Astrocytes express transient receptor potential channels (TRPCs), which have been implicated in C... more Astrocytes express transient receptor potential channels (TRPCs), which have been implicated in Ca 2ϩ influx triggered by intracellular Ca 2ϩ stores depletion, a phenomenon known as capacitative Ca 2ϩ entry. We studied the properties of capacitative Ca 2ϩ entry in astrocytes by means of single-cell Ca 2ϩ imaging with the aim of understanding the involvement of TRPCs in this function. We found that, in astrocytes, capacitative Ca 2ϩ entry is not attributable to TRPC opening because the TRPC-permeable ions Sr 2ϩ and Ba 2ϩ do not enter astrocytes during capacitative Ca 2ϩ entry. Instead, natively expressed oleyl-acetyl-glycerol (OAG) (a structural analog of DAG) -sensitive
Ca 2+ uptake into the endoplasmic reticulum (ER) is mediated by Ca 2+ ATPase isoforms, which are ... more Ca 2+ uptake into the endoplasmic reticulum (ER) is mediated by Ca 2+ ATPase isoforms, which are all selectively inhibited by nanomolar concentrations of thapsigargin. Using ATP/ Mg 2+ -dependent 45 Ca 2+ transport in rat brain microsomes, tissue sections, and permeabilized cells, as well as Ca 2+ imaging in living cells we distinguish two ER Ca 2+ pools in the rat CNS. Nanomolar levels of thapsigargin blocked one component of brain microsomal 45 Ca 2+ transport, which we designate as the thapsigargin-sensitive pool (TG-S). The remaining component was only inhibited by micromolar thapsigargin, and thus designated as thapsigargin resistant (TG-R). Ca 2+ ATPase and [ 32 P]phosphoenzyme assays also distinguished activities with differential sensitivities to thapsigargin. The TG-R Ca 2+ uptake displayed unique anion permeabilities, was inhibited by vanadate, but was unaffected by sulfhydryl reduction. Ca 2+ sequestered into the TG-R pool could not be released by inositol-1,4,5-trisphosphate, caffeine, or cyclic ADP-ribose. The TG-R Ca 2+ pool had a unique anatomical distribution in the brain, with selective enrichment in brainstem and spinal cord structures. Cell lines that expressed high levels of the TG-R pool required micromolar concentrations of thapsigargin to effectively raise cytoplasmic Ca 2+ levels. TG-R Ca 2+ accumulation represents a distinct Ca 2+ buffering pool in specific CNS regions with unique pharmacological sensitivities and anatomical distributions.
Dysregulated glucose metabolism and energy deficit is a characteristic of severe traumatic brain ... more Dysregulated glucose metabolism and energy deficit is a characteristic of severe traumatic brain injury (TBI) but its mechanism remains to be fully elucidated. Phosphorylation of pyruvate dehydrogenase (PDH) is the rate-limiting mitochondria enzyme reaction coupling glycolysis to the tricarboxylic acid cycle. Phosphorylation of PDH E1α1 subunit catalyzed by PDH kinase (PDK) inhibits PDH activity, effectively decoupling aerobic glycolysis whereas dephosphorylation of phosphorylated PDHE1α1 by PDH phosphatase (PDP) restores PDH activity. We recently reported altered expression and phosphorylation of pyruvate dehydrogenase (PDH) following TBI. However, little is known about PDK and PDP involvement. We determined PDK (PDK1-4) and PDP isoenzyme (PDP1-2) mRNA and protein expression in rat brain using immunohistochemistry and in situ hybridization techniques. We also quantified PDK and PDP mRNA and protein levels in rat brain following TBI using quantitative real-time PCR and Western blot, respectively. Controlled cortical impact-induced TBI (CCI-TBI) and craniotomy significantly enhanced PDK1-2 isoenzyme mRNA expression level but significantly suppressed PDP1 and PDK4 mRNA expression after the injury (4 h to 7 days). CCI-TBI and craniotomy also significantly increased PDK1-4 isoenzyme protein expression but suppressed PDP1-2 protein expression in rat brain. In summary, the divergent changes between PDK and PDP expression indicate imbalance between PDK and PDP activities that would favor increased PDHE1α1 phosphorylation and enzyme inhibition contributing to impaired oxidative glucose metabolism in TBI as well as craniotomy.
Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat b... more Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine [( 3H]NBI) the solubilized site is characterized both kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible, specific, and high affinity in nature. The KD and Bmax of guinea pig extracts are 0.13 +/- 0.02 nM and 133 +/- 18 fmol/mg protein, respectively, with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of [3H]NBI binding with the following order of potency, dilazep greater than hexobendine greater than dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding, and caffeine is without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. The binding of the adenosine A1 receptor agonist [3H]cyclohexyladenosine [( 3H]CHA) to the solubilized brain extract was also studied and compared with that of [3H]NBI. In contrast to the [3H]NBI binding site [3H]CHA binds to two apparent populations of adenosine receptor, a high-affinity site with a KD of 0.32 +/- 0.06 nM and a Bmax of 105 +/- 30 fmol/mg protein and a lower-affinity site with a KD of 5.50 +/- 0.52 nM and Bmax of 300 +/- 55 fmol/mg protein. The pharmacology of the [3H]CHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake [( 3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state.
Postoperative complications and nutritional deficits resulting from bariatric surgery can lead to... more Postoperative complications and nutritional deficits resulting from bariatric surgery can lead to severe vitamin-deficiency states, such as Wernicke's encephalopathy (WE). Patients with acute WE generally present with the classic clinical triad of inattentiveness, ataxia, and ophthalmoplegia. We describe a patient who presented with acute WE at 2 months after laparoscopic bariatric surgery. Initial MRI of the brain demonstrated the characteristic injuries of WE, and repeat imaging showed resolution after 4 months of thiamine supplementation , at which time the patient had normal gait but persistent memory deficits. Even with early recognition and aggressive therapy, acute WE commonly results in permanent disability due to the irreversible cytotoxic effects on specific regions of the brain. Since the clinical onset of acute WE follows a predictable time-course in postbariatric surgery patients with malnutrition, we recommend prevention by administration of parenteral thiamine beginning at 6 weeks postoperatively in malnourished patients.
Cerebral pyruvate depletion and lactate acidosis are common metabolic characteristics of patients... more Cerebral pyruvate depletion and lactate acidosis are common metabolic characteristics of patients with traumatic brain injury (TBI) and are associated with poor prognosis. Pyruvate dehydrogenase (PDH) is the rate-limiting enzyme coupling glycolysis to mitochondrial tricarboxylic acid (TCA) cycle. Brain PDH activity is regulated by its phosphorylation status and other effectors. Phosphorylation of PDH E1α1 subunit by PDH kinase inhibits PDH activity while dephosphorylation of phosphorylated PDHE1α1 by PDH phosphatase (PDP1) restores PDH activity. In situ hybridization showed that PDP1 mRNA is highly expressed in the cerebral cortex, hippocampus and thalamus of rat. Controlled cortical impact (CCI) induced a significant increase in PDP1 mRNA expression in ipsilateral cerebral cortex at 4 h (P<0.05) and 24 h post CCI (P<0.01) that returned to basal level 72 h post CCI. PDP1 mRNA level increased transiently in ipsilateral hippocampal dentate gyrus and CA1-3 subfields 4 h post CCI (P<0.01) but decreased significantly 24 h and 72 h (P<0.01) post CCI, coinciding with a marked increase in neuronal apoptosis in ipsilateral hippocampus 24 h post CCI. PDP1 mRNA expression in thalamus and other subcortical regions decreased persistently post CCI. Contralateral CCI and craniotomy showed similar effects on PDP1 mRNA expression as ipsilateral CCI. Because GFAP mRNA expression was induced in brain regions where PDP1 expression was altered, further study should determine the potential relationship between astrocyte activation, PDP1 alteration, and pyruvate metabolism following TBI.
Traditional analyses of calcium homeostasis have separately quantified either calcium accumulatio... more Traditional analyses of calcium homeostasis have separately quantified either calcium accumulation or release mechanisms. To define the system as a whole, however, requires multiple experimental techniques to examine both accumulation and release. Here we describe a technique that couples the simultaneous quantification of radio-labeled calcium accumulation in endoplasmic reticulum (ER) microsomes with the release of inorganic phosphate (Pi) by the hydrolytic activity of sarco-endoplasmic reticulum calcium ATPase (SERCA) all in the convenience of a 96-well format.
