Characterizing voltage-dependent Ca2+ channels coupled to VIP release and NO synthesis in enteric synaptosomes

2002 ◽  
Vol 283 (5) ◽  
pp. G1027-G1034 ◽  
Author(s):  
M. Kurjak ◽  
A. Sennefelder ◽  
M. Aigner ◽  
V. Schusdziarra ◽  
H. D. Allescher
Keyword(s):  
Nitric Oxide ◽  
No Synthase ◽  
Channel Blockers ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
P Type ◽  
Ca2 Channels

In enteric synaptosomes of the rat, the role of voltage-dependent Ca2+channels in K+-induced VIP release and nitric oxide (NO) synthesis was investigated. Basal VIP release was 39 ± 4 pg/mg, and cofactor-substituted NO synthase activity was 7.0 ± 0.8 fmol · mg−1 · min−1. K+ depolarization (65 mM) stimulated VIP release Ca2+ dependently (basal, 100%; K+, 172.2 ± 16.2%; P < 0.05, n = 5). K+-stimulated VIP release was reduced by blockers of the P-type (ω-agatoxin-IVA, 3 × 10−8 M) and N-type (ω-conotoxin-GVIA, 10−6 M) Ca2+ channels by ∼50 and 25%, respectively, but not by blockers of the L-type (isradipine, 10−8 M), Q-type (ω-conotoxin-MVIIC, 10−6 M), or T-type (Ni2+, 10−6 M) Ca2+ channels. In contrast, NO synthesis was suppressed by ω-agatoxin-IVA, ω-conotoxin-GVIA, and isradipine by ∼79, 70, and 70%, respectively, whereas Ni2+ and ω-conotoxin-MVIIC had no effect. These findings are suggestive of a coupling of depolarization-induced VIP release primarily to the P- and N-type Ca2+ channels, whereas NO synthesis is presumably dependent on Ca2+ influx not only via the P- and N- but also via the L-type Ca2+ channel. In contrast, none of the Ca2+ channel blockers affected VIP release evoked by exogenous NO, suggesting that NO induces VIP secretion by a different mechanism, presumably involving intracellular Ca2+ stores.

Role of Ca2+ and Na+ on luteinizing hormone release from the calf pituitary

1988 ◽  
Vol 255 (4) ◽  
pp. E469-E474
Author(s):  
J. P. Kile ◽  
M. S. Amoss
Keyword(s):  
Luteinizing Hormone ◽  
Ionophore A23187 ◽  
Channel Blockers ◽  
Hormone Release ◽  
Primary Cells ◽  
Rat Pituitary ◽  
Voltage Dependent ◽  
Lh Release ◽  
Ca2 Channels

It has been proposed that gonadotropin-releasing hormone (GnRH) stimulates Ca2+ entry by activation of voltage-independent, receptor-mediated Ca2+ channels in the rat gonadotroph. Little work has been done on the role of calcium in GnRH-induced luteinizing hormone (LH) release in species other than the rat. Therefore, this study was done to compare the effects of agents that alter Ca2+ or Na+ entry on LH release from calf anterior pituitary primary cells in culture. GnRH (100 ng/ml), Ca2+ ionophore A23187 (2.5 microM), and the depolarizing agent ouabain (0.1-10 microM) all produced significant increases (P less than 0.05) in LH release; these effects were significantly reduced when the cells were preincubated with the organic Ca2+ channel blockers nifedipine (1-10 microM) and verapamil (1-10 microM) and with Co2+ (0.01-1 mM). The effect of ouabain was inhibited by tetrodotoxin (TTX; 1-10 nM) as well as by nifedipine at 0.1-10 microM. In contrast to its effect on rat pituitary LH release, TTX significantly inhibited GnRH-stimulated LH release at 1-100 nM. These results suggest that GnRH-induced LH release may employ Ca2+ as a second messenger in bovine gonadotrophs and support recent speculation that GnRH-induced Ca2+ mobilization may in part be voltage dependent.

