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John N. Fain - One of the best experts on this subject based on the ideXlab platform.

  • Effect of K+-induced depolarization on carbachol-stimulated Inositol Tetrakisphosphate accumulation in rat cerebrocortical slices.
    Biochimica et biophysica acta, 1996
    Co-Authors: Marvin E. Myles, John N. Fain
    Abstract:

    Carbachol-stimulated accumulation of labeled IP4 or of total Ins 1,3,4,5-P4 in rat brain cortical slices was maximal in buffer containing 10 mM K+. Iso-osmotic elevation of extracellular K+ to 30 mM did not affect total Ins 1,3,4,5-P4 accumulation but did enhance carbachol stimulated Ins 1,4,5-P3 accumulation. Iso-osmotically elevated K+ suppressed carbachol stimulated accumulation of labeled IP4 while enhancing accumulation of labeled Inositol mono-, bis- and trisphosphates. High K+ alone increased basal accumulation of labeled Inositol mono-, bis- and trisphosphates, and total Ins 1,4,5-P3, while having no significant effect on accumulation of labeled IP4 or total Ins 1,3,4,5-P4. Long-term incubation with hyper-osmotically elevated K+ potentiated carbachol-stimulated Ins 1,3,4,5-P4 accumulation at 5 min. However, hyper-osmotically elevated K+ suppressed accumulation of labeled IP4 due to carbachol. These results indicate that there is no short-term effect of iso-osmotically elevated K+ on carbachol-stimulated total Ins 1,3,4,5-P4 accumulation. Furthermore, elevating K+ above 10 mM either iso-osmotically or hyper-osmotically suppresses carbachol stimulated accumulation of labeled IP4. The results suggest that the altered Na+/K+ ratio influenced the production of Inositol Tetrakisphosphates and emphasize the important role of cations such as Na+, K+, and Ca2+ in the receptor-mediated Inositol response. Moreover, the results underscore the unique ability of carbachol (a cholinergic agonist) to stimulate significant accumulation of Inositol Tetrakisphosphate.

  • Inhibition by NMDA of carbachol-stimulated Inositol Tetrakisphosphate accumulation in rat brain cortical slices.
    Neuropharmacology, 1996
    Co-Authors: Marvin E. Myles, Yesim Gokmen-polar, John N. Fain
    Abstract:

    The present studies examined the inhibitory effect of veratridine (a Na+ channel activator) on carbachol (a cholinergic agonist) stimulated Inositol 1,3,4,5-Tetrakisphosphate accumulation in rat brain cortical slices. Veratridine inhibited carbachol stimulation of Inositol 1,3,4,5-Tetrakisphosphate formation (after a delay of about 30 seconds) at 60 or 120 seconds when there was little inhibition of Inositol 1,4,5 trisphophate accumulation. The inhibitory effect of veratridine on carbachol stimulated Inositol 1,3,4,5-Tetrakisphosphate accumulation was abolished in the presence of ouabain or tetrodotoxin but was unaffected in low calcium conditions. Veratridine reduced the total ATP content and this effect was abolished by tetrodotoxin. The inhibitory effect of 10 but not 30 μM veratridine on Inositol 1,3,4,5-Tetrakisphosphate accumulation in the presence of carbachol was reversed by the presence of exogenous 8-bromo cyclic AMP or forskolin which activates adenylylcyclase. However, the decrease in brain slice ATP seen in the presence of veratridine was unaffected by forskolin. Our results are compatible with the hypothesis that veratridine inhibition of carbachol-stimulated Inositol 1,3,4,5-Tetrakisphosphate formation is due to depletion of ATP at the site of Ins 1,3,4,5-P4 formation from Ins 1,4,5-P3.

