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Neuropathic pain in patients carrying sodium channel gain-of-function mutations is generally refractory to pharmacotherapy. However, we have shown that pretreatment of cells with clinically achievable concentration of carbamazepine (CBZ; 30 μM) depolarizes the voltage dependence of activation in some NaV1.7 mutations such as S241T, a novel CBZ mode of action of this drug. CBZ reduces the excitability of dorsal root ganglion (DRG) neurons expressing NaV1.7-S241T mutant channels, and individuals carrying the S241T mutation respond to treatment with CBZ. Whether the novel activation-modulating activity of CBZ is specific to NaV1.7, and whether this pharmacogenomic approach can be extended to other sodium channel subtypes, are not known. We report here the novel NaV1.8-S242T mutation, which corresponds to the NaV1.7-S241T mutation, in a patient with neuropathic pain and diabetic peripheral neuropathy. Voltage-clamp recordings demonstrated hyperpolarized and accelerated activation of NaV1.8-S242T. Current-clamp recordings showed that NaV1.8-S242T channels render DRG neurons hyperexcitable. Structural modeling shows that despite a substantial difference in the primary amino acid sequence of NaV1.7 and NaV1.8, the S242 (NaV1.8) and S241 (NaV1.7) residues have similar position and orientation in the domain I S4-S5 linker of the channel. Pretreatment with a clinically achievable concentration of CBZ corrected the voltage dependence of activation of NaV1.8-S242T channels and reduced DRG neuron excitability as predicted from our pharmacogenomic model. These findings extend the novel activation modulation mode of action of CBZ to a second sodium channel subtype, NaV1.8.

Objective Painful small fibre neuropathy (SFN) represents a significant public health problem, with no cause apparent in one-half of cases (termed idiopathic, I-SFN). Gain-of-function mutations of sodium channel Na V 1.7 have recently been identified in nearly 30% of patients with biopsy-confirmed I-SFN. More recently, gain-of-function mutations of Na V 1.8 have been found in patients with I-SFN. These Na V 1.8 mutations accelerate recovery from inactivation, enhance the response to slow depolarisations, and enhance activation at the channel level, thereby producing hyperexcitability of small dorsal root ganglion (DRG) neurons, which include nociceptors, at the cellular level. Identification and functional profiling of additional Na V 1.8 variants are necessary to determine the spectrum of changes in channel properties that underlie DRG neuron hyperexcitability in these patients. Methods Two patients with painful SFN were evaluated by skin biopsy, quantitative sensory testing, nerve conduction studies, screening of genomic DNA for mutations in SCN9A and SCN10A and electrophysiological functional analysis. Results A novel sodium channel Na V 1.8 mutation G1662S was identified in both patients. Voltage-clamp analysis revealed that the Na V 1.8/G1662S substitution impairs fast-inactivation, depolarising the midpoint (V 1/2 ) by approximately 7 mV. Expression of G1662S mutant channels within DRG neurons rendered these cells hyperexcitable. Conclusions We report for the first time a mutation of Na V 1.8 which impairs inactivation, in patients with painful I-SFN. Together with our earlier results, our observations indicate that an array of Na V 1.8 mutations, which affect channel function in multiple ways, can contribute to the pathophysiology of painful peripheral neuropathy.

Sodium channel Na(v)1.7 is preferentially expressed in dorsal root ganglion (DRG) and sympathetic ganglion neurons. Gain-of-function Na(v)1.7 mutations/variants have been identified in the painful disorders inherited erythromelalgia and small-fiber neuropathy (SFN). DRG neurons transfected with these channel variants display depolarized resting potential, reduced current-threshold, increased firing-frequency and spontaneous firing. Whether the depolarizing shift in resting potential and enhanced spontaneous firing are due to persistent activity of variant channels, or to compensatory changes in other conductance(s) in response to expression of the variant channel, as shown in model systems, has not been studied. We examined the effect of wild-type Na(v)1.7 and a Na(v)1.7 mutant channel, D623N, associated with SFN, on resting potential and membrane potential during interspike intervals in DRG neurons. Resting potential in DRG neurons expressing D623N was depolarized compared to neurons expressing WT-Na(v)1.7. Exposure to TTX hyperpolarized resting potential by 7 mV, increased current-threshold, decreased firing-frequency, and reduced NMDG-induced-hyperpolarization in DRG neurons expressing D623N. To assess the contribution of depolarized resting potential to DRG neuron excitability, we mimicked the mutant channel's depolarizing effect by current injection to produce equivalent depolarization; the depolarization decreased current threshold and increased firing-frequency. Voltage-clamp using ramp or repetitive action potentials as commands showed that D623N channels enhance the TTX-sensitive inward current, persistent at subthreshold membrane voltages, as predicted by a Hodgkin-Huxley model. Our results demonstrate that a variant of Na(v)1.7 associated with painful neuropathy depolarizes resting membrane potential and produces an enhanced inward current during interspike intervals, thereby contributing to DRG neuron hyperexcitability.