Mechanisms of endothelial P2Y1- and P2Y2-mediated vasodilatation involve differential [Ca2+]i responses

2001 ◽  
Vol 281 (4) ◽  
pp. H1759-H1766 ◽  
Author(s):  
Sean P. Marrelli
Keyword(s):  
Nitric Oxide ◽  
Simultaneous Measurement ◽  
Cerebral Arteries ◽  
No Synthase ◽  
Fura 2 ◽  
Middle Cerebral Arteries ◽  
Intracellular Ca ◽  
The Difference ◽  
Stimulation Of

The present study was designed to evaluate the role of endothelial intracellular Ca2+ concentration ([Ca2+]i) in the difference between P2Y1- and P2Y2-mediated vasodilatations in cerebral arteries. Rat middle cerebral arteries were cannulated, pressurized, and luminally perfused. The endothelium was selectively loaded with fura 2, a fluorescent Ca2+indicator, for simultaneous measurement of endothelial [Ca2+]i and diameter. Luminal administration of 2-methylthioadenosine 5′-triphosphate (2-MeS-ATP), an endothelial P2Y1 agonist, resulted in purely nitric oxide (NO)-dependent dilation and [Ca2+]i increases up to ∼300 nM (resting [Ca2+]i = 145 nM). UTP, an endothelial P2Y2 agonist, resulted in dilations that were both endothelium-derived hyperpolarizing factor (EDHF)- and NO-dependent with [Ca2+]iincreases to >400 nM. In the presence of N G-nitro-l-arginine-indomethacin to inhibit NO synthase and cyclooxygenase, UTP resulted in an EDHF-dependent dilation alone. The [Ca2+]ithreshold for NO-dependent dilation was 220 vs. 340 nM for EDHF. In summary, the differences in the mechanism of vasodilatation resulting from stimulation of endothelial P2Y1 and P2Y2purinoceptors result in part from differential [Ca2+]i responses. Consistent with this finding, these studies also demonstrate a higher [Ca2+]i threshold for EDHF-dependent responses compared with NO.

Presenilin 1 Deficiency Alters the Activity of Voltage-Gated Ca2+ Channels in Cultured Cortical Neurons

2005 ◽  
Vol 94 (6) ◽  
pp. 4421-4429 ◽  
Author(s):  
David G. Cook ◽  
Xiaofan Li ◽  
Sheree D. Cherry ◽  
Angela R. Cantrell
Keyword(s):  
Cortical Neurons ◽  
Critical Role ◽  
Neuronal Firing ◽  
Voltage Gated ◽  
Voltage Dependent ◽  
Cultured Cortical Neurons ◽  
Intracellular Ca ◽  
Multiple Levels ◽  
P Type ◽  
Ca2 Channels

Presenilins 1 and 2 (PS1 and PS2, respectively) play a critical role in mediating γ-secretase cleavage of the amyloid precursor protein (APP). Numerous mutations in the presenilins are known to cause early-onset familial Alzheimer's disease (FAD). In addition, it is well established that PS1 deficiency leads to altered intracellular Ca2+ homeostasis involving endoplasmic reticulum Ca2+ stores. However, there has been little evidence suggesting Ca2+ signals from extracellular sources are influenced by PS1. Here we report that the Ca2+ currents carried by voltage-dependent Ca2+ channels are increased in PS1-deficient cortical neurons. This increase is mediated by a significant increase in the contributions of L- and P-type Ca2+ channels to the total voltage-mediated Ca2+ conductance in PS1 (−/−) neurons. In addition, chelating intracellular Ca2+ with 1,2-bis-( o-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA) produced an increase in Ca2+ current amplitude that was comparable to the increase caused by PS1 deficiency. In contrast to this, BAPTA had no effect on voltage-dependent Ca2+ conductances in PS1-deficient neurons. These data suggest that PS1 deficiency may influence voltage-gated Ca2+ channel function by means that involve intracellular Ca2+ signaling. These findings reveal that PS1 functions at multiple levels to regulate and stabilize intracellular Ca2+ levels that ultimately control neuronal firing behavior and influence synaptic transmission.