  • Inhibition by veratridine of carbachol-stimulated Inositol Tetrakisphosphate accumulation in rat brain cortical slices
    Neurochemical Research, 1995
    Co-Authors: Marvin E. Myles, Yesim Gokmen-polar, John N. Fain
    Abstract:

    The present studies examined the inhibitory effect of veratridine (a Na^+ channel activator) on carbachol (a cholinergic agonist) stimulated Inositol 1,3,4,5-Tetrakisphosphate accumulation in rat brain cortical slices. Veratridine inhibited carbachol stimulation of Inositol 1,3,4,5-Tetrakisphosphate formation (after a delay of about 30 seconds) at 60 or 120 seconds when there was little inhibition of Inositol 1,4,5 trisphophate accumulation. The inhibitory effect of veratridine on carbachol stimulated Inositol 1,3,4,5-Tetrakisphosphate accumulation was abolished in the presence of ouabain or tetrodotoxin but was unaffected in low calcium conditions. Veratridine reduced the total ATP content and this effect was abolished by tetrodotoxin. The inhibitory effect of 10 but not 30 μM veratridine on Inositol 1,3,4,5-Tetrakisphosphate accumulation in the presence of carbachol was reversed by the presence of exogenous 8-bromo cyclic AMP or forskolin which activates adenylylcyclase. However, the decrease in brain slice ATP seen in the presence of veratridine was unaffected by forskolin. Our results are compatible with the hypothesis that veratridine inhibition of carbachol-stimulated Inositol 1,3,4,5-Tetrakisphosphate formation is due to depletion of ATP at the site of Ins 1,3,4,5-P_4 formation from Ins 1,4,5-P_3.

Marvin E. Myles - One of the best experts on this subject based on the ideXlab platform.

  • Effect of K+-induced depolarization on carbachol-stimulated Inositol Tetrakisphosphate accumulation in rat cerebrocortical slices.
    Biochimica et biophysica acta, 1996
    Co-Authors: Marvin E. Myles, John N. Fain
    Abstract:

    Carbachol-stimulated accumulation of labeled IP4 or of total Ins 1,3,4,5-P4 in rat brain cortical slices was maximal in buffer containing 10 mM K+. Iso-osmotic elevation of extracellular K+ to 30 mM did not affect total Ins 1,3,4,5-P4 accumulation but did enhance carbachol stimulated Ins 1,4,5-P3 accumulation. Iso-osmotically elevated K+ suppressed carbachol stimulated accumulation of labeled IP4 while enhancing accumulation of labeled Inositol mono-, bis- and trisphosphates. High K+ alone increased basal accumulation of labeled Inositol mono-, bis- and trisphosphates, and total Ins 1,4,5-P3, while having no significant effect on accumulation of labeled IP4 or total Ins 1,3,4,5-P4. Long-term incubation with hyper-osmotically elevated K+ potentiated carbachol-stimulated Ins 1,3,4,5-P4 accumulation at 5 min. However, hyper-osmotically elevated K+ suppressed accumulation of labeled IP4 due to carbachol. These results indicate that there is no short-term effect of iso-osmotically elevated K+ on carbachol-stimulated total Ins 1,3,4,5-P4 accumulation. Furthermore, elevating K+ above 10 mM either iso-osmotically or hyper-osmotically suppresses carbachol stimulated accumulation of labeled IP4. The results suggest that the altered Na+/K+ ratio influenced the production of Inositol Tetrakisphosphates and emphasize the important role of cations such as Na+, K+, and Ca2+ in the receptor-mediated Inositol response. Moreover, the results underscore the unique ability of carbachol (a cholinergic agonist) to stimulate significant accumulation of Inositol Tetrakisphosphate.

  • Inhibition by NMDA of carbachol-stimulated Inositol Tetrakisphosphate accumulation in rat brain cortical slices.
    Neuropharmacology, 1996
    Co-Authors: Marvin E. Myles, Yesim Gokmen-polar, John N. Fain
    Abstract:

    The present studies examined the inhibitory effect of veratridine (a Na+ channel activator) on carbachol (a cholinergic agonist) stimulated Inositol 1,3,4,5-Tetrakisphosphate accumulation in rat brain cortical slices. Veratridine inhibited carbachol stimulation of Inositol 1,3,4,5-Tetrakisphosphate formation (after a delay of about 30 seconds) at 60 or 120 seconds when there was little inhibition of Inositol 1,4,5 trisphophate accumulation. The inhibitory effect of veratridine on carbachol stimulated Inositol 1,3,4,5-Tetrakisphosphate accumulation was abolished in the presence of ouabain or tetrodotoxin but was unaffected in low calcium conditions. Veratridine reduced the total ATP content and this effect was abolished by tetrodotoxin. The inhibitory effect of 10 but not 30 μM veratridine on Inositol 1,3,4,5-Tetrakisphosphate accumulation in the presence of carbachol was reversed by the presence of exogenous 8-bromo cyclic AMP or forskolin which activates adenylylcyclase. However, the decrease in brain slice ATP seen in the presence of veratridine was unaffected by forskolin. Our results are compatible with the hypothesis that veratridine inhibition of carbachol-stimulated Inositol 1,3,4,5-Tetrakisphosphate formation is due to depletion of ATP at the site of Ins 1,3,4,5-P4 formation from Ins 1,4,5-P3.

