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James A. Angus - One of the best experts on this subject based on the ideXlab platform.
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selectivity of Omega Conotoxin GVIA for n type calcium channels in rat isolated small mesenteric arteries
Clinical and Experimental Pharmacology and Physiology, 2010Co-Authors: Sarah L Whorlow, James A. Angus, Christine E. WrightAbstract:: 1. The selectivity of Omega-Conotoxin GVIA (Omega-CTX) for prejunctional N-type voltage-operated calcium channels (VOCC) was examined in rat isolated small mesenteric arteries mounted in a Mulvany-Halpern myograph. Contractile responses to perivascular nerve stimulation, noradrenaline (NA) and potassium (K+) were obtained before and after treatment with Omega-CTX. The effects of Omega-CTX were compared with those of felodipine, an L-type VOCC blocker. 2. Omega-CTX (3 nmol/L-10 mu mol/L) inhibited contractions to electrical field stimulation by up to 94%, compared with the corresponding time control group. Felodipine (0.1 mu mol/L) had little effect on the contractions to electrical stimulation compared with the vehicle-treated vessels. 3. Concentration-response curves to exogenous NA (0.1 mu mol/L) and contractions to a submaximal concentration of K+ (50 mmol/L) were unaffected by Omega-CTX (3 nmol/L-10 mu mol/L). In contrast, the maximum contraction to NA in vessels exposed to felodipine (0.1 mu mol/L) was reduced by 37%, and the contraction to K+ (62 mmol/L) was reduced by 84% compared with vehicle-treated arteries. 4. The results indicate that even at concentrations up to 10 mu mol/L (10 000-fold higher than required to inhibit prejunctional N-type VOCC), Omega-CTX inhibits only neurotransmitter release. Its effects are clearly different to felodipine as Omega-CTX has no effect on post-junctional alpha1-adrenoceptor-mediated vasoconstriction or direct smooth muscle depolarization considered to be mediated by L-type VOCC. Therefore, at least at the vascular neuroeffector junction, Omega-CTX appears to be highly selective for N-type VOCC with no effect on L-type VOCC.
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prolonged cardiovascular effects of the n type ca2 channel antagonist Omega Conotoxin GVIA in conscious rabbits
Journal of Cardiovascular Pharmacology, 1997Co-Authors: Christine E. Wright, James A. AngusAbstract:: Omega-Conotoxin GVIA (Omega-CTX) is an N-type Ca2+ channel antagonist that is considered to be only partially reversible in vitro. In vivo, its effects after 24 h are unknown. To assess the duration of action of this peptide in vivo, the effects of a single intravenous injection of Omega-CTX on mean arterial pressure (MAP), heart rate (HR), postural adaptation, and the baroreflex were investigated in conscious rabbits. MAP, HR, the baroreflex induced by i.v. glyceryl trinitrate (0.4-20 micrograms/kg) and phenylephrine (0.1-15 micrograms/kg) and orthostatic responses to 1 min 90 degrees head-up tilt were assessed before (0 h) and 2-168 h after administration of Omega-CTX (10 micrograms/kg i.v. bolus: n = 6-9) or vehicle (0.9% saline; n = 6). Acute phase I: By 2 h after Omega-CTX administration, MAP had decreased from 75 +/- 3 mm Hg to 60 +/- 2 mm Hg; HR increased from 220 +/- 7 beats/min to 249 +/- 5 beats/min (n = 9). There was marked attenuation of the baroreflex curve (HR range decreasing by 61%). By 24 h. MAP and HR had returned to control values, but the HR range was still 18% less than that of control. Phase II: MAP and HR then decreased steadily over the next 96 h to significantly lower values by 120 h after Omega-CTX administration (delta-8 +/- 2 mm Hg and -29 +/- 2 beats/min, respectively; n = 6). Thereafter, MAP and HR values increased and by 168 h these parameters, and the baroreflex, were similar to control values. In response to 90 degrees tilt, there was no change in MAP at 0 h; however, 1 h after Omega-CTX, significant postural hypotension was observed with decreases of 14 +/- 1 mm Hg(n = 9). Smaller orthostatic responses were still observed 48 h after Omega-CTX administration: however, by 72 h, head-up tilt no longer induced a significant change in MAP. In the vehicle-treatment group, there were no changes in cardiovascular parameters during 0-168 h. Thus Omega-CTX (10 micrograms/kg i.v.) causes acute hypotension, as well as postural hypotension, and has sympatholytic and vagolytic effects that are mostly reversed after 48 h in the conscious rabbit. However, a second hypotensive and bradycardic phase lasting a further 96 h ensues, suggesting that other prolonged effects from central neural or hormonal mechanisms or fluid shifts may occur.
