Calcium requirement for cGMP production during muscarinic activation of N1E-115 neuroblastoma cells

1995 ◽  
Vol 269 (4) ◽  
pp. C979-C985 ◽  
Author(s):  
S. H. Thompson ◽  
C. Mathes ◽  
A. A. Alousi
Keyword(s):  
Time Course ◽  
Neuroblastoma Cells ◽  
Large Contribution ◽  
Muscarinic Agonists ◽  
Strongly Coupled ◽  
Cgmp Production ◽  
Store Depletion ◽  
Calcium Requirement ◽  
Oxide Synthase ◽  
Ca2 Channels

Muscarinic agonists elicit large increases in intracellular Ca2+ and guanosine 3',5'-cyclic monophosphate (cGMP) in N1E-115 neuroblastoma cells. Both signals are blocked in cells loaded with the Ca2+ buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid showing that the increase in intracellular Ca2+ concentration ([Ca2+]i) is necessary to stimulate cGMP accumulation. Inhibition of nitric oxide synthase (NOS) blocks the cGMP response without affecting the peak amplitude of the intracellular Ca2+ signal, and it is concluded that Ca(2+)-dependent activation of NOS is required for cGMP production. cGMP accumulation is reduced by 60% when cells are bathed in Ca(2+)-free saline, but the peak change in [Ca2+]i is not affected. This suggests that Ca2+ influx is strongly coupled to the activation of cGMP production, even though it makes a smaller contribution to the intracellular Ca2+ signal than does Ca2+ release. Thapsigargin, which releases Ca2+ from intracellular stores, activates Ca2+ influx and increases cGMP. The cGMP increase is transient and follows approximately the same time course as Ca2+ store depletion. Ca2+ influx remains activated after store depletion, however, which indicates that influx alone cannot sustain cGMP production. It is concluded that summation of Ca2+ influx and Ca2+ release is necessary to reach a threshold Ca2+ level needed to stimulate cGMP accumulation. Because of the large contribution from Ca2+ influx, we suggest that NOS or a cofactor necessary for its activation may be located close to Ca2+ channels in the membrane.

scholarly journals The relationship between depletion of intracellular Ca2+ stores and activation of Ca2+ current by muscarinic receptors in neuroblastoma cells.

1995 ◽  
Vol 106 (5) ◽  
pp. 975-993 ◽  
Author(s):  
C Mathes ◽  
S H Thompson
Keyword(s):  
Muscarinic Receptors ◽  
Current Density ◽  
Time Course ◽  
Neuroblastoma Cells ◽  
Calcium Stores ◽  
Store Depletion ◽  
Selective Membrane ◽  
Current Activation ◽  
M1 Muscarinic ◽  
The Relationship

The relationship between the depletion of IP3-releasable intracellular Ca2+ stores and the activation of Ca(2+)-selective membrane current was determined during the stimulation of M1 muscarinic receptors in N1E-115 neuroblastoma cells. External Ca2+ is required for refilling Ca2+ stores and the voltage-independent, receptor-regulated Ca2+ current represents a significant Ca2+ source for refilling. The time course of Ca2+ store depletion was measured with fura-2 fluorescence imaging, and it was compared with the time course of Ca2+ current activation measured with nystatin patch voltage clamp. At the time of maximum current density (0.18 + .03 pA/pF; n = 48), the Ca2+ content of the IP3-releasable Ca2+ pool is reduced to 39 + 3% (n = 10) of its resting value. Calcium stores deplete rapidly, reaching a minimum Ca2+ content in 15-30 s. The activation of Ca2+ current is delayed by 10-15 s after the beginning of Ca2+ release and continues to gradually increase for nearly 60 s, long after Ca2+ release has peaked and subsided. The delay in the appearance of the current is consistent with the idea that the production and accumulation of a second messenger is the rate-limiting step in current activation. The time course of Ca2+ store depletion was also measured after adding thapsigargin to block intracellular Ca2+ ATPase. After 15 min in thapsigargin, IP3-releasable Ca2+ stores are depleted by > 90% and the Ca2+ current is maximal (0.19 + 0.05 pA/pF; n = 6). Intracellular loading with the Ca2+ buffer EGTA/AM (10 microM; 30 min) depletes IP3-releasable Ca2+ stores by between 25 and 50%, and it activates a voltage-independent inward current with properties similar to the current activated by agonist or thapsigargin. The current density after EGTA/AM loading (0.61 + 0.32 pA/pF; n = 4) is three times greater than the current density in response to agonist or thapsigargin. This could result from partial removal of Ca(2+)-dependent inactivation.