Stimulation of voltage-dependent Ca2+ channels by NO at rat myenteric neurons

2007 ◽  
Vol 293 (4) ◽  
pp. G886-G893 ◽  
Author(s):  
Mabruka Sitmo ◽  
Matthias Rehn ◽  
Martin Diener
Keyword(s):  
Enteric Neurons ◽  
Channel Blockers ◽  
No Donor ◽  
Inward Current ◽  
Myenteric Neurons ◽  
Whole Cell Patch Clamp ◽  
Target Organs ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
Ca2 Channels

The aim of the present study was to characterize the action of the neurotransmitter NO on rat myenteric neurons. A NO donor such as GEA 3162 (10−4 mol/l) induced an increase in the intracellular Ca2+ concentration as indicated by an increase in the fura 2 ratio in ganglia loaded with this Ca2+-sensitive fluorescent dye. The effect of GEA 3162 was strongly reduced in the absence of extracellular Ca2+, suggesting an influx of Ca2+ from the extracellular space evoked by NO. A similar nearly complete inhibition was observed in the presence of Ca2+ channel blockers such as Ni2+ (5 × 10−4 mol/l) or nifedipine (10−6 mol/l). Whole cell patch-clamp recordings confirmed the activation of voltage-dependent Ca2+ channels, measured as inward current carried by Ba2+, by the NO donor. The peak Ba2+-carried inward current increased from −100 ± 19 to −185 ± 34 pA in the presence of sodium nitroprusside (10−4 mol/l). The consequence was a hyperpolarization of the membrane, which was blocked by intracellular Cs+ and thus most probably reflects the activation of Ca2+-dependent K+ channels. Furthermore, at least two subtypes of NO synthases, NOS-1 (neuronal form) and NOS-3 (endothelial form), were found as transcripts in mRNA isolated from the rat myenteric ganglia. The expression of these NO synthases was confirmed immunohistochemically. These observations suggest that NO, released from nitrergic neurons within the enteric nervous system, not only affects target organs such as smooth muscle cells in the gut but has in addition profound effects on the enteric neurons themselves, the key players in the regulation of many gastrointestinal functions.

Nitric oxide induces [Ca2+]i oscillations in pituitary GH3 cells: involvement of IDR and ERG K+ currents

2006 ◽  
Vol 290 (1) ◽  
pp. C233-C243 ◽  
Author(s):  
Agnese Secondo ◽  
Anna Pannaccione ◽  
Mauro Cataldi ◽  
Rossana Sirabella ◽  
Luigi Formisano ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Inhibitory Effect ◽  
Receptor Blocker ◽  
Gh3 Cells ◽  
No Donors ◽  
Quiescent Cells ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
Trisphosphate Receptor

The role of nitric oxide (NO) in the occurrence of intracellular Ca2+ concentration ([Ca2+]i) oscillations in pituitary GH3 cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with l-arginine and nitro-l-arginine methyl ester (l-NAME), respectively. When NO synthesis was blocked with l-NAME (1 mM) [Ca2+]i, oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [Ca2+]i oscillations in response to the NO synthase (NOS) substrate l-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso- N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent Ca2+ channels (VDCC) blocker nimodipine (1 μM) or in Ca2+-free medium. Conversely, its effect was preserved when Ca2+ release from intracellular Ca2+ stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 μM) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 μM). These results suggest that NO induces the appearance of [Ca2+]i oscillations by determining Ca2+ influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated K+ channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating ( IDR) and ether-à-go-go-related gene ( ERG) hyperpolarization-evoked, deactivating K+ currents. Similar results were obtained when GH3 cells were treated with l-arginine. The present study suggests that in GH3 cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca2+]i oscillations through an inhibitory effect on IDR and on IERG.

Systemic and regional hemodynamics after nitric oxide synthase inhibition: role of a neurogenic mechanism

1994 ◽  
Vol 267 (1) ◽  
pp. R84-R88 ◽  
Author(s):  
M. Huang ◽  
M. L. Leblanc ◽  
R. L. Hester
Keyword(s):  
Nitric Oxide ◽  
Blood Pressure ◽  
Cardiac Output ◽  
No Synthase ◽  
Before And After ◽  
Regional Hemodynamics ◽  
Autonomic Blockade ◽  
Infusion Of Norepinephrine ◽  
Oxide Synthase

