Timothy Angelotti | Stanford University (original) (raw)

Papers by Timothy Angelotti

Frontiers in Molecular Biosciences, Aug 19, 2022

Oxford University Press eBooks, Dec 1, 2011

A&A practice, Jul 1, 2018

Bioconjugate Chemistry, Nov 1, 1991

FEBS Letters, Nov 18, 1996

ASA Newsletter, Jul 1, 2008

Anesthesia & Analgesia, Apr 1, 2003

Neuroscience, Jun 1, 2008

A&A practice, Oct 14, 2019

Digestive Diseases and Sciences, Jun 5, 2020

Gastrointestinal Endoscopy, Jun 1, 2020

Critical Care Medicine, Mar 1, 2017

The GABA$\rm{\sb{A}}$ receptor (GABAR) is a multi-subunit, ligand-gated ion channel which is modu... more The GABA$\rm{\sb{A}}$ receptor (GABAR) is a multi-subunit, ligand-gated ion channel which is modulated by numerous drugs and possibly, post-translation modification. Recent molecular cloning has revealed that multiple subunit families exist ($\alpha$, beta\betabeta, gamma\gammagamma, delta\deltadelta, and rho\rhorho), with multiple isoforms in each family. This thesis describes molecular and electrophysiological analyses of recombinant GABARs to determine what subunit combinations most closely resembled native GABARs, how phosphorylation modified GABAR function and expression, and what was the role of isoform diversity in functional GABAR heterogeneity. Expression of alpha1gamma2\alpha1\gamma2alpha1gamma2S, beta1gamma2\beta1\gamma2beta1gamma2S, alpha1beta1\alpha1\beta1alpha1beta1, and alpha1beta1gamma2\alpha1\beta1\gamma2alpha1beta1gamma2S GABAR subunit combinations in L929 fibroblasts revealed that the former two combinations did not assemble into functional ion channels, whereas the later two did assembly with high efficiency. These two GABAR populations could be distinguished by their diazepam sensitivities, GABA concentration-response curves, single-channel conductance levels, and gating kinetics. More importantly, alpha1beta1gamma2\alpha1\beta1\gamma2alpha1beta1gamma2S GABARs were formed preferentially over alpha1beta1\alpha1\beta1alpha1beta1 GABARs. Also, alpha1beta1gamma2\alpha1\beta1\gamma2alpha1beta1gamma2S GABARs closely resembled native GABARs with respect to main (29 pS) and subconductance (21 pS) levels and gating properties. Modulation of GABAR function by Protein Kinase A (PKA) phosphorylation was next examined by comparing GABARs containing either wild-type beta1\beta1beta1 subunits or mutant subunits ($\beta1$(S409A)), lacking the PKA phosphorylated serine residue. Expression of alpha1beta1gamma2\alpha1\beta1\gamma2alpha1beta1gamma2S, and not alpha1beta1\alpha1\beta1alpha1beta1(S409A)$\gamma$2S, GABARs in C$\alpha12$ cells (high kinase activity) caused an enhancement of GABA-evoked whole-cell current amplitude relative to either GABAR expressed in cells with low (RAB10) or intermediate (L929) kinase activities. This effect was not seen for either alpha1beta1\alpha1\beta1alpha1beta1 or alpha1beta1\alpha1\beta1alpha1beta1(S409A) GABARs. There were no differences in GABA pharmacology, single-channel conductance or gating properties and acute intracellular application of PKA did not duplicate the effect, suggesting that chronic elevation of cellular PKA enhanced GABAR current by possibly modifying receptor assembly or stability. A polymorphic form of the rat alpha6\alpha6alpha6 subunit cDNA was cloned (His$\sp{121}\rightarrow$ Gln); the amino acid change did not effect alpha6beta1gamma2\alpha6\beta1\gamma2alpha6beta1gamma2S GABAR pharmacology. However, a comparison of alpha1beta1gamma2\alpha1\beta1\gamma2alpha1beta1gamma2S and alpha6beta1gamma2\alpha6\beta1\gamma2alpha6beta1gamma2S GABARs revealed that the latter were more sensitive to GABA activation, insensitive to diazepam, had a larger single-channel conductance (33 pS), brief duration openings, and produced less whole-cell current on a per cell basis.Ph.D.PharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103902/1/9423134.pdfDescription of 9423134.pdf : Restricted to UM users only