scholarly journals Effect of Scrapie Infection on the Activity of Neuronal Nitric-oxide Synthase in Brain and Neuroblastoma Cells

1996 ◽  
Vol 271 (28) ◽  
pp. 16856-16861 ◽  
Author(s):  
Haim Ovadia ◽  
Hana Rosenmann ◽  
Elias Shezen ◽  
Michele Halimi ◽  
Ishai Ofran ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Neuroblastoma Cells ◽  
Neuronal Nitric Oxide Synthase ◽  
Scrapie Infection ◽  
Oxide Synthase

scholarly journals Interleukin-4 stimulates cGMP production by IFN-gamma-activated human monocytes. Involvement of the nitric oxide synthase pathway

1994 ◽  
Vol 269 (13) ◽  
pp. 9811-9816
Author(s):  
J.P. Kolb ◽  
N. Paul-Eugene ◽  
C. Damais ◽  
K. Yamaoka ◽  
J.C. Drapier ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Interleukin 4 ◽  
Human Monocytes ◽  
Ifn Gamma ◽  
Cgmp Production ◽  
Oxide Synthase

scholarly journals Time Course of Inducible Nitric Oxide Synthase Activity Following Endotoxin Administration in Dogs

Nitric Oxide ◽  
2001 ◽  
Vol 5 (2) ◽  
pp. 208-211 ◽  
Author(s):  
Jean-Charles Preiser ◽  
Haibo Zhang ◽  
Bernard Vray ◽  
Andreas Hrabak ◽  
Jean-Louis Vincent
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Time Course ◽  
Endotoxin Administration ◽  
Oxide Synthase ◽  
Nitric Oxide Synthase Activity ◽  
Inducible Nitric Oxide

Regulation of calcium current by voltage and cytoplasmic calcium in canine gastric smooth muscle

1992 ◽  
Vol 262 (3) ◽  
pp. C691-C700 ◽  
Author(s):  
F. Vogalis ◽  
N. G. Publicover ◽  
K. M. Sanders
Keyword(s):  
Time Course ◽  
Fast Component ◽  
Patch Clamp Technique ◽  
Similar Time ◽  
Whole Cell Patch Clamp ◽  
Gastric Smooth Muscle ◽  
Double Exponential ◽  
Circular Layer ◽  
Double Exponential Function ◽  
Ca2 Channels

The regulation of Ca2+ current by intracellular Ca2+ was studied in isolated myocytes from the circular layer of canine gastric antrum. Ca2+ current was measured with the whole cell patch-clamp technique, and changes in cytoplasmic Ca2+ ([Ca2+]i) were simultaneously measured with indo-1 fluorescence. Ca2+ currents were activated by depolarization and inactivated despite maintained depolarization. Ca2+ current inactivation was fit with a double exponential function. Using Ba2+ or Na+ as charge carriers removed the fast component of inactivation, whereas enhanced intracellular buffering of Ca2+ did not remove the fast component. Ca2+ currents were associated with a rise in [Ca2+]i. The decrease in [Ca2+]i following repolarization was exponential, and during the relaxation of [Ca2+]i, Ca2+ current was inactivated. The inward current recovered with a similar time course as the decrease in [Ca2+]i, suggesting that [Ca2+]i regulates the basal availability of Ca2+ channels. These data support the hypothesis that, although [Ca2+]i may influence the resting level of inactivation, it is the "submembrane" compartment of [Ca2+]i that regulates the development of inactivation.

Endothelial vasodilator production by uterine and systemic arteries. II. Pregnancy effects on NO synthase expression

1997 ◽  
Vol 272 (4) ◽  
pp. H1730-H1740 ◽  
Author(s):  
R. R. Magness ◽  
C. E. Shaw ◽  
T. M. Phernetton ◽  
J. Zheng ◽  
I. M. Bird
Keyword(s):  
Specific Activity ◽  
No Synthase ◽  
No Production ◽  
Tetraacetic Acid ◽  
Cgmp Production ◽  
Western Analysis ◽  
Systemic Artery ◽  
Pregnant Sheep ◽  
Oxide Synthase ◽  
Systemic Arteries