  • Inhibition by veratridine of carbachol-stimulated Inositol Tetrakisphosphate accumulation in rat brain cortical slices
    Neurochemical Research, 1995
    Co-Authors: Marvin E. Myles, Yesim Gokmen-polar, John N. Fain
    Abstract:

    The present studies examined the inhibitory effect of veratridine (a Na^+ channel activator) on carbachol (a cholinergic agonist) stimulated Inositol 1,3,4,5-Tetrakisphosphate accumulation in rat brain cortical slices. Veratridine inhibited carbachol stimulation of Inositol 1,3,4,5-Tetrakisphosphate formation (after a delay of about 30 seconds) at 60 or 120 seconds when there was little inhibition of Inositol 1,4,5 trisphophate accumulation. The inhibitory effect of veratridine on carbachol stimulated Inositol 1,3,4,5-Tetrakisphosphate accumulation was abolished in the presence of ouabain or tetrodotoxin but was unaffected in low calcium conditions. Veratridine reduced the total ATP content and this effect was abolished by tetrodotoxin. The inhibitory effect of 10 but not 30 μM veratridine on Inositol 1,3,4,5-Tetrakisphosphate accumulation in the presence of carbachol was reversed by the presence of exogenous 8-bromo cyclic AMP or forskolin which activates adenylylcyclase. However, the decrease in brain slice ATP seen in the presence of veratridine was unaffected by forskolin. Our results are compatible with the hypothesis that veratridine inhibition of carbachol-stimulated Inositol 1,3,4,5-Tetrakisphosphate formation is due to depletion of ATP at the site of Ins 1,3,4,5-P_4 formation from Ins 1,4,5-P_3.

Haruhiro Higashida - One of the best experts on this subject based on the ideXlab platform.

  • Inositol-1,3,4,5-Tetrakisphosphate Binding Sites in Control andras-Transformed NIH/3T3 Fibroblasts
    Biochemical and biophysical research communications, 1997
    Co-Authors: Megumi Taketo, Shigeru Yokoyama, Mitsunori Fukuda, Katsuhiko Mikoshiba, Haruhiro Higashida
    Abstract:

    Abstract Inositol-1,3,4,5-Tetrakisphosphate (Ins(1,3,4,5)P4) binding properties were investigated in NIH/3T3 fibroblasts and itsras-transformant (DT cells), in which Inositol Tetrakisphosphates induce Ca2+influx. [3H]Ins(1,3,4,5)P4bound to membranes of both types of cells with Kdvalues of 10.6 and 8.6 nM, respectively. The rank order of Inositol polyphosphates for displacing [3H]Ins(1,3,4,5)P4in DT cells was Ins(1,3,4,5)P4> Inositol-1,3,4,5,6-pentakisphosphate > Inositol hexakisphosphate > Inositol-1,4,5-trisphosphate. This order is similar to that reported in two Ras-GTPase-activating proteins, GAP1IP4BPand GAP1m, which are also the Ins(1,3,4,5)P4binding proteins. Northern blot analysis revealed that NIH/3T3 and DT cells expressed mRNA species that were hybridizable with GAP1mcDNA. These results suggest that parental andras-transformed NIH/3T3 fibroblasts possess GAP1-like proteins, which may be responsible for triggering Inositol Tetrakisphosphate-dependent Ca2influx.