Astrocytes express transient receptor potential channels (TRPCs), which have been implicated in C... more Astrocytes express transient receptor potential channels (TRPCs), which have been implicated in Ca 2ϩ influx triggered by intracellular Ca 2ϩ stores depletion, a phenomenon known as capacitative Ca 2ϩ entry. We studied the properties of capacitative Ca 2ϩ entry in astrocytes by means of single-cell Ca 2ϩ imaging with the aim of understanding the involvement of TRPCs in this function. We found that, in astrocytes, capacitative Ca 2ϩ entry is not attributable to TRPC opening because the TRPC-permeable ions Sr 2ϩ and Ba 2ϩ do not enter astrocytes during capacitative Ca 2ϩ entry. Instead, natively expressed oleyl-acetyl-glycerol (OAG) (a structural analog of DAG) -sensitive
Ca 2+ uptake into the endoplasmic reticulum (ER) is mediated by Ca 2+ ATPase isoforms, which are ... more Ca 2+ uptake into the endoplasmic reticulum (ER) is mediated by Ca 2+ ATPase isoforms, which are all selectively inhibited by nanomolar concentrations of thapsigargin. Using ATP/ Mg 2+ -dependent 45 Ca 2+ transport in rat brain microsomes, tissue sections, and permeabilized cells, as well as Ca 2+ imaging in living cells we distinguish two ER Ca 2+ pools in the rat CNS. Nanomolar levels of thapsigargin blocked one component of brain microsomal 45 Ca 2+ transport, which we designate as the thapsigargin-sensitive pool (TG-S). The remaining component was only inhibited by micromolar thapsigargin, and thus designated as thapsigargin resistant (TG-R). Ca 2+ ATPase and [ 32 P]phosphoenzyme assays also distinguished activities with differential sensitivities to thapsigargin. The TG-R Ca 2+ uptake displayed unique anion permeabilities, was inhibited by vanadate, but was unaffected by sulfhydryl reduction. Ca 2+ sequestered into the TG-R pool could not be released by inositol-1,4,5-trisphosphate, caffeine, or cyclic ADP-ribose. The TG-R Ca 2+ pool had a unique anatomical distribution in the brain, with selective enrichment in brainstem and spinal cord structures. Cell lines that expressed high levels of the TG-R pool required micromolar concentrations of thapsigargin to effectively raise cytoplasmic Ca 2+ levels. TG-R Ca 2+ accumulation represents a distinct Ca 2+ buffering pool in specific CNS regions with unique pharmacological sensitivities and anatomical distributions.
Dysregulated glucose metabolism and energy deficit is a characteristic of severe traumatic brain ... more Dysregulated glucose metabolism and energy deficit is a characteristic of severe traumatic brain injury (TBI) but its mechanism remains to be fully elucidated. Phosphorylation of pyruvate dehydrogenase (PDH) is the rate-limiting mitochondria enzyme reaction coupling glycolysis to the tricarboxylic acid cycle. Phosphorylation of PDH E1α1 subunit catalyzed by PDH kinase (PDK) inhibits PDH activity, effectively decoupling aerobic glycolysis whereas dephosphorylation of phosphorylated PDHE1α1 by PDH phosphatase (PDP) restores PDH activity. We recently reported altered expression and phosphorylation of pyruvate dehydrogenase (PDH) following TBI. However, little is known about PDK and PDP involvement. We determined PDK (PDK1-4) and PDP isoenzyme (PDP1-2) mRNA and protein expression in rat brain using immunohistochemistry and in situ hybridization techniques. We also quantified PDK and PDP mRNA and protein levels in rat brain following TBI using quantitative real-time PCR and Western blot, respectively. Controlled cortical impact-induced TBI (CCI-TBI) and craniotomy significantly enhanced PDK1-2 isoenzyme mRNA expression level but significantly suppressed PDP1 and PDK4 mRNA expression after the injury (4 h to 7 days). CCI-TBI and craniotomy also significantly increased PDK1-4 isoenzyme protein expression but suppressed PDP1-2 protein expression in rat brain. In summary, the divergent changes between PDK and PDP expression indicate imbalance between PDK and PDP activities that would favor increased PDHE1α1 phosphorylation and enzyme inhibition contributing to impaired oxidative glucose metabolism in TBI as well as craniotomy.
Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat b... more Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine [( 3H]NBI) the solubilized site is characterized both kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible, specific, and high affinity in nature. The KD and Bmax of guinea pig extracts are 0.13 +/- 0.02 nM and 133 +/- 18 fmol/mg protein, respectively, with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of [3H]NBI binding with the following order of potency, dilazep greater than hexobendine greater than dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding, and caffeine is without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. The binding of the adenosine A1 receptor agonist [3H]cyclohexyladenosine [( 3H]CHA) to the solubilized brain extract was also studied and compared with that of [3H]NBI. In contrast to the [3H]NBI binding site [3H]CHA binds to two apparent populations of adenosine receptor, a high-affinity site with a KD of 0.32 +/- 0.06 nM and a Bmax of 105 +/- 30 fmol/mg protein and a lower-affinity site with a KD of 5.50 +/- 0.52 nM and Bmax of 300 +/- 55 fmol/mg protein. The pharmacology of the [3H]CHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake [( 3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state.
Postoperative complications and nutritional deficits resulting from bariatric surgery can lead to... more Postoperative complications and nutritional deficits resulting from bariatric surgery can lead to severe vitamin-deficiency states, such as Wernicke's encephalopathy (WE). Patients with acute WE generally present with the classic clinical triad of inattentiveness, ataxia, and ophthalmoplegia. We describe a patient who presented with acute WE at 2 months after laparoscopic bariatric surgery. Initial MRI of the brain demonstrated the characteristic injuries of WE, and repeat imaging showed resolution after 4 months of thiamine supplementation , at which time the patient had normal gait but persistent memory deficits. Even with early recognition and aggressive therapy, acute WE commonly results in permanent disability due to the irreversible cytotoxic effects on specific regions of the brain. Since the clinical onset of acute WE follows a predictable time-course in postbariatric surgery patients with malnutrition, we recommend prevention by administration of parenteral thiamine beginning at 6 weeks postoperatively in malnourished patients.
Cerebral pyruvate depletion and lactate acidosis are common metabolic characteristics of patients... more Cerebral pyruvate depletion and lactate acidosis are common metabolic characteristics of patients with traumatic brain injury (TBI) and are associated with poor prognosis. Pyruvate dehydrogenase (PDH) is the rate-limiting enzyme coupling glycolysis to mitochondrial tricarboxylic acid (TCA) cycle. Brain PDH activity is regulated by its phosphorylation status and other effectors. Phosphorylation of PDH E1α1 subunit by PDH kinase inhibits PDH activity while dephosphorylation of phosphorylated PDHE1α1 by PDH phosphatase (PDP1) restores PDH activity. In situ hybridization showed that PDP1 mRNA is highly expressed in the cerebral cortex, hippocampus and thalamus of rat. Controlled cortical impact (CCI) induced a significant increase in PDP1 mRNA expression in ipsilateral cerebral cortex at 4 h (P<0.05) and 24 h post CCI (P<0.01) that returned to basal level 72 h post CCI. PDP1 mRNA level increased transiently in ipsilateral hippocampal dentate gyrus and CA1-3 subfields 4 h post CCI (P<0.01) but decreased significantly 24 h and 72 h (P<0.01) post CCI, coinciding with a marked increase in neuronal apoptosis in ipsilateral hippocampus 24 h post CCI. PDP1 mRNA expression in thalamus and other subcortical regions decreased persistently post CCI. Contralateral CCI and craniotomy showed similar effects on PDP1 mRNA expression as ipsilateral CCI. Because GFAP mRNA expression was induced in brain regions where PDP1 expression was altered, further study should determine the potential relationship between astrocyte activation, PDP1 alteration, and pyruvate metabolism following TBI.
Traditional analyses of calcium homeostasis have separately quantified either calcium accumulatio... more Traditional analyses of calcium homeostasis have separately quantified either calcium accumulation or release mechanisms. To define the system as a whole, however, requires multiple experimental techniques to examine both accumulation and release. Here we describe a technique that couples the simultaneous quantification of radio-labeled calcium accumulation in endoplasmic reticulum (ER) microsomes with the release of inorganic phosphate (Pi) by the hydrolytic activity of sarco-endoplasmic reticulum calcium ATPase (SERCA) all in the convenience of a 96-well format.
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Papers by Ajay Verma