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Omega Conotoxin GVIA and prazosin but not felodipine cause postural hypotension in rabbits
Clinical and Experimental Pharmacology and Physiology, 1995Co-Authors: Anna L Hawkes, James A. Angus, Christine E. WrightAbstract:1. The aim was to compare the effect of N-type calcium channel blockade by Omega-Conotoxin GVIA (Omega-CTX) with alpha 1-adrenoceptor or L-type calcium channel blockade on postural adaptation in conscious rabbits. 2. Orthostatic responses were assessed by rapidly tilting the rabbits through 90 degrees for 1 min. Tilts were performed before, 30 and 60 min after i.v. bolus administration of vehicle (propylene glycol 0.17 mL/kg; n = 8), prazosin (0.5 mg/kg; n = 8), felodipine (30 micrograms/kg; n = 8) or Omega-CTX (3 & 7 micrograms/kg; n = 9). 3. Prazosin, felodipine or Omega-CTX caused significant falls in mean arterial pressure (MAP) with corresponding increases in heart rate (HR). Vehicle administration had no effect on MAP but caused a small fall in HR. 4. Before drug or vehicle administration, a small rise in MAP and HR occurred in response to tilt in all rabbits. In the vehicle treatment group, similar responses were observed to tilt at 30 and 60 min. Postural hypotension was observed in the prazosin treatment group, but not following administration of felodipine. Tilts 30 and 60 min after Omega-CTX (3 micrograms/kg) caused an increase in HR but no change in MAP, different to the small pressor response observed following vehicle administration. However, following administration of Omega-CTX 7 micrograms/kg (total dose, 10 micrograms/kg), significant falls in MAP with tachycardia were observed in response to tilt. 5. In conclusion, orthostatic hypotension was observed following acute alpha 1-adrenoceptor or N-type calcium channel blockade in the conscious rabbit. These findings are compatible with the expectation that agents which are directly sympatholytic interfere with postural adaptation. In contrast, L-type calcium channel antagonism with felodipine did not elicit postural hypotension.
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hemodynamic and autonomic reflex effects of chronic n type ca2 channel blockade with Omega Conotoxin GVIA in conscious normotensive and hypertensive rabbits
Journal of Cardiovascular Pharmacology, 1995Co-Authors: Christine E. Wright, James A. AngusAbstract:: The effects of chronic administration of Omega-Conotoxin GVIA (Omega-CTX), an N-type Ca2+ channel blocker, on hemodynamics and autonomic reflexes were studied in conscious normotensive (sham) and hypertensive (wrap) New Zealand white rabbits. During surgery, a pulsed Doppler-flow probe was implanted around the lower abdominal aorta, and both kidneys were wrapped in cellophane (wrap) or left undisturbed (sham). Rabbits were studied 4 weeks later on 5 consecutive days. On days 1-4, hemodynamics, the baroreceptor-heart-rate (HR) reflex induced by drugs and the Bezold-Jarisch-like reflex evoked by serotonin, were measured before and 2 h after administration of Omega-CTX (10 micrograms/kg i.v. bolus). On day 5, hemodynamics and reflexes were again assessed, but no further Omega-CTX was given. On day 1, Omega-CTX caused falls in mean arterial pressure (MAP) of 17 +/- 3 and 27 +/- 5 mm Hg in sham (n = 6) and wrap (n = 11) rabbits, respectively, with tachycardia. Hindquarter vascular conductance (HVC) increased > 35% in both groups. On days 2-4, Omega-CTX still caused falls in MAP; however it was progressively less than on day 1. HR and HVC did not change. After Omega-CTX on day 1, there was marked attenuation of the sympathetic components and decrease in the vagal components of the baroreceptor-HR reflex curves, with HR range decreasing by > 61% in sham and wrap rabbits. These curves remained similar over the next 4 days, and were unaffected by further Omega-CTX. However, Omega-CTX had no effect on the vagally mediated Bezold-Jarisch-like reflex. In auxiliary experiments in vitro, Omega-CTX (1 nM) inhibited sympathetic, but not vagal, responses to electrical nerve stimulation of rabbit-isolated right atria. Thus, Omega-CTX is a potent hypotensive agent in normotensive and hypertensive rabbits, predominantly via a peripheral sympatholytic action with no effect on vagal-cardiac efferent activity. However, it may affect the vagal component of the baroreceptor-HR reflex by an unknown central mechanism.