  • Ca2+ Influx Gated by Inositol-3,4,5,6-Tetrakisphosphate in NIH/3T3 Fibroblasts
    Biochemical and biophysical research communications, 1994
    Co-Authors: Minako Hashii, Masato Hirata, Shoichiro Ozaki, Yoshinori Nozawa, Haruhiro Higashida
    Abstract:

    Abstract The functional role of three Inositol Tetrakisphosphate isomers on Ca 2+ influx was examined by intracellular application in NIH/3T3 mouse fibroblasts. Infusion of Inositol-1,3,4,5-Tetrakisphosphate (Ins(1,3,4,5)P 4 ), Inositol-l,3,4,6-Tetrakisphosphate, and Inositol-3,4,5,6-Tetrakisphosphate (Ins(3,4,5,6)P 4 ) from the patch pipette into the cytoplasm produced a transient increase in cytoplasmic free Ca 2+ concentration by fura-2 recording in extracellular Ca 2+ -dependent fashion. Stimulation of NIH/3T3 cells with bradykinin increased the levels of Ins(1,3,4,5)P 4 and Ins(3,4,5,6)P 4 . These results suggest that mouse fibroblast cells possess a Ca 2+ influx pathway gated occuring Inositol Tetrakisphosphates in response to stimulation with bradykinin.

  • bradykinin induced cytosolic ca2 oscillations and Inositol Tetrakisphosphate induced ca2 influx in voltage clamped ras transformed nih 3t3 fibroblasts
    Journal of Biological Chemistry, 1993
    Co-Authors: Minako Hashii, Yoshinori Nozawa, Haruhiro Higashida
    Abstract:

    Abstract Microspectrofluorometry (fura-2) was combined with the whole-cell patch-clamp technique to study bradykinin-activated calcium (Ca2+) influx in single control or v-Ki-ras-transformed NIH/3T3 (DT) fibroblasts. Application of bradykinin on DT cells, but not on control NIH/3T3 cells, evoked cytosolic Ca2+ oscillations in the presence of extracellular Ca2+, but not in the absence of external Ca2+. This effect of zero external Ca2+ concentration could be mimicked by holding at depolarized membrane potentials. Cytosolic Ca2+ oscillations observed at holding potentials of -20 to -80 mV were terminated by holding at -10 mV or more depolarized potentials. The frequency of Ca2+ oscillations increased with membrane hyperpolarization. Bradykinin significantly enhanced the hyperpolarization-induced increases in the intracellular free Ca2+ concentration ([Ca2+]i) upon membrane hyperpolarization only in DT cells but not in control cells. No [Ca2+]i increase upon hyperpolarization was observed in bradykinin-stimulated DT cells in the absence of external Ca2+, suggesting that bradykinin activates Ca2+ influx. [Ca2+]i increased upon application of Inositol 1,3,4,5-Tetrakisphosphate (Ins(1,3,4,5)P4) into control and DT cells in an extracellular Ca(2+)-dependent manner, indicating that NIH/3T3 fibroblasts have an Ins(1,3,4,5)P4-gated Ca2+ influx pathway. Ins-(1,3,4,5)P4, however, produced the sustained [Ca2+]i increase in DT cells, but not in control NIH/3T3 cells, suggesting that ras may lock the Ca2+ influx pathway at the activated state. Cytosolic Ca2+ oscillations, bradykinin-enhanced Ca2+ influx, and Ins(1,3,4,5)P4-induced Ca2+ influx were all similar in that activity was increased by membrane hyperpolarization. The results suggest that bradykinin-induced cytosolic Ca2+ oscillations in ras-transformed NIH/3T3 cells are maintained by bradykinin-activated continuous Ca2+ influx which may use Ins(1,3,4,5)P4 as an intracellular messenger.

Yesim Gokmen-polar - One of the best experts on this subject based on the ideXlab platform.

  • Inhibition by NMDA of carbachol-stimulated Inositol Tetrakisphosphate accumulation in rat brain cortical slices.
    Neuropharmacology, 1996
    Co-Authors: Marvin E. Myles, Yesim Gokmen-polar, John N. Fain
    Abstract:

    The present studies examined the inhibitory effect of veratridine (a Na+ channel activator) on carbachol (a cholinergic agonist) stimulated Inositol 1,3,4,5-Tetrakisphosphate accumulation in rat brain cortical slices. Veratridine inhibited carbachol stimulation of Inositol 1,3,4,5-Tetrakisphosphate formation (after a delay of about 30 seconds) at 60 or 120 seconds when there was little inhibition of Inositol 1,4,5 trisphophate accumulation. The inhibitory effect of veratridine on carbachol stimulated Inositol 1,3,4,5-Tetrakisphosphate accumulation was abolished in the presence of ouabain or tetrodotoxin but was unaffected in low calcium conditions. Veratridine reduced the total ATP content and this effect was abolished by tetrodotoxin. The inhibitory effect of 10 but not 30 μM veratridine on Inositol 1,3,4,5-Tetrakisphosphate accumulation in the presence of carbachol was reversed by the presence of exogenous 8-bromo cyclic AMP or forskolin which activates adenylylcyclase. However, the decrease in brain slice ATP seen in the presence of veratridine was unaffected by forskolin. Our results are compatible with the hypothesis that veratridine inhibition of carbachol-stimulated Inositol 1,3,4,5-Tetrakisphosphate formation is due to depletion of ATP at the site of Ins 1,3,4,5-P4 formation from Ins 1,4,5-P3.

  • Inhibition by veratridine of carbachol-stimulated Inositol Tetrakisphosphate accumulation in rat brain cortical slices
    Neurochemical Research, 1995
    Co-Authors: Marvin E. Myles, Yesim Gokmen-polar, John N. Fain
    Abstract:

    The present studies examined the inhibitory effect of veratridine (a Na^+ channel activator) on carbachol (a cholinergic agonist) stimulated Inositol 1,3,4,5-Tetrakisphosphate accumulation in rat brain cortical slices. Veratridine inhibited carbachol stimulation of Inositol 1,3,4,5-Tetrakisphosphate formation (after a delay of about 30 seconds) at 60 or 120 seconds when there was little inhibition of Inositol 1,4,5 trisphophate accumulation. The inhibitory effect of veratridine on carbachol stimulated Inositol 1,3,4,5-Tetrakisphosphate accumulation was abolished in the presence of ouabain or tetrodotoxin but was unaffected in low calcium conditions. Veratridine reduced the total ATP content and this effect was abolished by tetrodotoxin. The inhibitory effect of 10 but not 30 μM veratridine on Inositol 1,3,4,5-Tetrakisphosphate accumulation in the presence of carbachol was reversed by the presence of exogenous 8-bromo cyclic AMP or forskolin which activates adenylylcyclase. However, the decrease in brain slice ATP seen in the presence of veratridine was unaffected by forskolin. Our results are compatible with the hypothesis that veratridine inhibition of carbachol-stimulated Inositol 1,3,4,5-Tetrakisphosphate formation is due to depletion of ATP at the site of Ins 1,3,4,5-P_4 formation from Ins 1,4,5-P_3.

Maria Delivoria-papadopoulos - One of the best experts on this subject based on the ideXlab platform.

  • Inositol Tetrakisphosphate (IP4)- and Inositol Triphosphate (IP3)-Dependent Ca^2+ Influx in Cortical Neuronal Nuclei of Newborn Piglets Following Graded Hypoxia
    Neurochemical Research, 2004
    Co-Authors: Om P. Mishra, Maria Delivoria-papadopoulos
    Abstract:

    Previous studies have shown that hypoxia results in a modification of the binding characteristics of the neuronal nuclear membrane Inositol Tetrakisphosphate (IP_4) and Inositol triphosphate (IP_3) receptors. The present study tests the hypothesis that hypoxia-induced modification of the IP_4 and IP_3 receptors results in increased IP_4 and IP_3 dependent Ca^2+ influx in neuronal nuclei as a function of the degree of cerebral tissue hypoxia in newborn piglets. Studies were performed in piglets, 3–5 days old, divided into normoxic (N = 5) and hypoxic (N = 6) groups. The hypoxic group was exposed to decreased Fio_2 ranging from 0.15 to 0.05 for 1 h. Brain tissue hypoxia was documented biochemically by determining ATP and phosphocreatine (PCr) levels. Neuronal nuclei were isolated and ^45Ca^2+ influx was determined in a medium containing 50 mM Tris buffer (pH 7.4), neuronal nuclei (150 μg protein), 1 μM ^45Ca^2+, with or without 10 μM IP_4 or IP_3. In normoxic and hypoxic groups, ATP levels were 4.27 ± 0.80 and 1.40 ± 0.69 μmoles/g brain, respectively, P < .001 (ranging from 4.78 to 0.82). PCr levels were 3.40 ± 0.99 and 0.91 ± 0.57 μmoles/g brain, respectively, P < .001 (raning from 4.07 to 0.60). During hypoxia, IP_4-dependent intranuclear ^45Ca^2+ influx increased from 3.39 ± 0.64 in normoxic nuclei to 13.30 ± 2.18 pM/mg protein in hypoxic nuclei (P < .01). There was an inverse correlation between the ^45Ca^2+ influx in neuronal nuclei and the levels of cerebral tissue ATP (r = 0.83) and PCr (r = 0.85). Similarly, IP_3-dependent intranuclear ^45Ca^2+ influx increased from 2.26 ± 0.38 pmoles/mg protein in normoxic nuclei to 11.12 ± 1.65 pmoles/mg protein in hypoxic nuclei and showed an inverse correlation between ^45Ca^2+ influx in neuronal nuclei and the levels of cerebral tissue ATP (r = 0.86) and PCr (r = 0.71). The data demonstrate that there is an IP_4- as well as IP_3-dependent increase in nuclear Ca^2+ influx with increasing cerebral tissue hypoxia, suggesting a hypoxia-induced modification of the nuclear membrane IP_4 and IP_3 receptors. We propose that there is a specific level of tissue hypoxia that results in a critical increase of intranuclear Ca^2+ that leads to altered transcription of apoptotic genes and activation of nuclear endonucleases resulting in hypoxia-induced programmed neuronal death.