Pregnancy is characterized by elevations in uterine but not omental artery nitric oxide synthase (NOS)-specific activity. We hypothesized that increases in NO production during pregnancy are associated with elevations in protein expression of the constitutive isoform, endothelial cell NOS (ecNOS), in uterine but not systemic arteries. Arterial NOS-specific activity and guanosine 3',5'-cyclic monophosphate (cGMP) production were tested in pregnant sheep in the presence or absence [+5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] of Ca2+. With the use of Western analysis, ecNOS and neuronal NOS (nNOS) constitutive isoform expressions were evaluated in intact and denuded [vascular smooth muscle (VSM)] uterine and systemic (omental and renal) arteries as well as in isolated endothelium-derived proteins from nonpregnant and pregnant sheep. Uterine and omental artery NOS activity and cGMP production were inhibited 75-85% by Ca2+ removal. ecNOS was localized only in uterine and systemic artery endothelium (not VSM) by immunohistochemistry and Western analysis; nNOS was not detected. Compared with nonpregnant ewes, pregnancy increased expression of ecNOS in uterine [2.1- to 4.2-fold (P < 0.0001)] and omental [1.3- to 2.2-fold (P = 0.032)] but not renal (P = 0.1367) artery endothelium. Increases in uterine were greater than in omental artery endothelium. Levels of plasma and urinary cGMP were elevated (P < 0.01) proportionally (1.8- to 2.0-fold) in pregnant versus nonpregnant ewes. During pregnancy, expression of uterine artery endothelium-derived (not VSM) ecNOS constitutive isoform is increased, whereas expression in systemic vessels shows little or no change.

Cd(2+)-induced injury in CNS white matter

1996 ◽  
Vol 76 (5) ◽  
pp. 3264-3273 ◽  
Author(s):  
R. Fern ◽  
J. A. Black ◽  
B. R. Ransom ◽  
S. G. Waxman
Keyword(s):  
White Matter ◽  
Optic Nerve ◽  
Mitochondrial Respiration ◽  
Time Course ◽  
White Matter Injury ◽  
Neurological Injury ◽  
Antimycin A ◽  
Optic Nerves ◽  
Voltage Gated ◽  
Ca2 Channels

1. The affect of extracellular Cd2+ on CNS white matter was studied using an isolated rat optic nerve preparation. A 100-min exposure to 200 microM Cd2+ reduced the area of the compound action potential (CAP) recorded from the optic nerve to 32.6 +/- 3.8% (mean +/- SE) of the preexposure area, compared with a reduction to 74.9 +/- 2.9% after 100 min in control conditions (P > 0.001). This CAP reduction was not reversed after 120 min of reperfusion with Cd(2+)-free solution, or by perfusion with Cd2+ chelators. 2. Cd(2+)-induced CAP loss occurred in the absence of extracellular Ca2+. Increasing extracellular Ca2+ concentration to 16 mM, however, prevented Cd(2+)-induced CAP loss. Once evident, Cd(2+)-induced CAP reduction could not subsequently be reversed by addition of 16 mM Ca2+. 3. Low concentrations of Cd2+ (60 microM) did not significantly reduce CAP area. This concentration of Cd2+ combined with high extracellular K+ (30 mM) caused CAP loss that was blocked by 10 microM nifedipine, an antagonist of L-type voltage-gated Ca2+ channels. 4. Treatment with pharmacological inhibitors of membrane proteins known to be inhibited by Cd2+ did not affect the CAP. These included inhibitors of voltage-gated Ca2+ channels, Ca(2+)-activated K+ channels, Ca(2+)-ATPase and the Na+/Ca2+ exchanger. 5. Treatment with pharmacological agents that inhibit calmodulin or disrupt tubulin, two intracellular proteins affected by Cd2+, did not affect CAP area. 6. The effect of Cd2+ was not prevented by pretreatment with (+)-cyanidanol-3, an agent that prevents Cd(2+)-induced lipid peroxidation. 7. Treatment with antimycin A, a inhibitor of mitochondrial respiration, resulted in irreversible CAP reduction with a time course and extent similar to that produced by 200 microM Cd2+. Cd(2+)-induced CAP reduction was prevented by 1 mM cysteine, which prevents Cd(2+)-induced disruption of mitochondrial respiration. 8. The ultrastructure of optic nerves exposed to 200 microM Cd2+ for 100 min was characterized by swollen mitochondria with disrupted cristae and dissolution of microtubules, which were replaced by flocculent debris. Occasional regions of axonal swelling and empty spaces beneath the myelin also were found. Qualitatively similar changes in mitochondria and cytoskeletal elements were found in optic nerves exposed to antimycin A for 100 min. Astrocytes also displayed disrupted mitochondria and had an electron-lucent appearance under both conditions. 9. The neurological injury produced by exposure to Cd2+ is characterized by lesions of CNS white matter. Our results indicate that Cd(2+)-induced white matter injury in vitro results largely from disruption of mitochondrial respiration after Cd2+ influx through routes that include voltage-gated Ca2+ channels.