PubMed, 1996

While GABAergic inhibition plays a major role in the regulation of neuronal excitability, a role ... more While GABAergic inhibition plays a major role in the regulation of neuronal excitability, a role for altered GABAergic inhibition in the pathogenesis of epilepsy remains to be proven. The demonstration that GABAA receptors are composed of multiple subunits and that the properties and pharmacology of GABAA receptors are different for different subunit combinations, suggests that GABAA receptor heterogeneity may be of importance in determining the properties of GABAergic inhibition in different regions of the nervous system. While it is clear that GABAA receptor heterogeneity is present in the nervous system, a role for receptor heterogeneity in the pathogenesis of epilepsy remains uncertain. GABAA receptor heterogeneity may have implications for the treatment of epilepsy. It is quite possible that drugs which regulate GABAergic function may have variable efficacy in different regions of the nervous system due to expression of receptors with subunits that have different sensitivity to allosteric regulators. In situ hybridization studies indicate the colocalization of alpha 1 beta 1 gamma 2L and delta subunit mRNAs in hippocampal dentate gyrus granule cells while only the alpha 1, beta 1 and gamma 2L and not the delta subunit mRNAs colocalize in the pyramidal cells of the hippocampus. The reduced rate of acute desensitization and the slow recovery of GABA-evoked currents typical of delta-containing subunit combinations could generate tonic inhibition via long-lasting IPSPs in the dentate gyrus and thus play a role in preventing seizures. By the same rationale, a reduction in the level of expression of the delta subunit mRNA in the dentate gyrus or its absence as in the hippocampal pyramidal cells could be associated with a reduced seizure threshold. Furthermore, it is likely that there are developmental changes in the stoichiometry or subunit composition of GABAA receptors rendering the developing nervous system more or less sensitive to the effects of GABAergic anticonvulsant drugs. In addition to the heterogeneous expression of GABAA receptors, other issues concerning the regulation of GABAergic function are of potential importance. The regulatory events that control the expression of specific receptor subtypes and levels of GABA receptors are unknown. To understand the role of GABAA receptor heterogeneity in the pathogenesis of epilepsy will require the combination of biophysical and molecular biological techniques. It will be important to determine not only whether the properties of GABAA receptors have been altered in a specific form of epilepsy but also whether gene expression has been altered.

PubMed, Nov 1, 1992

Single-channel recordings from excised outside-out patches were obtained from Chinese hamster ova... more Single-channel recordings from excised outside-out patches were obtained from Chinese hamster ovary cells stably transfected with plasmids containing bovine gamma-aminobutyric acid (GABA) type A (GABAA) receptor channel alpha 1 and beta 1 subunit cDNAs. The predominant or main conductance level recorded had a 17-pS chord conductance. There were minor contributions made from 25-pS and 11-pS conductance levels. Average open duration, burst duration, and openings/burst did not change as the GABA concentration was increased from 5 to 25 microM. However, opening frequency increased from 11.0 to 19.5 openings/sec. Pentobarbital increased average channel open duration without increasing opening frequency, whereas picrotoxin slightly reduced average channel open duration and reduced opening frequency. Open duration frequency distributions were fitted best with the sum of two exponential functions, suggesting that the alpha 1 beta 1 GABAA receptor channel had at least two open states. The time constants and relative proportions of the two components did not vary when GABA concentration was increased from 5 to 25 microM. Closed duration distributions of closures between main conductance level openings were fitted best with multiple exponential functions, suggesting that the alpha 1 beta 1 GABAA receptor channel had several closed states. Burst duration frequency distributions were fitted best with two exponential functions whose time constants and relative proportions did not change with GABA concentration. A gating kinetic scheme for the alpha 1 beta 1 GABAA receptor channel was proposed that consisted of a single binding site for GABA and at least two open and five closed states. The kinetic properties of the alpha 1 beta 1 main conductance level differed from those of the spinal cord neuron (native) 27-pS main conductance level and the 19-pS subconductance level. The native main conductance and subconductance levels were characterized by longer openings and at least three open states. Based on the aforementioned observations, it appears that different subunit combinations produce receptor channels with different kinetic properties, but the basic mechanism of regulation by pentobarbital and picrotoxin may be similar for the different receptor channels. Also, it is unlikely that the 19-pS substate of the native GABAA receptor is produced by an alpha 1 beta 1 dimer.

