Yashanad Mhaskar

Abnormal serotonin receptor expression in DBA/2 mice associated with susceptibility to sudden death due to respiratory arrest

Previous studies indicate that DBA/2 mice may be a useful model of human sudden unexpected death in epilepsy (SUDEP), since these mice exhibit generalized convulsive seizures followed by respiratory arrest (RA). Respiratory deficits, following generalized convulsive seizures, are observed prior to SUDEP in patients. RA that occurs in DBA/2 mice following sound-induced seizures can be prevented by treatments that activate serotonin (5-HT) receptors. 5-HT receptor subtypes in brainstem respiratory centers are important in regulating normal respiration. The present study compared the expression of 5-HT subtype receptor proteins in excised brainstem tissue, containing the rostral ventral medulla respiratory region in DBA/2 mice vs. seizure-resistant C57BL/6J mice, using Western blot analysis. The results indicate that expression of specific 5-HT(2C), 5-HT(3), and 5-HT(4) receptor proteins in the brainstem tissue of DBA/2 mice is significantly diminished, while expression of the 5-HT(2B) receptors is significantly enhanced as compared to C57BL/6J mice. No difference in expression of 5-HT transporter protein is seen. These findings suggest that the DBA/2 mice are susceptible to RA, in part, because of the altered expression of 5-HT receptors. Preliminary studies indicate that 5-HT(2C) receptors may be particularly important, since a 5-HT(2C) agonist is very effective in blocking RA in DBA/2 mice.

Differences in serotonin receptor expression in the brainstem may explain the differential ability of a serotonin agonist to block seizure-induced sudden death in DBA/2 vs. DBA/1 mice

DBA mice are models of sudden unexpected death in epilepsy (SUDEP) that exhibit audiogenic generalized convulsive seizures (GCS), ending in death due to respiratory arrest (RA). Serotonin (5-HT) normally enhances respiration in response to elevated CO(2) levels, which occur during GCS in patients. Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), blocks GCS-induced SUDEP in both DBA/2 and DBA/1 mice. This study examined the effects of a 5-HT(2B/2C) agonist (m-chlorophenylpiperazine, mCPP) to test the generality of serotonergic effects on DBA mice. In DBA/2 mice mCPP pre-treatment [5 or 10 (but not 2) mg/kg, i.p.] significantly reduced RA incidence without blocking seizure susceptibility. However, in DBA/1 mice mCPP in doses up to 40mg/kg was ineffective in blocking seizure-induced RA, and 60mg/kg was toxic. The cause of this strain difference was perplexing. Previous studies showed that brainstem 5-HT receptor protein expression was abnormal in DBA/2 mice. Therefore, expression of 5-HT receptor proteins in the medial-caudal brainstem of DBA/1 mice was evaluated using Western blots. In DBA1/mice 5-HT(2C) and 5-HT(3B) receptor expression levels were significantly reduced, as seen previously in DBA/2 mice. However, 5-HT(2B) receptor expression was also reduced in DBA/1 mice, contrasting with the 5-HT(2B) receptor elevation seen in DBA/2 mice. This difference may explain the differential effects of the 5-HT(2B/2C) agonist in these SUDEP models. mCPP blocked RA in DBA/2 mice and concomitantly reduced tonic seizures, which also occurs. Fluoxetine is the only agent tested that blocks RA selectively in these SUDEP models, which may be clinically relevant.

Regulation of brain 6-phosphofructo-1-kinase: effects of aging, fructose-2,6-bisphosphate, and regional subunit distribution

Total 6-phosphofructo-1-kinase (PFK) activity, amounts of each type of PFK subunit, and levels of fructose-2,6-P2 in the cerebral cortex, midbrain, pons-medulla, and cerebellum of 3, 12, and 25 month rats were measured. Further, the role of fructose-2,6-P2 in the regulation of brain PFK activity was examined. A positive correlation was found to exist between the reported losses of glucose utilization as measured by 2-deoxy-D-glucose uptake and PFK activity in each region. That is, both parameters decreased to their lowest level by 12 months of age and remained decreased and fairly constant thereafter. Fructose-2,6-P2 levels did not appear to directly correlate with regional changes in glucose utilization. Also, region-specific and age-related alterations of the PFK subunits were found although these changes apparently did not correlate with decreased glucose utilization. Brain PFK is apparently saturated with fructose-2,6-P2 due to the high endogenous levels, and it contains a large proportion of the C-type subunit which dampens catalytic efficiency. Consequently, brain PFK could exist in a conformational state such that it can readily consume fructose-6-P rather than in an inhibited state requiring activation. This may explain, in part, the ability of brain to efficiently but conservatively utilize available glucose in energy production.