  • Inositol Tetrakisphosphate (IP4)- and Inositol triphosphate (IP3)-dependent Ca2+ influx in cortical neuronal nuclei of newborn piglets following graded hypoxia.
    Neurochemical research, 2004
    Co-Authors: Om P. Mishra, Maria Delivoria-papadopoulos
    Abstract:

    Previous studies have shown that hypoxia results in a modification of the binding characteristics of the neuronal nuclear membrane Inositol Tetrakisphosphate (IP4) and Inositol triphosphate (IP3) receptors. The present study tests the hypothesis that hypoxia-induced modification of the IP4 and IP3 receptors results in increased IP4 and IP3 dependent Ca2+ influx in neuronal nuclei as a function of the degree of cerebral tissue hypoxia in newborn piglets. Studies were performed in piglets, 3–5 days old, divided into normoxic (N = 5) and hypoxic (N = 6) groups. The hypoxic group was exposed to decreased Fio2 ranging from 0.15 to 0.05 for 1 h. Brain tissue hypoxia was documented biochemically by determining ATP and phosphocreatine (PCr) levels. Neuronal nuclei were isolated and 45Ca2+ influx was determined in a medium containing 50 mM Tris buffer (pH 7.4), neuronal nuclei (150 μg protein), 1 μM 45Ca2+, with or without 10 μM IP4 or IP3. In normoxic and hypoxic groups, ATP levels were 4.27 ± 0.80 and 1.40 ± 0.69 μmoles/g brain, respectively, P < .001 (ranging from 4.78 to 0.82). PCr levels were 3.40 ± 0.99 and 0.91 ± 0.57 μmoles/g brain, respectively, P < .001 (raning from 4.07 to 0.60). During hypoxia, IP4-dependent intranuclear 45Ca2+ influx increased from 3.39 ± 0.64 in normoxic nuclei to 13.30 ± 2.18 pM/mg protein in hypoxic nuclei (P < .01). There was an inverse correlation between the 45Ca2+ influx in neuronal nuclei and the levels of cerebral tissue ATP (r = 0.83) and PCr (r = 0.85). Similarly, IP3-dependent intranuclear 45Ca2+ influx increased from 2.26 ± 0.38 pmoles/mg protein in normoxic nuclei to 11.12 ± 1.65 pmoles/mg protein in hypoxic nuclei and showed an inverse correlation between 45Ca2+ influx in neuronal nuclei and the levels of cerebral tissue ATP (r = 0.86) and PCr (r = 0.71). The data demonstrate that there is an IP4- as well as IP3-dependent increase in nuclear Ca2+ influx with increasing cerebral tissue hypoxia, suggesting a hypoxia-induced modification of the nuclear membrane IP4 and IP3 receptors. We propose that there is a specific level of tissue hypoxia that results in a critical increase of intranuclear Ca2+ that leads to altered transcription of apoptotic genes and activation of nuclear endonucleases resulting in hypoxia-induced programmed neuronal death.