Cholinergic modulation of the Ca2+ response to bradykinin in neuroblastoma cells

1997 ◽  
Vol 273 (2) ◽  
pp. C612-C617 ◽  
Author(s):  
J. S. Coggan ◽  
S. H. Thompson
Keyword(s):  
G Protein ◽  
Neuroblastoma Cells ◽  
Cholinergic Receptor ◽  
Unique Property ◽  
Cholinergic Stimulation ◽  
Early Step ◽  
Store Depletion ◽  
Mouse Neuroblastoma ◽  
Inositol 1,4,5 Trisphosphate ◽  
Heterologous Desensitization

Fura 2 imaging was used to measure intracellular Ca2+ signals in N1E-115 mouse neuroblastoma cells during combined activation of bradykinin (BK) and cholinergic receptors. BK and carbachol (CCh) both activate phospholipase C (PLC) and cause Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores. The Ca2+ signal in response to CCh is prolonged by the activation of Ca2+ influx, but BK does not appear to activate the influx pathway. When BK and CCh are applied together (BK+CCh), the Ca2+ response is composed of both Ca2+ release and Ca2+ influx. Ca2+ influx is also activated by BK+CCh in a subset of cells that does not respond with a intracellular Ca2+ concentration increase when CCh is presented by itself. This suggests that CCh stimulates a Ca(2+)-silent cholinergic receptor that is not coupled to Ca2+ release but acts synergistically with BK receptors to activate Ca2+ influx. Pertussis toxin reduces influx without affecting release, indicating that the G protein that modulates the influx pathway is different from the G protein responsible for activating PLC. Cholinergic stimulation also causes progressive heterologous desensitization of BK-evoked Ca2+ release. Desensitization has the unique property of continuing to develop after the cholinergic agonist is removed and the cholinergic Ca2+ response has fully recovered. Heterologous desensitization is not the result of Ca2+ store depletion or a long-lasting inhibition of PLC or IP3-dependent Ca2+ release. Instead, it appears to involve an early step in the BK-signaling cascade, possibly at the level of the B2 receptor or associated G proteins.

Nitric oxide-dependent and -independent components of cerebrovasodilation elicited by hypercapnia

1994 ◽  
Vol 266 (2) ◽  
pp. R546-R552 ◽  
Author(s):  
C. Iadecola ◽  
F. Zhang
Keyword(s):  
Nitric Oxide ◽  
Methyl Ester ◽  
Time Course ◽  
Slow Time ◽  
Independent Components ◽  
Arterial Partial Pressure ◽  
Partial Pressure Of Co2 ◽  
Nos Inhibitor ◽  
Oxide Synthase ◽  
Doppler Flowmeter

We studied the effect of nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, on the increases in cerebral blood flow (CBF) elicited by stepwise elevations in arterial partial pressure of CO2 (PaCO2) from normocapnia up to 204 mmHg. Rats were anesthetized with halothane and ventilated. CBF was monitored over the parietal cortex using a laser-Doppler flowmeter. Increasing levels of hypercapnia elicited graded elevations in CBF that reached a plateau at PaCO2 = 82 +/- 1 mmHg (CBF +215 +/- 25%; n = 8; P < 0.05, analysis of variance). L-NAME (40 mg/kg i.v.; n = 8), but not nitro-D-arginine methyl ester (n = 8), reduced resting CBF (-42 +/- 4%) and attenuated the increase in CBF elicited by hypercapnia. The attenuation occurred only at PaCO2 40-80 mmHg and was maximal (-75 +/- 8%; P < 0.05) at 54 +/- 2 mmHg. At PaCO2 > or = 100 mmHg, L-NAME (40-80 mg/kg) did not attenuate the response (P > 0.05). Reduction of resting CBF (-50 +/- 4%; n = 6) by administration of chloralose (20-40 mg/kg i.v.) did not attenuate the CBF response to hypercapnia (P > 0.05). We also found that the attenuation by L-NAME of resting CBF (n = 5) and of the cerebrovasodilation elicited by hypercapnia (n = 6) has a relatively slow time course, the effects reaching a maximum 45-60 min after intravenous administration of the drug. We conclude that L-NAME does not attenuate the CBF response to CO2 uniformly at all levels of hypercapnia.(ABSTRACT TRUNCATED AT 250 WORDS)

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