PubMed, Dec 1, 1993

alpha 1, beta 1, and gamma 2S gamma-aminobutyric acid (GABA) type A receptor (GABAR) subunit cDNA... more alpha 1, beta 1, and gamma 2S gamma-aminobutyric acid (GABA) type A receptor (GABAR) subunit cDNAs were transiently expressed in derivative cell lines of mouse L929 fibroblasts, which possessed different levels of the catalytic subunit of cAMP-dependent protein kinase (PKA). These cell lines included L929 (intermediate levels of kinase), C alpha 12 (elevated levels of kinase), and RAB10 (low levels of kinase) cells. Pharmacological analysis of GABA-evoked whole-cell currents revealed that, compared with expression in L929 and RAB10 cells, expression of alpha 1 beta 1 gamma 2S GABARs in C alpha 12 cells produced a selective enhancement of single whole-cell current amplitudes. No other pharmacological properties (Hill slope, EC50, or diazepam sensitivity) of the expressed alpha 1 beta 1 gamma 2S GABARs were modified. The GABAR current enhancement in C alpha 12 cells was blocked by substitution of a beta 1 subunit mutated at the PKA consensus phosphorylation site, Ser409 [beta 1(S409A)], for the wild-type beta subunit. Interestingly, enhancement was specific for GABARs containing all three subunits, because it was not seen after expression of alpha 1 beta 1 or alpha 1 beta 1 (S409A) GABAR subunit combinations. Single-channel conductance and gating properties were not different for alpha 1 beta 1 gamma 2S or alpha 1 beta 1 (S409A) gamma 2S GABARs expressed in each cell line, suggesting that PKA did not enhance whole-cell currents by altering these properties of GABARs. These results suggested that unlike acute application of PKA, which has been shown to produce a decrease in GABAR current, chronic elevation of PKA activity can result in enhancement of GABAR currents. More importantly, this effect occurred only with GABARs composed of alpha 1 beta 1 gamma 2S subunits and not alpha 1 beta 1 subunits and was mediated by a single amino acid residue (Ser409) of the beta 1 subunit.

The Journal of Neuroscience, Apr 1, 1993

Journal of Biological Chemistry, Nov 1, 1995

The type I cGMP-dependent protein kinases (cGK I␣ and I␤) form homodimers (subunit M r ϳ 76,000),... more The type I cGMP-dependent protein kinases (cGK I␣ and I␤) form homodimers (subunit M r ϳ 76,000), presumably through conserved, amino-terminal leucine zipper motifs. Type II cGMP-dependent protein kinase (cGK II) has been reported to be monomeric (M r ϳ 86,000), but recent cloning and sequencing of mouse brain cGK II cDNA revealed a leucine zipper motif near its amino terminus. In the present study, recombinant mouse brain cGK II was expressed, purified, and characterized. Sucrose gradient centrifugation and gel filtration chromatography were used to determine M r values for holoenzymes of cGK I␣ (168,000) and cGK II (152,500), which suggest that both are dimers. Native cGK I␣ possessed significantly lower K a values for cGMP (8-fold) and ␤-phenyl-1,N 2-etheno-cGMP (300-fold) than did recombinant cGK II. Conversely, the Sp-and Rp-isomers of 8-(4-chloro-phenylthio)-guanosine-3,5-cyclic monophosphorothioate demonstrated selectivity toward cGK II in assays of kinase activation or inhibition, respectively. A peptide substrate derived from histone f 2B had a 20-fold greater V max /K m ratio for cGK I␣ than for cGK II, whereas a peptide based upon a cAMP response element binding protein phosphorylation site exhibited a greater V max /K m ratio for cGK II. Finally, gel filtration of extracts of mouse intestine partially resolved two cGK activities, one of which had properties similar to those demonstrated by recombinant cGK II. The combined results show that both cGK I and cGK II form homodimers but possess distinct cyclic nucleotide and substrate specificities. Appreciation of cGMP as a distinct intracellular second messenger was closely followed by an intensive search for effector proteins in various organisms. Subsequently, a cGMP-dependent protein kinase (cGK) 1 was discovered in arthropods (1), * This work was supported by National Institutes of Health Grants GM 38788 and MH 42652 (to M. D. U.) and DK 40029 (to J. D. C.

Anesthesia & Analgesia, 2007

Sodium azide (NaN 3 ) is a highly reactive, crystalline solid found in almost every new car as th... more Sodium azide (NaN 3 ) is a highly reactive, crystalline solid found in almost every new car as the gas-generating chemical for airbag deployment (1). Similar to hydrogen cyanide, absorption of NaN 3 leads to inhibition of cytochrome c oxidase and eventual cell death. In addition ...