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Omega Conotoxin GVIA the n type calcium channel inhibitor is sympatholytic but not vagolytic consequences for hemodynamics and autonomic reflexes in conscious rabbits
Journal of Cardiovascular Pharmacology, 1990Co-Authors: Didier Pruneau, James A. AngusAbstract:: We investigated the effects of the N-type calcium channel blocking agent, Omega-Conotoxin GVIA, on the resting hemodynamics and on some autonomic reflexes in the conscious rabbit. Omega-Conotoxin 3 and 10 micrograms/kg i.v. reduced mean arterial blood pressure by 16 +/- 2 and 25 +/- 3 mm Hg, respectively, over 30 min accompanied by a tachycardia. Renal vascular conductance (Doppler flowmeter) increased by 27.6 +/- 3.7 and 38.6 +/- 10.3% at 30 min after Omega-Conotoxin 3 and 10 micrograms/kg, respectively. Vasodilatation was also observed but to a lesser extent in the hindquarter and mesenteric vascular beds. The baroreceptor-heart rate reflex was evoked by a drug method (bolus injection of sodium nitroprusside and phenylephrine) and by inflation of perivascular balloons implanted on the thoracic vena cava and aorta. Omega-Conotoxin (3 micrograms/kg) abolished the sympathetic component of the cardiac baroreceptor reflex without affecting vagal efferent activity. In addition, marked vagal-mediated bradycardia from (a) the "Bezold-Jarisch-like" reflex evoked by serotonin (1-10 micrograms/kg i.v.) and (b) the nasopharyngeal reflex evoked by cigarette smoke were unaffected by Omega-Conotoxin (3-10 micrograms/kg). We conclude that Omega-Conotoxin-induced N-type calcium channel blockade abolishes sympathetic but not vagal cardiac efferent activity. The hypotension and peripheral vasodilatation are probably due to the prejunctional sympatholytic action of the peptide. These N-type calcium channels are thus limited to the sympathetic varicosities in the rabbit.
Christine E. Wright - One of the best experts on this subject based on the ideXlab platform.
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selectivity of Omega Conotoxin GVIA for n type calcium channels in rat isolated small mesenteric arteries
Clinical and Experimental Pharmacology and Physiology, 2010Co-Authors: Sarah L Whorlow, James A. Angus, Christine E. WrightAbstract:: 1. The selectivity of Omega-Conotoxin GVIA (Omega-CTX) for prejunctional N-type voltage-operated calcium channels (VOCC) was examined in rat isolated small mesenteric arteries mounted in a Mulvany-Halpern myograph. Contractile responses to perivascular nerve stimulation, noradrenaline (NA) and potassium (K+) were obtained before and after treatment with Omega-CTX. The effects of Omega-CTX were compared with those of felodipine, an L-type VOCC blocker. 2. Omega-CTX (3 nmol/L-10 mu mol/L) inhibited contractions to electrical field stimulation by up to 94%, compared with the corresponding time control group. Felodipine (0.1 mu mol/L) had little effect on the contractions to electrical stimulation compared with the vehicle-treated vessels. 3. Concentration-response curves to exogenous NA (0.1 mu mol/L) and contractions to a submaximal concentration of K+ (50 mmol/L) were unaffected by Omega-CTX (3 nmol/L-10 mu mol/L). In contrast, the maximum contraction to NA in vessels exposed to felodipine (0.1 mu mol/L) was reduced by 37%, and the contraction to K+ (62 mmol/L) was reduced by 84% compared with vehicle-treated arteries. 4. The results indicate that even at concentrations up to 10 mu mol/L (10 000-fold higher than required to inhibit prejunctional N-type VOCC), Omega-CTX inhibits only neurotransmitter release. Its effects are clearly different to felodipine as Omega-CTX has no effect on post-junctional alpha1-adrenoceptor-mediated vasoconstriction or direct smooth muscle depolarization considered to be mediated by L-type VOCC. Therefore, at least at the vascular neuroeffector junction, Omega-CTX appears to be highly selective for N-type VOCC with no effect on L-type VOCC.