Characterization of phosphofructokinase-deficient canine erythrocytes

SummaryDogs homozygously affected with muscle-type phosphofructokinase (PFK) deficiency had about 20% of normal erythrocyte PFK activity and exhibited a compensated haemolytic anaemia. Erythrocyte glucose-6-phosphate and fructose-6-phosphate concentrations were increased and dihydroxyacetone phosphate and 2,3-bisphosphoglycerate values were below normal in affected dogs. Other intermediates distal to the PFK step were not significantly below normal and fructose-1,6-bisphosphate was even above normal. Erythrocyte ATP was higher than normal in affected dogs owing to the reticulocytes present. Abnormal adenylate metabolism was demonstrated by low ATP/AMP and ADP/AMP ratios and the inability to maintain ATP content when affected erythrocytes were incubated with cyanide. Glucose-1,6-bisphosphate content was normal, and fructose-2,6-bisphosphate content in affected canine erythrocytes was higher than normal. Studies of erythrocyte PFK isozymes revealed altered enzyme kinetic properties in affected dogs which appeared to be due to the loss of the M-type subunit.

The subunit proportions and kinetic properties of 6-phosphofructo-1-kinase isozymes from rat heart atria and ventricle progressively change during aging.

Relative to 2–3 month rats, total 6-phosphofructo-1-kinase (PFK) activity in heart atria from 12 month rats declined 31%; but, by 24 months it was decreased by only 13%. PFK activities from 12 and 24 month ventricles relative to the 2–3 month rat were decreased by 40% and 30%, respectively. This change in PFK activity in each heart region was associated with alterations of subunit composition. In heart atria from 12 and 24 month rats when compared to 3 month rats, the levels of L-type subunit were not significantly different; but the levels of the M-type subunit were decreased by 43% and 38%, respectively. With respect to levels in 2–3 month atria, the C-type subunit in 12 month atria decreased by 27%; and at 24 months it increased by 31%. Making the same comparison for the heart ventricle at 12 and 24 months, L-type subunit decreased by 30% and 24% respectively; M-type subunit decreased by approximately 47%; and the C-type subunit increased 1.9 and 4.7 fold, respectively. These age-related changes of subunit composition in atrial and ventricular PFK isozyme pools led to changes in their kinetic and regulatory properties suggesting that the aged rat could exhibit a diminished capacity to produce ATP from glucose.

Presence of a truncated M-type subunit and altered kinetic properties of 6-phosphofructo-1-kinase isozymes in the brain of a dog affected by glycogen storage disease type VII

6-Phosphofructo-1-kinase (PFK) activity in the brain of a dog affected by glycogen storage disease type VII was only 31% of the PFK activity in the normal dog brain. PFK in the normal dog brain was composed of L-type, M-type and C-type subunits with apparent molecular weights of 78,000, 86,000, and 88,000, respectively, and subunit proportions (L:M:C) of 27:49:24. PFK in the affected dog brain was composed of nearly equal levels of the normal L-type and C-type subunits, but a normal M-type subunit was not detected. Using antidog muscle PFK IgG, immunoblots of gels containing partially purified PFK from the affected dog brain revealed a small amount of immunoreactive protein with an apparent molecular weight of 84,000, suggesting the presence of a truncated M-type subunit. Kinetic studies indicated that the PFK isozymes in the affected dog brain exhibited significantly different kinetic regulatory properties when compared to the PFK isozyme pool in the normal dog brain.

Developmental changes of 6-phosphofructo-1-kinase subunit levels in erythrocytes from normal dogs and dogs affected by glycogen storage disease type VII

1. The subunit proportions (L:M:C) of the PFK isozymes from normal adult erythrocytes were 2:86:12. Affected adult erythrocyte 6-phosphofructo-1-kinase (PFK) isozymes contained normal L-type (31%) and C-type (61%) subunits as well as a small amount (8%) of truncated M-type subunit. 2. When measured within 24 hr of birth, both normal and affected dog erythrocytes contained high PFK activities due to elevated levels of the L-type subunit. As the dogs matured, PFK activity decreased due to a greater than 99% loss of the L-type subunit. 3. By 2 weeks of age, the M-type and C-type subunits in normal dog PFK isozymes increased several-fold and attained near adult levels. 4. During post-natal development, the L-type subunit from affected dog erythrocytes decreased more rapidly than from normal dog erythrocytes; but it was maintained at a higher level in the affected adult erythrocytes. Also, in the affected dog erythrocytes, truncated M-type subunits were detected; and the initially high levels of the C-type subunit decreased approximately 50% after 4 weeks.