The FASEB Journal, Apr 1, 2012

Frontiers in Molecular Biosciences, Aug 19, 2022

Oxford University Press eBooks, Dec 1, 2011

A&A practice, Jul 1, 2018

Bioconjugate Chemistry, Nov 1, 1991

FEBS Letters, Nov 18, 1996

ASA Newsletter, Jul 1, 2008

Anesthesia & Analgesia, Apr 1, 2003

Neuroscience, Jun 1, 2008

A&A practice, Oct 14, 2019

Digestive Diseases and Sciences, Jun 5, 2020

Gastrointestinal Endoscopy, Jun 1, 2020

Critical Care Medicine, Mar 1, 2017

The GABA$\rm{\sb{A}}$ receptor (GABAR) is a multi-subunit, ligand-gated ion channel which is modu... more The GABA$\rm{\sb{A}}$ receptor (GABAR) is a multi-subunit, ligand-gated ion channel which is modulated by numerous drugs and possibly, post-translation modification. Recent molecular cloning has revealed that multiple subunit families exist ($\alpha$, beta\betabeta, gamma\gammagamma, delta\deltadelta, and rho\rhorho), with multiple isoforms in each family. This thesis describes molecular and electrophysiological analyses of recombinant GABARs to determine what subunit combinations most closely resembled native GABARs, how phosphorylation modified GABAR function and expression, and what was the role of isoform diversity in functional GABAR heterogeneity. Expression of alpha1gamma2\alpha1\gamma2alpha1gamma2S, beta1gamma2\beta1\gamma2beta1gamma2S, alpha1beta1\alpha1\beta1alpha1beta1, and alpha1beta1gamma2\alpha1\beta1\gamma2alpha1beta1gamma2S GABAR subunit combinations in L929 fibroblasts revealed that the former two combinations did not assemble into functional ion channels, whereas the later two did assembly with high efficiency. These two GABAR populations could be distinguished by their diazepam sensitivities, GABA concentration-response curves, single-channel conductance levels, and gating kinetics. More importantly, alpha1beta1gamma2\alpha1\beta1\gamma2alpha1beta1gamma2S GABARs were formed preferentially over alpha1beta1\alpha1\beta1alpha1beta1 GABARs. Also, alpha1beta1gamma2\alpha1\beta1\gamma2alpha1beta1gamma2S GABARs closely resembled native GABARs with respect to main (29 pS) and subconductance (21 pS) levels and gating properties. Modulation of GABAR function by Protein Kinase A (PKA) phosphorylation was next examined by comparing GABARs containing either wild-type beta1\beta1beta1 subunits or mutant subunits ($\beta1$(S409A)), lacking the PKA phosphorylated serine residue. Expression of alpha1beta1gamma2\alpha1\beta1\gamma2alpha1beta1gamma2S, and not alpha1beta1\alpha1\beta1alpha1beta1(S409A)$\gamma$2S, GABARs in C$\alpha12$ cells (high kinase activity) caused an enhancement of GABA-evoked whole-cell current amplitude relative to either GABAR expressed in cells with low (RAB10) or intermediate (L929) kinase activities. This effect was not seen for either alpha1beta1\alpha1\beta1alpha1beta1 or alpha1beta1\alpha1\beta1alpha1beta1(S409A) GABARs. There were no differences in GABA pharmacology, single-channel conductance or gating properties and acute intracellular application of PKA did not duplicate the effect, suggesting that chronic elevation of cellular PKA enhanced GABAR current by possibly modifying receptor assembly or stability. A polymorphic form of the rat alpha6\alpha6alpha6 subunit cDNA was cloned (His$\sp{121}\rightarrow$ Gln); the amino acid change did not effect alpha6beta1gamma2\alpha6\beta1\gamma2alpha6beta1gamma2S GABAR pharmacology. However, a comparison of alpha1beta1gamma2\alpha1\beta1\gamma2alpha1beta1gamma2S and alpha6beta1gamma2\alpha6\beta1\gamma2alpha6beta1gamma2S GABARs revealed that the latter were more sensitive to GABA activation, insensitive to diazepam, had a larger single-channel conductance (33 pS), brief duration openings, and produced less whole-cell current on a per cell basis.Ph.D.PharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103902/1/9423134.pdfDescription of 9423134.pdf : Restricted to UM users only