Background: Nav1.8 sodium channels, encoded by SCN10A, are preferentially expressed in nociceptive neurons and play an important role in human pain. Although rare gain-of-function variants in SCN10A have been identified in individuals with painful peripheral neuropathies, whether more common variants in SCN10A can have an effect at the channel level and at the dorsal root ganglion, neuronal level leading to a pain disorder or an altered normal pain threshold has not been determined. Results: Candidate single nucleotide polymorphism association approach together with experimental pain testing in human subjects was used to explore possible common SCN10A missense variants that might affect human pain sensitivity. We demonstrated an association between rs6795970 (G > A; p.Ala1073Val) and higher thresholds for mechanical pain in a discovery cohort (496 subjects) and confirmed it in a larger replication cohort (1005 female subjects). Functional assessments showed that although the minor allele shifts channel activation by −4.3 mV, a proexcitatory attribute, it accelerates inactivation, an antiexcitatory attribute, with the net effect being reduced repetitive firing of dorsal root ganglion neurons, consistent with lower mechanical pain sensitivity. Conclusions: At the association and mechanistic levels, the SCN10A single nucleotide polymorphism rs6795970 biases human pain sensitivity.

Gain-of-function mutations of NaV1.7 have been shown to produce two distinct disorders: NaV1.7 mutations that enhance activation produce inherited erythromelalgia (IEM), characterized by burning pain in the extremities; NaV1.7 mutations that impair inactivation produce a different, nonoverlapping syndrome, paroxysmal extreme pain disorder (PEPD), characterized by rectal, periocular, and perimandibular pain. Here we report a novel NaV1.7 mutation associated with a mixed clinical phenotype with characteristics of IEM and PEPD, with an alanine 1632 substitution by glutamate (A1632E) in domain IV S4–S5 linker. Patch-clamp analysis shows that A1632E produces changes in channel function seen in both IEM and PEPD mutations: A1632E hyperpolarizes (−7 mV) the voltage dependence of activation, slows deactivation, and enhances ramp responses, as observed in NaV1.7 mutations that produce IEM. A1632E depolarizes (+17mV) the voltage dependence of fast inactivation, slows fast inactivation, and prevents full inactivation, resulting in persistent inward currents similar to PEPD mutations. Using current clamp, we show that A1632E renders dorsal root ganglion (DRG) and trigeminal ganglion neurons hyperexcitable. These results demonstrate a NaV1.7 mutant with biophysical characteristics common to PEPD (impaired fast inactivation) and IEM (hyperpolarized activation, slow deactivation, and enhanced ramp currents) associated with a clinical phenotype with characteristics of both IEM and PEPD and show that this mutation renders DRG and trigeminal ganglion neurons hyperexcitable. These observations indicate that IEM and PEPD mutants are part of a physiological continuum that can produce a continuum of clinical phenotypes.