The study tested the hypothesis that the increase in blood pressure and decrease in cardiac output after nitric oxide (NO) synthase inhibition with N omega-nitro-L-arginine methyl ester (L-NAME) was partially mediated by a neurogenic mechanism. Rats were anesthetized with Inactin (thiobutabarbital), and a control blood pressure was measured for 30 min. Cardiac output and tissue flows were measured with radioactive microspheres. All measurements of pressure and flows were made before and after NO synthase inhibition (20 mg/kg L-NAME) in a group of control animals and in a second group of animals in which the autonomic nervous system was blocked by 20 mg/kg hexamethonium. In this group of animals, an intravenous infusion of norepinephrine (20-140 ng/min) was used to maintain normal blood pressure. L-NAME treatment resulted in a significant increase in mean arterial pressure in both groups. L-NAME treatment decreased cardiac output approximately 50% in both the intact and autonomic blocked animals (P < 0.05). Autonomic blockade alone had no effect on tissue flows. L-NAME treatment caused a significant decrease in renal, hepatic artery, stomach, intestinal, and testicular blood flow in both groups. These results demonstrate that the increase in blood pressure and decreases in cardiac output and tissue flows after L-NAME treatment are not dependent on a neurogenic mechanism.

Bitter stimuli induce Ca2+ signaling and CCK release in enteroendocrine STC-1 cells: role of L-type voltage-sensitive Ca2+ channels

2006 ◽  
Vol 291 (4) ◽  
pp. C726-C739 ◽  
Author(s):  
Monica C. Chen ◽  
S. Vincent Wu ◽  
Joseph R. Reeve ◽  
Enrique Rozengurt
Keyword(s):  
Endocrine Cells ◽  
Bitter Taste ◽  
Dependent Manner ◽  
Gi Tract ◽  
Intracellular Ca ◽  
Α Subunits ◽  
Ca2 Channels ◽  
Cck Release

We previously demonstrated the expression of bitter taste receptors of the type 2 family (T2R) and the α-subunits of the G protein gustducin (Gαgust) in the rodent gastrointestinal (GI) tract and in GI endocrine cells. In this study, we characterized mechanisms of Ca2+ fluxes induced by two distinct T2R ligands: denatonium benzoate (DB) and phenylthiocarbamide (PTC), in mouse enteroendocrine cell line STC-1. Both DB and PTC induced a marked increase in intracellular [Ca2+] ([Ca2+]i) in a dose- and time-dependent manner. Chelating extracellular Ca2+ with EGTA blocked the increase in [Ca2+]i induced by either DB or PTC but, in contrast, did not prevent the effect induced by bombesin. Thapsigargin blocked the transient increase in [Ca2+]i induced by bombesin, but did not attenuate the [Ca2+]i increase elicited by DB or PTC. These results indicate that Ca2+ influx mediates the increase in [Ca2+]i induced by DB and PTC in STC-1 cells. Preincubation with the L-type voltage-sensitive Ca2+ channel (L-type VSCC) blockers nitrendipine or diltiazem for 30 min inhibited the increase in [Ca2+]i elicited by DB or PTC. Furthermore, exposure to the L-type VSCCs opener BAY K 8644 potentiated the increase in [Ca2+]i induced by DB and PTC. Stimulation with DB also induced a marked increase in the release of cholecystokinin from STC-1 cells, an effect also abrogated by prior exposure to EGTA or L-type VSCC blockers. Collectively, our results demonstrate that bitter tastants increase [Ca2+]i and cholecystokinin release through Ca2+ influx mediated by the opening of L-type VSCCs in enteroendocrine STC-1 cells.

L-type Ca2+ channels in fetal and adult ovine cerebral arteries

2002 ◽  
Vol 282 (1) ◽  
pp. R131-R138 ◽  
Author(s):  
Arlin B. Blood ◽  
Yu Zhao ◽  
Wen Long ◽  
Lubo Zhang ◽  
Lawrence D. Longo
Keyword(s):  
Carotid Arteries ◽  
Cerebral Arteries ◽  
Bay K 8644 ◽  
Channel Density ◽  
Immunoblotting Assay ◽  
Intracellular Ca ◽  
Common Carotid Arteries ◽  
Fetal Vessels ◽  
Ca2 Channels