PubMed, 1996

While GABAergic inhibition plays a major role in the regulation of neuronal excitability, a role ... more While GABAergic inhibition plays a major role in the regulation of neuronal excitability, a role for altered GABAergic inhibition in the pathogenesis of epilepsy remains to be proven. The demonstration that GABAA receptors are composed of multiple subunits and that the properties and pharmacology of GABAA receptors are different for different subunit combinations, suggests that GABAA receptor heterogeneity may be of importance in determining the properties of GABAergic inhibition in different regions of the nervous system. While it is clear that GABAA receptor heterogeneity is present in the nervous system, a role for receptor heterogeneity in the pathogenesis of epilepsy remains uncertain. GABAA receptor heterogeneity may have implications for the treatment of epilepsy. It is quite possible that drugs which regulate GABAergic function may have variable efficacy in different regions of the nervous system due to expression of receptors with subunits that have different sensitivity to allosteric regulators. In situ hybridization studies indicate the colocalization of alpha 1 beta 1 gamma 2L and delta subunit mRNAs in hippocampal dentate gyrus granule cells while only the alpha 1, beta 1 and gamma 2L and not the delta subunit mRNAs colocalize in the pyramidal cells of the hippocampus. The reduced rate of acute desensitization and the slow recovery of GABA-evoked currents typical of delta-containing subunit combinations could generate tonic inhibition via long-lasting IPSPs in the dentate gyrus and thus play a role in preventing seizures. By the same rationale, a reduction in the level of expression of the delta subunit mRNA in the dentate gyrus or its absence as in the hippocampal pyramidal cells could be associated with a reduced seizure threshold. Furthermore, it is likely that there are developmental changes in the stoichiometry or subunit composition of GABAA receptors rendering the developing nervous system more or less sensitive to the effects of GABAergic anticonvulsant drugs. In addition to the heterogeneous expression of GABAA receptors, other issues concerning the regulation of GABAergic function are of potential importance. The regulatory events that control the expression of specific receptor subtypes and levels of GABA receptors are unknown. To understand the role of GABAA receptor heterogeneity in the pathogenesis of epilepsy will require the combination of biophysical and molecular biological techniques. It will be important to determine not only whether the properties of GABAA receptors have been altered in a specific form of epilepsy but also whether gene expression has been altered.

PubMed, Nov 1, 1992

Single-channel recordings from excised outside-out patches were obtained from Chinese hamster ova... more Single-channel recordings from excised outside-out patches were obtained from Chinese hamster ovary cells stably transfected with plasmids containing bovine gamma-aminobutyric acid (GABA) type A (GABAA) receptor channel alpha 1 and beta 1 subunit cDNAs. The predominant or main conductance level recorded had a 17-pS chord conductance. There were minor contributions made from 25-pS and 11-pS conductance levels. Average open duration, burst duration, and openings/burst did not change as the GABA concentration was increased from 5 to 25 microM. However, opening frequency increased from 11.0 to 19.5 openings/sec. Pentobarbital increased average channel open duration without increasing opening frequency, whereas picrotoxin slightly reduced average channel open duration and reduced opening frequency. Open duration frequency distributions were fitted best with the sum of two exponential functions, suggesting that the alpha 1 beta 1 GABAA receptor channel had at least two open states. The time constants and relative proportions of the two components did not vary when GABA concentration was increased from 5 to 25 microM. Closed duration distributions of closures between main conductance level openings were fitted best with multiple exponential functions, suggesting that the alpha 1 beta 1 GABAA receptor channel had several closed states. Burst duration frequency distributions were fitted best with two exponential functions whose time constants and relative proportions did not change with GABA concentration. A gating kinetic scheme for the alpha 1 beta 1 GABAA receptor channel was proposed that consisted of a single binding site for GABA and at least two open and five closed states. The kinetic properties of the alpha 1 beta 1 main conductance level differed from those of the spinal cord neuron (native) 27-pS main conductance level and the 19-pS subconductance level. The native main conductance and subconductance levels were characterized by longer openings and at least three open states. Based on the aforementioned observations, it appears that different subunit combinations produce receptor channels with different kinetic properties, but the basic mechanism of regulation by pentobarbital and picrotoxin may be similar for the different receptor channels. Also, it is unlikely that the 19-pS substate of the native GABAA receptor is produced by an alpha 1 beta 1 dimer.