Alteration of 6-phosphofructo-1-kinase subunits during neonatal maturation of the rat cochlear cells.

During postnatal development of rat cochlear cells and the onset of hearing (10-23 days), the increasing endocochlear potential and energy requirements are largely provided by increased glucose utilization. It is well established that the ability of maturing rat tissues to use glucose is directly related to alteration of 6-phosphofructo-1-kinase (PFK) subunits. To gain insight into the alteration of PFK subunit levels in the cochlea from 6 to 60 days of age, PFK subunit types were measured in sections of paraffin-embedded temporal bone using IgG specific for each type of PFK subunit and quantified by computer image analysis. Although the L-type and C-type subunits did not exhibit statistically significant changes in the cochlear structures during maturation, the levels of M-type subunit in the stria vascularis cells, spiral ligament cell types I, II, and III, outer hair cells, inner hair cells, and support cells significantly increased. Also, the type IV and V spiral ligament fibrocytes during this period did not exhibit significant alterations of the M-type subunit. These data suggest that during neonatal development of the cochlear, the elevated levels of the M-type subunit are associated with increased glucose utilization and the onset of hearing.

Alteration of 6-phosphofructo-1-kinase subunit protein, synthesis rates, and mRNA during rat neonatal development

For the three 6-phosphofructo-1-kinase (PFK) subunits in heart, skeletal muscle, liver and kidney, developmentally-associated changes in protein, mRNA and apparent synthesis rates were observed. During neonatal maturation, all three phenomena for the M-type in heart and skeletal muscle exhibited large increases. Also, during neonatal development, the L-type and C-type subunits were unaffected in heart but disappeared from skeletal muscle. In the newborn liver and kidney, the amounts of each type of PFK subunit protein were nearly identical. During neonatal development, the levels of all three PFK subunit proteins in kidney increased more than twofold; and this was associated with a similar increase in apparent subunit synthesis rates and mRNA levels. During liver neonatal development, the L-type subunit protein, synthesis and mRNA levels also increased more than twofold. However, during hepatic maturation, M-type subunit protein, synthesis and mRNA levels were unchanged and apparently unaffected. The C-type subunit protein during neonatal liver development decreased approximately 80% as did its apparent synthesis rate. These data suggest that regulation of the alteration of the PFK subunit proteins during neonatal maturation can vary among these tissues and is not the same for each subunit type. Different mechanisms, such as transcription, translation, and mRNA stability could be involved.

Alteration of the levels of the M-type 6-phosphofructo-1-kinase mRNA isoforms during neonatal maturation of heart, brain and muscle.

During muscle, heart, and brain neonatal maturation, the capacity to utilize glucose in energy metabolism is directly related to the extent of accumulation of the 6-phosphofructo-1-kinase (PFK) M-type subunit. Neonatal development of other organs, such as liver and kidney, which are not characterized by large increases in the capacity to use glucose do not exhibit large increases in the M-type subunit protein. The presence of the M-type subunit in a PFK isozyme pool fosters a higher affinity utilization of carbohydrate and increased responsiveness to the levels of regulatory metabolites. To better appreciate this phenomenon, which is vital for normal development, the different isoforms of the M-type subunit mRNAs and alteration of their levels during maturation have been examined. Further, the potential promoter regions, i.e., the regions upstream from the sites of initiation of transcription, which are involved in expression of the different M-type subunit mRNA isoforms have been isolated, sequenced, and examined for possible transcription factor interaction sites. Using cDNA libraries produced from adult rat brain or skeletal muscle RNA, two primary forms of rat M-type subunit cDNAs were detected. Although the translated regions of these mRNAs were essentially identical, the 5′-untranslated region (5′-UTR) exhibited different lengths (90 or 59 bp) and sequences. Each M-type subunit cDNA had 10 common nucleotides immediately upstream from the initiator ATG, and the remaining 5′-UTRs had insignificant identity. A genomic fragment which interacted with probes complimentary to the sequences of the 5′-UTR of each M-type subunit mRNA isoform was isolated and sequenced by primer walking. It was discovered that the 5′-UTR of one of the mRNAs (proximal mRNA) was located immediately upstream from exon I and was apparently transcribed without splicing. Subsequently, the initial bp in the sequence of the other mRNA isoform (distal mRNA) was located 4010 bp upstream from the ATG in exon 1. Employing Reverse Transcription-Polymerase Chain Reaction using total RNA and scanning densitometry, the relative levels of the proximal and distal mRNAs during neonatal maturation of brain, heart, and muscle were measured. In these tissues, both forms of M-type subunit mRNAs were present, and during maturation tissue-specific differences were noted.