Voltage-gated sodium channel Nav1.7 is a central player in human pain. Mutations in Nav1.7 produce several pain syndromes, including inherited erythromelalgia (IEM), a disorder in which gain-of-function mutations render dorsal root ganglia (DRG) neurons hyperexcitable. Although patients with IEM suffer from episodes of intense burning pain triggered by warmth, the effects of increased temperature on DRG neurons expressing mutant Nav1.7 channels have not been well documented. Here, using structural modeling, voltage-clamp, current-clamp, and multielectrode array recordings, we have studied a newly identified Nav1.7 mutation, Ala1632Gly, from a multigeneration family with IEM. Structural modeling suggests that Ala1632 is a molecular hinge and that the Ala1632Gly mutation may affect channel gating. Voltage-clamp recordings revealed that the Nav1.7-A1632G mutation hyperpolarizes activation and depolarizes fast-inactivation, both gain-of-function attributes at the channel level. Whole-cell current-clamp recordings demonstrated increased spontaneous firing, lower current threshold, and enhanced evoked firing in rat DRG neurons expressing Nav1.7-A1632G mutant channels. Multielectrode array recordings further revealed that intact rat DRG neurons expressing Nav1.7-A1632G mutant channels are more active than those expressing Nav1.7 WT channels. We also showed that physiologically relevant thermal stimuli markedly increase the mean firing frequencies and the number of active rat DRG neurons expressing Nav1.7-A1632G mutant channels, whereas the same thermal stimuli only increase these parameters slightly in rat DRG neurons expressing Nav1.7 WT channels. The response of DRG neurons expressing Nav1.7-A1632G mutant channels upon increase in temperature suggests a cellular basis for warmth-triggered pain in IEM. SIGNIFICANCE STATEMENT Inherited erythromelalgia (IEM), a severe pain syndrome characterized by episodes of intense burning pain triggered by warmth, is caused by mutations in sodium channel Nav1.7, which are preferentially expressed in sensory and sympathetic neurons. More than 20 gain-of-function Nav1.7 mutations have been identified from IEM patients, but the question of how warmth triggers episodes of pain in IEM has not been well addressed. Combining multielectrode array, voltage-clamp, and current-clamp recordings, we assessed a newly identified IEM mutation (Nav1.7-A1632G) from a multigeneration family. Our data demonstrate gain-of-function attributes at the channel level and differential effects of physiologically relevant thermal stimuli on the excitability of DRG neurons expressing mutant and WT Nav1.7 channels, suggesting a cellular mechanism for warmth-triggered pain episodes in IEM patients.

Recent studies indicate that the actions of arginine vasopressin (AVP) and other agonists that stimulate electrogenic sodium transport in renal epithelial A6 cells are linked to a Ca 2+ -mobilizing signal transduction mechanism that involves generation of inositol trisphosphate. Since diacylglycerol is the other product in this pathway, studies were performed to determine the possible role of PKC in the stimulation of sodium transport. AVP induced a biphasic increase in diacylglycerol generation, characterized by an initial rapid rise and then a sustained elevation, and PKC activation, reflected by phosphorylation of a specific 80 kDa myristoylated alanine-rich PKC substrate (MARCKS). To determine the PKC isoform(s) involved in this process, immunoblot analysis was performed using antisera that recognize both classical PKC isoforms, XPKC-I and XPCK-II, cloned from Xenopus oocytes. The transcripts of both isoforms were expressed in the A6 cell. Since protein recognized by antisera was translocated from cytosol to the particulate fraction after exposure to AVP, one or both isoforms were activated in the A6 cell. Further studies showed that cyclohexyladenosine and insulin, additional agonists of sodium transport in A6 cells, also stimulated phosphorylation of MARCKS. These results argue that Ca 2+ -dependent PKC is involved in the action of AVP, and that of other agonists, which stimulate sodium transport.

Vasopressin is known to activate two types of cell surface receptors; V2, coupled to adenylate cyclase, and V1, linked to a Ca(2+)-dependent transduction system. We investigated whether arginine vasopressin (AVP) stimulation of electrogenic sodium transport in A6 cells, derived from Xenopus laevis, is mediated by activation of either one or both types of AVP-specific receptors. AVP caused a rapid increase in electrogenic sodium transport, reflected by the transepithelial potential difference (VT) and equivalent short circuit current (Ieq) measurements. AVP also rapidly increased intracellular Ca2+ (Ca2+i) and total inositol trisphosphate. The increase in Ieq was dependent on the rise in (Ca2+i), because 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) dose-dependently inhibited the Ieq response. There was no evidence, however, that activation of adenylate cyclase mediated AVP-stimulated Ieq; transport was not inhibited after AVP-induced activation of adenylate cyclase was abolished by 2',5'-dideoxyadenosine or when cAMP-dependent protein kinase (PKA) activity was abolished by the specific PKA inhibitor IP20. Further studies showed that although both forskolin and 8-(4-chlorophenylthio)-cAMP stimulated Ieq, this occurred by mechanisms independent of PKA activation. These results indicate that AVP-stimulated Na+ transport is mediated by a V1 receptor and a Ca(2+)-dependent mechanism.