PubMed, Dec 1, 1993

alpha 1, beta 1, and gamma 2S gamma-aminobutyric acid (GABA) type A receptor (GABAR) subunit cDNA... more alpha 1, beta 1, and gamma 2S gamma-aminobutyric acid (GABA) type A receptor (GABAR) subunit cDNAs were transiently expressed in derivative cell lines of mouse L929 fibroblasts, which possessed different levels of the catalytic subunit of cAMP-dependent protein kinase (PKA). These cell lines included L929 (intermediate levels of kinase), C alpha 12 (elevated levels of kinase), and RAB10 (low levels of kinase) cells. Pharmacological analysis of GABA-evoked whole-cell currents revealed that, compared with expression in L929 and RAB10 cells, expression of alpha 1 beta 1 gamma 2S GABARs in C alpha 12 cells produced a selective enhancement of single whole-cell current amplitudes. No other pharmacological properties (Hill slope, EC50, or diazepam sensitivity) of the expressed alpha 1 beta 1 gamma 2S GABARs were modified. The GABAR current enhancement in C alpha 12 cells was blocked by substitution of a beta 1 subunit mutated at the PKA consensus phosphorylation site, Ser409 [beta 1(S409A)], for the wild-type beta subunit. Interestingly, enhancement was specific for GABARs containing all three subunits, because it was not seen after expression of alpha 1 beta 1 or alpha 1 beta 1 (S409A) GABAR subunit combinations. Single-channel conductance and gating properties were not different for alpha 1 beta 1 gamma 2S or alpha 1 beta 1 (S409A) gamma 2S GABARs expressed in each cell line, suggesting that PKA did not enhance whole-cell currents by altering these properties of GABARs. These results suggested that unlike acute application of PKA, which has been shown to produce a decrease in GABAR current, chronic elevation of PKA activity can result in enhancement of GABAR currents. More importantly, this effect occurred only with GABARs composed of alpha 1 beta 1 gamma 2S subunits and not alpha 1 beta 1 subunits and was mediated by a single amino acid residue (Ser409) of the beta 1 subunit.

The Journal of Neuroscience, Apr 1, 1993

Journal of Biological Chemistry, Nov 1, 1995

The type I cGMP-dependent protein kinases (cGK I␣ and I␤) form homodimers (subunit M r ϳ 76,000),... more The type I cGMP-dependent protein kinases (cGK I␣ and I␤) form homodimers (subunit M r ϳ 76,000), presumably through conserved, amino-terminal leucine zipper motifs. Type II cGMP-dependent protein kinase (cGK II) has been reported to be monomeric (M r ϳ 86,000), but recent cloning and sequencing of mouse brain cGK II cDNA revealed a leucine zipper motif near its amino terminus. In the present study, recombinant mouse brain cGK II was expressed, purified, and characterized. Sucrose gradient centrifugation and gel filtration chromatography were used to determine M r values for holoenzymes of cGK I␣ (168,000) and cGK II (152,500), which suggest that both are dimers. Native cGK I␣ possessed significantly lower K a values for cGMP (8-fold) and ␤-phenyl-1,N 2-etheno-cGMP (300-fold) than did recombinant cGK II. Conversely, the Sp-and Rp-isomers of 8-(4-chloro-phenylthio)-guanosine-3,5-cyclic monophosphorothioate demonstrated selectivity toward cGK II in assays of kinase activation or inhibition, respectively. A peptide substrate derived from histone f 2B had a 20-fold greater V max /K m ratio for cGK I␣ than for cGK II, whereas a peptide based upon a cAMP response element binding protein phosphorylation site exhibited a greater V max /K m ratio for cGK II. Finally, gel filtration of extracts of mouse intestine partially resolved two cGK activities, one of which had properties similar to those demonstrated by recombinant cGK II. The combined results show that both cGK I and cGK II form homodimers but possess distinct cyclic nucleotide and substrate specificities. Appreciation of cGMP as a distinct intracellular second messenger was closely followed by an intensive search for effector proteins in various organisms. Subsequently, a cGMP-dependent protein kinase (cGK) 1 was discovered in arthropods (1), * This work was supported by National Institutes of Health Grants GM 38788 and MH 42652 (to M. D. U.) and DK 40029 (to J. D. C.

Anesthesia & Analgesia, 2007

Sodium azide (NaN 3 ) is a highly reactive, crystalline solid found in almost every new car as th... more Sodium azide (NaN 3 ) is a highly reactive, crystalline solid found in almost every new car as the gas-generating chemical for airbag deployment (1). Similar to hydrogen cyanide, absorption of NaN 3 leads to inhibition of cytochrome c oxidase and eventual cell death. In addition ...

The FASEB Journal, Apr 1, 2012