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prolonged cardiovascular effects of the n type ca2 channel antagonist Omega Conotoxin GVIA in conscious rabbits
Journal of Cardiovascular Pharmacology, 1997Co-Authors: Christine E. Wright, James A. AngusAbstract:: Omega-Conotoxin GVIA (Omega-CTX) is an N-type Ca2+ channel antagonist that is considered to be only partially reversible in vitro. In vivo, its effects after 24 h are unknown. To assess the duration of action of this peptide in vivo, the effects of a single intravenous injection of Omega-CTX on mean arterial pressure (MAP), heart rate (HR), postural adaptation, and the baroreflex were investigated in conscious rabbits. MAP, HR, the baroreflex induced by i.v. glyceryl trinitrate (0.4-20 micrograms/kg) and phenylephrine (0.1-15 micrograms/kg) and orthostatic responses to 1 min 90 degrees head-up tilt were assessed before (0 h) and 2-168 h after administration of Omega-CTX (10 micrograms/kg i.v. bolus: n = 6-9) or vehicle (0.9% saline; n = 6). Acute phase I: By 2 h after Omega-CTX administration, MAP had decreased from 75 +/- 3 mm Hg to 60 +/- 2 mm Hg; HR increased from 220 +/- 7 beats/min to 249 +/- 5 beats/min (n = 9). There was marked attenuation of the baroreflex curve (HR range decreasing by 61%). By 24 h. MAP and HR had returned to control values, but the HR range was still 18% less than that of control. Phase II: MAP and HR then decreased steadily over the next 96 h to significantly lower values by 120 h after Omega-CTX administration (delta-8 +/- 2 mm Hg and -29 +/- 2 beats/min, respectively; n = 6). Thereafter, MAP and HR values increased and by 168 h these parameters, and the baroreflex, were similar to control values. In response to 90 degrees tilt, there was no change in MAP at 0 h; however, 1 h after Omega-CTX, significant postural hypotension was observed with decreases of 14 +/- 1 mm Hg(n = 9). Smaller orthostatic responses were still observed 48 h after Omega-CTX administration: however, by 72 h, head-up tilt no longer induced a significant change in MAP. In the vehicle-treatment group, there were no changes in cardiovascular parameters during 0-168 h. Thus Omega-CTX (10 micrograms/kg i.v.) causes acute hypotension, as well as postural hypotension, and has sympatholytic and vagolytic effects that are mostly reversed after 48 h in the conscious rabbit. However, a second hypotensive and bradycardic phase lasting a further 96 h ensues, suggesting that other prolonged effects from central neural or hormonal mechanisms or fluid shifts may occur.
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Omega Conotoxin GVIA and prazosin but not felodipine cause postural hypotension in rabbits
Clinical and Experimental Pharmacology and Physiology, 1995Co-Authors: Anna L Hawkes, James A. Angus, Christine E. WrightAbstract:1. The aim was to compare the effect of N-type calcium channel blockade by Omega-Conotoxin GVIA (Omega-CTX) with alpha 1-adrenoceptor or L-type calcium channel blockade on postural adaptation in conscious rabbits. 2. Orthostatic responses were assessed by rapidly tilting the rabbits through 90 degrees for 1 min. Tilts were performed before, 30 and 60 min after i.v. bolus administration of vehicle (propylene glycol 0.17 mL/kg; n = 8), prazosin (0.5 mg/kg; n = 8), felodipine (30 micrograms/kg; n = 8) or Omega-CTX (3 & 7 micrograms/kg; n = 9). 3. Prazosin, felodipine or Omega-CTX caused significant falls in mean arterial pressure (MAP) with corresponding increases in heart rate (HR). Vehicle administration had no effect on MAP but caused a small fall in HR. 4. Before drug or vehicle administration, a small rise in MAP and HR occurred in response to tilt in all rabbits. In the vehicle treatment group, similar responses were observed to tilt at 30 and 60 min. Postural hypotension was observed in the prazosin treatment group, but not following administration of felodipine. Tilts 30 and 60 min after Omega-CTX (3 micrograms/kg) caused an increase in HR but no change in MAP, different to the small pressor response observed following vehicle administration. However, following administration of Omega-CTX 7 micrograms/kg (total dose, 10 micrograms/kg), significant falls in MAP with tachycardia were observed in response to tilt. 5. In conclusion, orthostatic hypotension was observed following acute alpha 1-adrenoceptor or N-type calcium channel blockade in the conscious rabbit. These findings are compatible with the expectation that agents which are directly sympatholytic interfere with postural adaptation. In contrast, L-type calcium channel antagonism with felodipine did not elicit postural hypotension.