Gangliosides are implicated in cell signal transduction. Prior to investigating this phenomenon in macrophages, the in situ accessibility of gangliosides to macromolecules was assessed for peritoneal macrophages isolated from normal C3H/HeN and endotoxin-hyporesponsive C3H/HeJ mice. C3H/HeJ resident and thioglycolate-elicited macrophage ganglioside patterns are the same as normal strains, and no strain differences in galactose oxidase accessibility for resident or thioglycolate-elicited macrophage gangliosides were found. The only gangliosides accessible to galactose oxidase in resident macrophages are GM1a structures. In thioglycolate-elicited macrophages, an additional ganglioside is accessible. For Escherichia coli-activated macrophages, where ganglioside distribution differs between strains, a difference in galactose oxidase-accessible gangliosides also exists. Escherichia coli-activated C3H/HeN patterns show three triplets absent in C3H/HeJ patterns. There were no differences in ganglioside accessibility to Vibrio cholerae sialidase between the thioglycolate-elicited C3H/HeJ and C3H/HeN macrophages. However, despite differences in sialidase-sensitive ganglioside content between E.coli-activated macrophages of these strains, sialidase accessibility for E.coli-activated macrophages was also similar. Sialidase-susceptible GM3 was cryptic in either strain under all conditions examined. The accessibility of murine macrophage gangliosides to galactose oxidase or sialidase was independent of their sialic acid species and chain length of the ceramide fatty acid. With the exception of GM3, major murine macrophage gangliosides are accessible in situ to macromolecules, especially to exogenous pathogenic bacterial sialidase which can alter macrophage cell surface characteristics. Altered macrophage ganglioside accessibility appears sometimes as a consequence, but not a cause, of C3H/HeJ endotoxin hyporesponsiveness.

The NaV1.7 sodium channel is preferentially expressed within dorsal root ganglion and sympathetic ganglion neurons and their small-diameter peripheral axons. Gain-of-function variants of NaV1.7 have recently been described in patients with painful small fibre neuropathy and no other apparent cause. Here, we describe a novel syndrome of pain, dysautonomia, small hands and small feet in a kindred carrying a novel NaV1.7 mutation. A 35-year-old male presented with erythema and burning pain in the hands since early childhood, later disseminating to the feet, cheeks and ears. He also experienced progressive muscle cramps, profound sweating, bowel disturbances (diarrhoea or constipation), episodic dry eyes and mouth, hot flashes, and erectile dysfunction. Neurological examination was normal. Physical examination was remarkable in revealing small hands and feet (acromesomelia). Blood examination and nerve conduction studies were unremarkable. Intra-epidermal nerve fibre density was significantly reduced compared to age- and sex-matched normative values. The patient's brother and father reported similar complaints including distal extremity redness and pain, and demonstrated comparable distal limb under-development. Quantitative sensory testing revealed impaired warmth sensation in the proband, father and brother. Genetic analysis revealed a novel missense mutation in the SCN9A gene encoding sodium channel NaV1.7 (G856D; c.2567G > A) in all three affected subjects, but not in unaffected family members. Functional analysis demonstrated that the mutation hyperpolarizes (−9.3 mV) channel activation, depolarizes (+6.2 mV) steady-state fast-inactivation, slows deactivation and enhances persistent current and the response to slow ramp stimuli by 10- to 11-fold compared with wild-type NaV1.7 channels. Current-clamp analysis of dorsal root ganglion neurons transfected with G856D mutant channels demonstrated depolarized resting potential, reduced current threshold, increased repetitive firing in response to suprathreshold stimulation and increased spontaneous firing. Our results demonstrate that the G856D mutation produces DRG neuron hyperexcitability which underlies pain in this kindred, and suggest that small peripheral nerve fibre dysfunction due to this mutation may have contributed to distal limb under-development in this novel syndrome. * Abbreviations : DRG : dorsal root ganglion