Recently, we reported that, whereas in cerebral arteries of the adult a majority of norepinephrine (NE)-induced increase in intracellular Ca2+ concentration ([Ca2+]i) comes from release of the sarcoplasmic reticulum (SR) Ca2+ stores, in the fetus the SR Ca2+ stores are relatively small, and NE-induced increase in [Ca2+]i results mainly from activation of plasma membrane L-type Ca2+ channels (20). In an effort to establish further the role of L-type Ca2+ channels in the developing cerebral arteries, we tested the hypothesis that, in the fetus, increased reliance on plasmalemmal L-type Ca2+ channels is mediated, in part, by increased L-type Ca2+ channel density. We used3H-labeled (+)isopropyl-4-(2,1,3-benzoxadiazol-4-y1)-1,4-dihydro-(2,6-dimethyl-5-methoxycarbonyl)pyridine-3-carboxylate (PN200–110, isradipine) to measure L-type Ca2+ channel density (Bmax) in the cerebral arteries, common carotid artery (CCA), and descending aortae of fetal (∼140 gestation days), newborn (7–10 days), and adult sheep. In the cerebral and common carotid arteries, Bmax values (fmol/mg protein) of fetuses and newborns were significantly greater than those of adults. Western immunoblotting assay also revealed that the density of L-type Ca2+ channel protein in the cerebral arteries and CCA was about twofold greater in the fetus than the adult. Finally, compared with the adult, fetal cerebral arteries demonstrated a significantly greater maximum tension and [Ca2+]i in response to stimulation with the L-type Ca2+ channel agonist Bay K 8644. In addition, Bay K 8644-stimulated fetal vessels demonstrated a maximal tension and [Ca2+]isimilar to that observed in response to stimulation with 10−4 NE. These results support the idea that fetal cerebrovascular smooth muscle relies more on extracellular Ca2+ and L-type Ca2+ channels for contraction than does the adult and that this increased reliance is mediated, in part, by greater L-type Ca2+ channel density. This may have important implications in the regulation of cerebral blood flow in the developing organism.

Release of nitric oxide and prostaglandin H2 to acetylcholine in skeletal muscle venules

1997 ◽  
Vol 272 (6) ◽  
pp. H2541-H2546 ◽  
Author(s):  
G. Dornyei ◽  
G. Kaley ◽  
A. Koller
Keyword(s):  
Nitric Oxide ◽  
Skeletal Muscle ◽  
Perfusion Pressure ◽  
Thromboxane A2 ◽  
No Synthase ◽  
Gracilis Muscle ◽  
Vasoactive Agents ◽  
Before And After ◽  
Higher Doses

The role of endothelium in regulating venular resistance is not well characterized. Thus we aimed to elucidate the endothelium-derived factors involved in the mediation of responses of rat gracilis muscle venules to acetylcholine (ACh) and other vasoactive agents. Changes in diameter of perfusion pressure (7.5 mmHg)- and norepinephrine (10(-6) M)-constricted venules (approximately 225 microns in diam) to cumulative doses of ACh (10(-9) to 10(-4) M) and sodium nitroprusside (SNP, 10(-9) to 10(-4) M), before and after endothelium removal or application of various inhibitors, were measured. Lower doses of ACh elicited dilations (up to 42.1 +/- 4.7%), whereas higher doses of ACh resulted in smaller dilations or even constrictions. Endothelium removal abolished both ACh-induced dilation and constriction. In the presence of indomethacin (2.8 x 10(-5) M), a cyclooxygenase blocker, or SQ-29548 (10(-6) M), a thromboxane A2-prostaglandin H2 (PGH2) receptor antagonist, higher doses of ACh caused further dilation (up to 72.7 +/- 7%) instead of constriction. Similarly, lower doses of arachidonic acid (10(-9) to 10(-6) M) elicited dilations that were diminished at higher doses. These reduced responses were, however, reversed to substantial dilation by SQ-29548. The nitric oxide (NO) synthase blocker, N omega-nitro-L-arginine (L-NNA, 10(-4) M), significantly reduced the dilation to ACh (from 30.6 +/- 5.5 to 5.4 +/- 1.4% at 10(-6) M ACh). In contrast, L-NNA did not affect dilation to SNP. Thus ACh elicits the release of both NO and PGH2 from the venular endothelium.

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