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hemodynamic and autonomic reflex effects of chronic n type ca2 channel blockade with Omega Conotoxin GVIA in conscious normotensive and hypertensive rabbits
Journal of Cardiovascular Pharmacology, 1995Co-Authors: Christine E. Wright, James A. AngusAbstract:: The effects of chronic administration of Omega-Conotoxin GVIA (Omega-CTX), an N-type Ca2+ channel blocker, on hemodynamics and autonomic reflexes were studied in conscious normotensive (sham) and hypertensive (wrap) New Zealand white rabbits. During surgery, a pulsed Doppler-flow probe was implanted around the lower abdominal aorta, and both kidneys were wrapped in cellophane (wrap) or left undisturbed (sham). Rabbits were studied 4 weeks later on 5 consecutive days. On days 1-4, hemodynamics, the baroreceptor-heart-rate (HR) reflex induced by drugs and the Bezold-Jarisch-like reflex evoked by serotonin, were measured before and 2 h after administration of Omega-CTX (10 micrograms/kg i.v. bolus). On day 5, hemodynamics and reflexes were again assessed, but no further Omega-CTX was given. On day 1, Omega-CTX caused falls in mean arterial pressure (MAP) of 17 +/- 3 and 27 +/- 5 mm Hg in sham (n = 6) and wrap (n = 11) rabbits, respectively, with tachycardia. Hindquarter vascular conductance (HVC) increased > 35% in both groups. On days 2-4, Omega-CTX still caused falls in MAP; however it was progressively less than on day 1. HR and HVC did not change. After Omega-CTX on day 1, there was marked attenuation of the sympathetic components and decrease in the vagal components of the baroreceptor-HR reflex curves, with HR range decreasing by > 61% in sham and wrap rabbits. These curves remained similar over the next 4 days, and were unaffected by further Omega-CTX. However, Omega-CTX had no effect on the vagally mediated Bezold-Jarisch-like reflex. In auxiliary experiments in vitro, Omega-CTX (1 nM) inhibited sympathetic, but not vagal, responses to electrical nerve stimulation of rabbit-isolated right atria. Thus, Omega-CTX is a potent hypotensive agent in normotensive and hypertensive rabbits, predominantly via a peripheral sympatholytic action with no effect on vagal-cardiac efferent activity. However, it may affect the vagal component of the baroreceptor-HR reflex by an unknown central mechanism.
Seiji Ichida - One of the best experts on this subject based on the ideXlab platform.
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Antigen Selectivity Characteristic of Polyclonal Antibodies Against Omega-Conotoxin GVIA and N-Type Voltage-Dependent Calcium Channels
Neurochemical Research, 2003Co-Authors: Seiji Ichida, Kouichiro Imoto, W. Sugimoto, M. Uematsu, K. Komoike, M. Mori, T. Minami, T Wada, Koji KakutaniAbstract:The antibodies against Omega-Conotoxin GVIA (ω-CTX GVIA; N-type voltage-dependent calcium channel [VDCC] blocker) and B_1Nt (N-terminal segment [residues 1–13] of BI α_1 subunits of VDCCs) were prepared, and the selectivity for each antigen ω-CTX GVIA and B1Nt was investigated. For the antigen selectivity of anti–ω-CTX GVIA antibody against ω-CTX GVIA, ELISA, and immunoprecipitation were used. The reactions for ELISA and immunoprecipitation were observed except when antibody IgG purified by Protein A–Sepharose CL-4B from nonimmunized serum (purified NI-Ab) was used. The specific reactions were inhibited by 10 nM ω-CTX GVIA, but not by ω-CTX SVIB (N-type VDCC blocker), ω-CTX MVIIC (N- and P-type VDCC blocker), or ω-Aga IVA (P-type VDCC blocker). For the antigen selectivity of the anti-B1Nt antibody, analyses by ELISA, immunoprecipitation, and Western blotting were conducted. The reactions were observed except when NI-Ab was used. The ELISA and immunoprecipitation reactions were inhibited by the antigen peptide B1Nt, and the IC_50 values were about 1.2 × 1028 and 1.3 × 1028 M, respectively. The bands of 210 and 190 kD by Western blotting of crude membranes from chick brain were also inhibited by 1 μM B1Nt. These results suggest that the antibodies prepared against ω-CTX GVIA and B1Nt in this work have high selectivity for their antigen. Therefore we assume that the antibodies against ω-CTX GVIA and B1Nt are useful tools for the analyses of the function and distribution of N-type VDCCs. The anti ω-CTX GVIA antibody must also be useful for the radioimmunoassay of ω-CTX GVIA.