WEHI-3 cells, derived from a BALB/c mouse, are a myclomonocytic leukemic cell line with macrophage-like properties. We have isolated, purified and characterized the monosialogangliosides from WEHI-3 cells by 1D-HPTLC, 2D-HPTLC, enzymatic degradation, HPTLC-immunostaining, gas liquid chromatography and fast atom bombardment-mass spectrometry (FAB-MS). Quantitative 2D-HPTLC shows two monosialogangliosides are the major components, constituting 77% of the total, with a third monosialoganglioside being 3%. The two major components were identified as (NeuAc)G M1b and (NeuAc)G M1b -GalNAc and the minor component as (NeuAc)G M1b -GalNAc-Gal. The presence of G M1b in this myelomonocytic cell line is consistent with its presence in other murine immune cells and tissues. G M1b -GalNAc and GM 1b -GalNAc-Gal have been reported in T-lineage cells but not is resident or stimulated murine macrophages. Each of these monosialogangliosides belongs to the asialoG M1 synthetic pathway. Preliminary results indicate a disialo member of this pathway. G Dlc , may also be present as a minor component. This ganglioside pathway, containing species which are not sialylated on the internal galactose, appears to be dominant in and may be characteristic of murine immune cells.

Properties of ion channels are affected by the background of the cells in which they are expressed. Thus, it is important for investigators interested in neuronal function to study these proteins in post-mitotic neurons. However, post-mitotic neurons, and many cell lines, are difficult to transfect by standard methods. Here we provide detailed protocols for two different procedures, biolistic and electroporation, which have been used to transfect peripheral sensory neurons from mice or rats with expression constructs of voltage-gated sodium channels. Neurons can be prepared, transfected and currents recorded within 48 h. Using these methods, primary sensory neurons can be transfected with an efficiency of 5-20%, which has permitted studying biophysical properties of sodium channels and their naturally occurring mutants in a native neuronal cell background. Although we have used sodium channels for the examples that we show here, these methods can also be used to study other types of molecules.

Gain-of-function mutations of NaV1.7 have been shown to produce two distinct disorders: NaV1.7 mutations which enhance activation produce inherited erythromelalgia (IEM), characterized by burning pain in the extremities; NaV1.7 mutations which impair inactivation produce a different, non-overlapping syndrome, paroxysmal extreme pain disorder (PEPD), characterized by rectal, periocular and perimandibular pain. Here we report a novel NaV1.7 mutation associated with a mixed clinical phenotype with characteristics of IEM and PEPD, with an alanine 1632 substitution by glutamate (A1632E) in domain IV S4-S5 linker. Patch-clamp analysis shows that A1632E produces changes in channel function seen in both IEM and PEPD mutations: A1632E hyperpolarizes (-7 mV) the voltage-dependence of activation, slows deactivation, and enhances ramp responses, as observed in NaV1.7 mutations that produce IEM. A1632E depolarizes (+17mV) the voltage-dependence of fast-inactivation, slows fast-inactivation and prevents full inactivation, resulting in persistent inward currents similar to PEPD mutations. Using current-clamp, we show that A1632E renders dorsal root ganglion (DRG) and trigeminal ganglion neurons hyperexcitable. These results demonstrate a NaV1.7 mutant with biophysical characteristics common to PEPD (impaired fast-inactivation) and IEM (hyperpolarized activation, slow deactivation and enhanced ramp currents) associated with a clinical phenotype with characteristics of both IEM and PEPD, and show that this mutation renders DRG and trigeminal ganglion neurons hyperexcitable. These observations indicate that IEM and PEPD mutants are part of a physiological continuum that can produce a continuum of clinical phenotypes.

There are conflicting reports in the literature regarding the adenosine receptor that mediates the increase in sodium transport in the A6 cell. In this study we used specific A1 and A2 adenosine receptor agonists and antagonists, as well as two different subclones of the A6 cell, to determine which adenosine receptor mediates the increase in sodium transport. In the A6S2 subclone, basolateral and apical N 6-cyclohexyladenosine (CHA), a selective A1 receptor agonist, stimulated sodium transport at a threshold concentration −7 M, whereas CGS-21680, a selective A2 receptor agonist, had a threshold concentration that was at least 10−5 M. The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) was found to have a nonspecific effect on CHA-stimulated sodium transport, whereas the A2 receptor antagonist 8-(3-chlorostyryl)caffeine (CSC) had no effect. As with the A6S2 subclone, basolateral and apical CHA stimulated sodium transport at a nanomolar concentration in the A6C1 subclone and the threshold concentration for CGS-21680 was in the high micromolar range. Concurrent with the increase in sodium transport, CHA also stimulated anion secretion in the A6C1 subclone. These data demonstrate that adenosine increases sodium transport via the A1 receptor in different subclones of the A6 cell, including a subclone capable of anion secretion.

