994

Anoxic injury of rat optic nerve: ultrastructural evidence for coupling between Na+ influx and Ca(2+)-mediated injury in myelinated CNS axons.

Physiological studies in the anoxic rat optic nerve indicate that irreversible loss of function, measured by the compound action potential, is due to depolarization and run-down of the transmembrane Na+ gradient which triggers Ca2+ entry through reverse Na(+)-Ca2+ exchange. EM studies in the anoxic optic nerve have demonstrated characteristic changes, including mitochondrial swelling and dissolution of cristae, submyelinic vacuoles, detachment of perinodal oligodendrocyte-axon loops, and severe cytoskeletal damage with loss of microtubules and neurofilaments within the axoplasm. To further examine the coupling between Na+ influx and Ca(2+)-mediated injury in myelinated axons within anoxic white matter, we have examined the ultrastructural effects of tetrodotoxin (TTX), in the anoxic optic nerve. Optic nerves, maintained in an interface brain slice chamber, were exposed to a 60-min period of anoxia. TTX (1 microM) was introduced 10 min before the onset of anoxia. Nerves were examined at the end of the anoxic period, or after 80 min in 1 microM TTX for normoxic controls. Under normoxic conditions, optic nerve axons exposed to TTX exhibited a normal ultrastructure. In optic nerves exposed to TTX studied at the end of a 60-min period of anoxia, mitochondria showed swelling and loss of cristae, and terminal oligodendroglial loops were detached from the nodal axon membrane. Cytoskeletal architecture was preserved in anoxic optic nerve axons treated with TTX, and axonal microtubules and neurofilaments maintained their continuity. Submyelinic empty spaces were not present. Perinodal astrocyte processes often appeared to be replaced by cellular remnants containing multiple membranous profiles; clusters of shrunken astrocytic processes were present between myelinated axons.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

B2 bradykinin receptors in NG108-15 cells: cDNA cloning and functional expression.

Two distinct cDNAs encoding bradykinin receptors (BKRs) were cloned from NG108-15 neuroblastoma-glioma hybrid cells. One was identical with rat uterus B2 BKR, whereas the other one (mBKR) had 91% amino acid homology to the rat B2 BKR and 82% homology to human B2 BKR. Southern blot analysis and genomic DNA cloning revealed that mBKR is derived from the mouse genome. The mBKR, expressed in Xenopus oocytes and COS-7 cells, produced functional BKRs that exhibited the properties of smooth muscle type B2 BKR. These results suggest that both the rat and mouse B2 BKRs of the smooth muscle type are expressed in NG108-15 cells.

Amino Acid Sequence↗

PubMed1994

Type II sodium channels in spinal cord astrocytes in situ: immunocytochemical observations.

The expression of sodium channel alpha-subunit isoforms in astrocytes in adult rat spinal cord and optic nerve was examined utilizing immunocytochemical methods with antibodies generated against conserved and subtype-specific sequences of the sodium channel. In adult rat spinal cord, astrocytes within the dorsal and ventral funiculi were immunolabelled with antibody SP20, which recognizes a conserved sequence within sodium channel types I, II, and III. In addition, astrocytes within these spinal cord white matter tracts were immunostained with antibody SP11-II, which recognizes sodium channel type II. Antibodies SP11-I and SP32-III, which are directed against subtype-specific sequences in sodium channel types I and III, respectively, did not label astrocytes in the dorsal and ventral funiculi of the spinal cord. In optic nerves, astrocytes were immunostained with antibody SP20. However, no detectable labelling of cells within the optic nerve was observed with antibodies SP11-I, SP11-II, and SP32-III. These observations demonstrate that sodium channel II is expressed by astrocytes in spinal cord white matter. Moreover, these data suggest that regional factors regulate the level of sodium channel isoform expression in astrocytes.

Animals↗

PubMed1994

Sodium channel mRNAs I, II and III in the CNS: cell-specific expression.

The cellular localization of rat brain sodium channel alpha-subunit mRNAs I, II and III in the central nervous system (CNS) was examined by non-isotope in situ hybridization cytochemistry utilizing two independent sets of isoform-specific RNA probes, one set recognizing sodium channel isoforms in the coding region and the other in the non-coding region of the sodium channel messages. The independent sets of probes demonstrated qualitatively similar patterns of sodium channel mRNA expression. In the hippocampus, sodium channel mRNA I was very weakly expressed in the pyramidal layer and in the granular layer of the dentate gyrus; in contrast, sodium channel mRNA II was strongly expressed by neurons in these regions. Sodium channel mRNA III exhibited low-to-moderate expression in some neurons of the pyramidal layer of the hippocampus and granular layer of the dentate gyrus, and was not detectable in others. In the cerebellum, sodium channel mRNA I was moderately expressed in some Purkinje cells, weakly expressed in scattered cells in the molecular layer and negligibly expressed in the granular layer. Sodium channel mRNA II was strongly expressed in Purkinje and granule cells, and was moderately expressed in some cells in the molecular layer. Sodium channel mRNA III was generally not detectable in the cerebellum. In the spinal cord, motor neurons and scattered neurons throughout the gray matter exhibited moderate-to-strong expression of both sodium channel mRNA I and II. A population of cells in the spinal zone of Lissauer showed heavy expression of mRNA II, but not mRNA I. Sodium channel mRNA III was not detectable in spinal cord neurons. These observations are consistent with a general regional distribution of sodium channel message isoforms, with mRNA II being preferentially expressed in rostral regions of the CNS and mRNA I in caudal regions. However, the results also indicate that different cell types, within a given region, display different patterns of sodium channel mRNA expression. Moreover, these data suggest that individual neurons may express multiple forms of sodium channel mRNA.

Animals↗