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characteristics of the inhibitory effect of calmodulin on specific 125i Omega Conotoxin GVIA binding to crude membranes from chick brain
Neurochemical Research, 2000Co-Authors: Seiji Ichida, Kensuke Sugihara, Kouichiro Imoto, Tetsuyuki Wada, Norihisa Fujita, Zhang Yuan, Hitoshi SohmaAbstract:The characteristics of the inhibitory effect of calcium ion (Ca2+)/calmodulin (CaM) on specific [125I]-Omega-Conotoxin GVIA (125I-ω-CTX) binding and on the labeling of 125I-ω-CTX to crude membranes from chick brain were investigated. The inhibitory effect of Ca2+/CaM depended on the concentrations of free Ca2+ and CaM. The IC50 values for free Ca2+ and CaM were about 2.0 × 10−8 M and 3.0 μg protein/ml, respectively. The inhibitory effect of Ca2+/CaM was attenuated by the CaM antagonists W-7, prenylamine and CaM-kinase II fragment (290–309), but not by the calcineurin inhibitor FK506. Ca2+/CaM also inhibited the labeling of a 135-kDa band (which was considered to be part of N-type Ca2+ channel α1 subunits) with 125I-ω-CTX using a cross-linker. These results suggest that Ca2+/CaM affects specific 125I-ω-CTX binding sites, probably N-type Ca2+ channel α1 subunits, in crude membranes from chick whole brain.
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calcium calmodulin inhibits the binding of specific 125i Omega Conotoxin GVIA to chick brain membranes
Neurochemical Research, 2000Co-Authors: Seiji Ichida, Kensuke Sugihara, Kouichiro Imoto, Tetsuyuki Wada, Zhang Yuan, Hitoshi SohmaAbstract:The effect of Ca2+/calmodulin (CaM) on the specific binding of [125I]Omega-Conotoxin GVIA (125I-ω-CTX) to crude membranes from chick brain was investigated. When we examined the effects of the activation of various endogenous protein kinases on specific [125I]ω-CTX binding to crude membranes, we observed that Ca2+/CaM had an inhibitory effect regardless of whether or not the standard medium contained ATP (0.5 mM). Ca2+/CaM also had an inhibitory effect in a simple binding-assay medium containing HEPES-HCl buffer, BSA, Ca2+ and CaM, and this effect was dependent on the concentration of Ca2+. The effect of Ca2+/CaM was attenuated by the CaM antagonists W-7 and CaM-kinase II fragment (290–309). An experiment with modified ELISA using purified anti ω-CTX antibody indicated that Ca2+/CaM did not affect the direct binding of [125I]ω-CTX and CaM. These results suggest that Ca2+/CaM either directly or indirectly affects specific [125I]ω-CTX binding sites, probably N-type Ca2+ channels in crude membranes from chick whole brain.