Adenosine stimulation of Na + transport by an A 1 receptor and a [Ca 2+ ] i -dependent mechanism. Studies were performed to determine the primary signal transduction mechanism that mediates adenosine stimulation of electrogenic sodium transport in renal epithelial cells. Experiments were performed on cultured amphibian A 6 cells with an adenosine analogue that preferentially binds to the A 1 receptor, cyclohexyladenosine (CHA). Sodium transport was assessed by the equivalent short circuit current (Ieq). CHA was found to stimulate Ieq via activation of an A 1 receptor because (1) the threshold concentration was 1 nM compared to that of 10 µM for the specific A 2 agonist CGS21680, (2) CHA inhibited vasopressin (AVP)-stimulated cAMP production by a pertussis toxinsensitive mechanism, and (3) the action of CHA was inhibited by the A 1 antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). CHA increased intracellular Ca 2+ ([Ca 2+ ] i ) and stimulated phosphoinositide turnover at concentrations that increased Ieq and in a time course that paralleled the increase in Ieq. Ion transport was stimulated by a Ca 2+ -dependent mechanism because the CHA induced increase in Ieq was inhibited by chelating [Ca 2+ ] i with 5,5′dimethyl BAPTA in a dose-dependent manner, with a K i of approximately 10 µM. The increase in Ieq was also dose-dependently inhibited by the specific PKC inhibitors dihydroxychlorpromazine and chelerythrine, and by trifluoperazine which inhibits PKC and calmodulin. Further studies indicated that CHA-stimulated Ieq was independent of cAMP generation because CHA did not induce an increase in cAMP accummulation parallel to the increase in Ieq in a dose-response analysis, and the adenylate cyclase inhibitor 2′,5′ dideoxyadenosine (DDA) did not affect the CHA-induced increase in Ieq. These studies indicate, therefore, that adenosine stimulation of Ieq occurs, at least in part, through calcium-dependent signal transduction events and not through regulation of adenylate cyclase.

The major neutral and acidic cerebellar lipids were studied in audiogenic seizure (AGS)-resistant C57BL 6 (B6) and AGS-susceptible DBA 2J (D2) mice. These cerebellar lipids were also studied in the D2.B6-Iasb congenic mice and in the B6D2F1 hybrids that are mostly AGS resistant. Except for the Iasb gene, which inhibits AGS susceptibility, the D2.B6-Iasb congenic mice are genetically similar to the D2 mice. Because subtle abnormalities in the chemical structure of membranes may underlie epilepsy, we wanted to determine if AGS susceptibility was associated with abnormalities in the distribution of cerebellar lipids. A new method for the analysis of total brain lipids was used in this study. Slight, but significant, differences were found between the B6 and D2 strains for the concentrations (μg/100 mg wet weight) of sphingomyelin, phosphatidylcholine, and cerebrosides. However, these differences may not be associated with differences in AGS susceptibility since the concentrations of these lipids in the AGS-resistant D2.B6-Iasb and B6D2F1 mice were more similar to the D2 than the B6 concentrations. The expression of Iasb is not responsible for the lipid differences found between the B6 and D2 mice.

To examine the effect of antiglycolipid antibodies on demyelination, myelinated cultures of embryonic mouse spinal cords were treated by antigalactocerebroside (anti-GC), anti-GM1; and anti-GM4 antisera, and the lipid composition of the cultures were studied. The anti-GC antiserum-treated cultures, which exhibited severe morphologic signs of demyelination, revealed a significant reduction of cerebroside. The anti-GM4 or anti-GM1 antiserum-treated cultures, which exhibited mild degrees of demyelination, also had low contents of cerebroside. These results support our previous data showing that antiglycolipid antibodies cause demyelination in cultured mouse spinal cords, and suggest a possible role of myelin-specific glycolipids in the demyelination process.