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Calcium/Calmodulin Inhibits the Binding of Specific [125I]Omega-Conotoxin GVIA to Chick Brain Membranes
Neurochemical Research, 2000Co-Authors: Seiji Ichida, Kensuke Sugihara, Kouichiro Imoto, Tetsuyuki Wada, Hitoshi SohmaAbstract:The effect of Ca^2+/calmodulin (CaM) on the specific binding of [^125I]Omega-Conotoxin GVIA (^125I-ω-CTX) to crude membranes from chick brain was investigated. When we examined the effects of the activation of various endogenous protein kinases on specific [^125I]ω-CTX binding to crude membranes, we observed that Ca^2+/CaM had an inhibitory effect regardless of whether or not the standard medium contained ATP (0.5 mM). Ca^2+/CaM also had an inhibitory effect in a simple binding-assay medium containing HEPES-HCl buffer, BSA, Ca^2+ and CaM, and this effect was dependent on the concentration of Ca^2+. The effect of Ca^2+/CaM was attenuated by the CaM antagonists W-7 and CaM-kinase II fragment (290–309). An experiment with modified ELISA using purified anti ω-CTX antibody indicated that Ca^2+/CaM did not affect the direct binding of [^125I]ω-CTX and CaM. These results suggest that Ca^2+/CaM either directly or indirectly affects specific [^125I]ω-CTX binding sites, probably N-type Ca^2+ channels in crude membranes from chick whole brain.
Koji Kakutani - One of the best experts on this subject based on the ideXlab platform.
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Antigen Selectivity Characteristic of Polyclonal Antibodies Against Omega-Conotoxin GVIA and N-Type Voltage-Dependent Calcium Channels
Neurochemical Research, 2003Co-Authors: Seiji Ichida, Kouichiro Imoto, W. Sugimoto, M. Uematsu, K. Komoike, M. Mori, T. Minami, T Wada, Koji KakutaniAbstract:The antibodies against Omega-Conotoxin GVIA (ω-CTX GVIA; N-type voltage-dependent calcium channel [VDCC] blocker) and B_1Nt (N-terminal segment [residues 1–13] of BI α_1 subunits of VDCCs) were prepared, and the selectivity for each antigen ω-CTX GVIA and B1Nt was investigated. For the antigen selectivity of anti–ω-CTX GVIA antibody against ω-CTX GVIA, ELISA, and immunoprecipitation were used. The reactions for ELISA and immunoprecipitation were observed except when antibody IgG purified by Protein A–Sepharose CL-4B from nonimmunized serum (purified NI-Ab) was used. The specific reactions were inhibited by 10 nM ω-CTX GVIA, but not by ω-CTX SVIB (N-type VDCC blocker), ω-CTX MVIIC (N- and P-type VDCC blocker), or ω-Aga IVA (P-type VDCC blocker). For the antigen selectivity of the anti-B1Nt antibody, analyses by ELISA, immunoprecipitation, and Western blotting were conducted. The reactions were observed except when NI-Ab was used. The ELISA and immunoprecipitation reactions were inhibited by the antigen peptide B1Nt, and the IC_50 values were about 1.2 × 1028 and 1.3 × 1028 M, respectively. The bands of 210 and 190 kD by Western blotting of crude membranes from chick brain were also inhibited by 1 μM B1Nt. These results suggest that the antibodies prepared against ω-CTX GVIA and B1Nt in this work have high selectivity for their antigen. Therefore we assume that the antibodies against ω-CTX GVIA and B1Nt are useful tools for the analyses of the function and distribution of N-type VDCCs. The anti ω-CTX GVIA antibody must also be useful for the radioimmunoassay of ω-CTX GVIA.
Vladimir J Basus - One of the best experts on this subject based on the ideXlab platform.
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solution structure of Omega Conotoxin GVIA using 2 d nmr spectroscopy and relaxation matrix analysis
Biochemistry, 1993Co-Authors: Jonathan H Davis, G P Miljanich, Erin K Bradley, Laszlo Nadasdi, J Ramachandran, Vladimir J BasusAbstract:: We report here the solution structure of Omega-Conotoxin GVIA, a peptide antagonist of the N-type neuronal voltage-sensitive calcium channel. The structure was determined using two-dimensional NMR in combination with distance geometry and restrained molecular dynamics. The full relaxation matrix analysis program MARDIGRAS was used to generate maximum and minimum distance restraints from the crosspeak intensities in NOESY spectra. The 187 restraints obtained were used in conjunction with 23 angle restraints from vicinal coupling constants as input for the structure calculations. The backbones of the best 21 structures match with an average pairwise RMSD of 0.58 A. The structures contain a short segment of triple-stranded beta-sheet involving residues 6-8, 18-21, and 24-27, making this the smallest published peptide structure to contain a triple-stranded beta-sheet. Conotoxins have been shown to be effective neuroprotective agents in animal models of brain ischemia. Our results should aid in the design of novel nonpeptide compounds with potential therapeutic utility.