We have studied the lipid composition of PC12 pheochromocytoma cells cultured in the presence and absence of nerve growth factor (NGF). Neutral and acidic lipid fractions were isolated by column chromatography on DEAE-Sephadex and analyzed by high-performance thin-layer chromatography (HPTLC). The total lipid concentration was approximately 220 micrograms/mg of protein, and the concentration of neutral glycolipids was 1.6-1.8 microgram/mg of protein for both NGF-treated and untreated cells. The neutral glycolipid fraction contained a major component, which accounted for approximately 80% of the total and which was characterized as globoside on the basis of HPTLC mobility, carbohydrate analysis, fast atom bombardment mass spectrometry, and mild acid hydrolysis. The major fatty acids of globoside were C16:0 (10%), C18:0 (16%), C22:0 (23%), C24:1 (17%), and C24:0 (24%). C18 sphingenine accounted for almost all of the long-chain bases. The other neutral glycolipids were tentatively identified as glucosylceramide (15%), lactosylceramide (4%), and globotriosylceramide (4.5%). The concentration of ganglioside sialic acid was approximately 0.34 and 0.18 microgram/mg of protein for cells grown in the presence and absence of NGF, respectively. Although there was an increase in ganglioside concentration in NGF-treated cells, NGF did not produce any differential effects on the relative proportions of the individual gangliosides. Several of the gangliosides appear to contain fucose, and one of these was tentatively identified as fucosyl-GM1. Brain-type gangliosides of the ganglio series were also detected by an HPTLC-immunostaining method. However, the fatty acid and long chain base compositions of PC12 cell gangliosides (and their TLC mobility) differ from those of brain gangliosides.(ABSTRACT TRUNCATED AT 250 WORDS)

We have examined the ganglioside composition of 30-day and 60-day postnatal rat oligodendroglia, adult bovine oligodendroglia, gray matter, white matter, and myelin and also the total lipid composition of the oligodendroglial preparations. The ganglioside patterns of rat and bovine oligodendroglia, as previously found for human oligodendroglia, were more complex than those of myelin. These data indicate that oligodendroglial perikarya can synthesize many brain type gangliosides, not all of which are incorporated into the compact myelin. Alternatively, the ganglioside composition of myelin may be altered in situ by the myelin-associated neuraminidase. In these two species, as in human, GM4 appears specific to oligodendroglia and myelin, while GD3 and GM3 are enriched in oligodendroglia but not myelin. In bovine oligodendrocytes GD3 is the major ganglioside. The total lipid concentration, as well as the percentage of cholesterol, sphingomyelin, phosphatidylinositol, and phosphatidylserine, differ for 30- and 60-day-old rat oligodendroglia and may be developmentally correlated with changes in myelin composition during myelinogenesis. There are also marked differences in the lipid composition of bovine oligodendroglia compared to rat oligodendroglia, with the former having more galactolipid and less ethanolamine phosphoglycerides.

We have devised a high performance thin-layer chromatography (HPTLC) densitometry method to resolve the major lipid classes of brain tissue. We used DEAE-Sephadex column chromatography to separate the total lipid into neutral and acidic lipid fractions. The lipid fractions were then spotted on separate HPTLC plates and chromatographed in one dimension using two solvent systems. Quantitation was by in situ densitometry with absolute amounts of the lipid classes determined from co-chromatographed standards. An internal standard was also used to improve the precision. The individual lipid classes of rat whole brain, human brain gray and white matter, rat and bovine myelin, and bovine oligodendroglia were quantitated. Human brain phosphatidylethanolamine plasmalogen was also quantitated. Sensitivity was increased by using the cupric acetate charring reagent, which we found to be more sensitive than the conventional sulfuric acid-dichromate reagent. Total lipid (less than 400 micrograms) was quantitated from 5 mg of tissue wet weight. The limit of detection, on HPTLC, for the individual lipid classes was below 20 ng.

Measurement of cAMP-dependent protein kinase activity using a fluorescent-labeled Kemptide. Background Traditional protein kinase assays include the use of [ 32 P] labeled ATP as phosphate donor and a substrate protein or peptide as phosphoreceptor. Since this approach has a number of drawbacks in addition to generating ionizing radiation, several non-isotopic methods have been developed. Although shown to reflect the activity of purified enzymes, none have been demonstrated to detect physiological changes in endogenous enzyme activity in cell homogenates. Methods Studies were performed to examine the kinetics, reproducibility, and optimal assay conditions of a novel non-radioisotopic kinase assay that detects PKA activity by phosphorylation of the peptide substrate Kemptide covalently bound to a fluorescent molecule (f-Kemptide). Basal and agonist-induced PKA activity in epithelial cell homogenates was measured. Results The kinetics of f-Kemptide were similar to the standard radioisotopic method with intraassay and interassay variations of 5.6 ± 0.8% and 14.3 ± 2.6%, respectively. Neither fluorescence quenching nor enhancing effects were found with consistent amounts of homogenate protein. Specific PKA activity was determined as the IP 20 -inhibitable fraction to account for nonspecific phosphorylation, perhaps due to S6 kinase or a similar enzyme. The basal activity of 38% of total PKA in A6 cells increased by 84% after exposure to vasopressin and by 58% after short exposure to forskolin. In T84 cells exposed to VIP there was a 360% increase over basal activity. Conclusions These results show that f-Kemptide exhibits acceptable kinetics, and that the assay system can quantitatively and reproducibly measure basal and stimulated PKA activity in cell homogenates.

The ganglioside distribution of Tay—Sachs brain was re-examined in detail. In both the gray and white matter, the levels of lipid-bound sialic acid were increased 6- and 10-fold, respectively, over normal infant brain, and approximately 90% of the total ganglioside was G M2 . The level of G M2 was increased about 90 times in gray matter and 220 times in white matter in comparison with that in normal controls. The level of G D1a -GaINAc was increased 19 times and 10 times in gray and white matter, respectively. The concentration of G D2 was increased about 4-fold in Tay-S-Sachs white matter. In addition, the G M3 level was increased 2.7 and 3.5 times and the G D3 level 2 and 2.4 times over normal gray and white matter, respectively. However, the levels of other complex gangliosides such as G M1 , G D1a , G D1b , G T1b and G Q1b decreased remarkably. Since G M2 , G D2 , G D1a -GalNAc and a recently characterized ganglioside G M1b -GalNAc possess a common N-acetylgalactosaminyl terminal structure, their accumulation in Tay—Sachs brains is therefore consistent with the known hexosaminidase A deficiency. However, the accumulation of hexosamine-free G M3 and G D3 is not. The in vitro incorporation of N-acetylgalactosamine into G M3 to form G M2 was examined in a rat brain microsomal fraction in the presence of large amounts of other glycolipids. Acidic glycolipids were slightly stimulating and then became increasingly inhibitory when the molar ratio of lipid to substrate G M3 exceeded 10 to 1. Neutral glycolipids and the phospholipid, phosphatidylcholine, were inhibitory at all levels tested. The data suggest that the accumulation of G M3 and G D3 in Tay—Sachs brains could be due to an inhibition of N-acetylgalactosaminyl-transferas by high levels of glycolipids, and the inhibition is not due to chelation of the obligate divalent cation necessary for the activity of this enzyme. The inhibition of this enzyme may also be responsible for the decreased levels of other complex gangliosides.

Developmental changes in ganglioside composition and biosynthesis were studied in rat brain between embryonic day (E) 14 and birth. In E14 brains, GM3 and GD3 were predominant. At E16, “b” series gangliosides, such as GD1b, GT1b, and GQ1b, increased in content. After E18, “a” series gangliosides such as GM1, GD1a, and GT1a increased in content, and the content of GM3 and GD3 markedly decreased. Because of these changes in composition, we determined the activities, in homogenates of embryonic brains, of two key enzymes of ganglioside synthesis: sialyltransferase for the synthesis of GD3 from GM3 and N-acetylgalactosaminyltransferase for GM2 synthesis from GM3. The sialyltransferase activity (GM3 GD3) was constant between E14 and E18 but decreased rapidly from E18 to birth. In contrast, the N-acetylgalacto-saminyltransferase activity (GM3 ± GM2) increased between E14 and E18 but was constant from E18 to birth. These changes in ganglioside composition and enzymatic activities indicate that during development there is a shift from synthesis of the simplest gangliosides of the “a” and “b” pathways to synthesis of the more complex gangliosides.

Bovine buttermilk contains a unique ganglioside, 9-O-acetyl-GD3. In order to isolate large quantities of this ganglioside, a simplified isolation scheme which consists of several ion-exchange and silica gel column chromatographic procedures was devised. The isolated 9-O-acetyl-GD3 was characterized on the basis of its thin-layer chromatographic behavior, its immunoreactivity with a specific monoclonal antibody, JONES, and by conversion to authentic GD3 by mild base treatment.