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.

Role of iPLA2 and store-operated channels in agonist-induced Ca2+ influx and constriction in cerebral, mesenteric, and carotid arteries

2008 ◽  
Vol 294 (3) ◽  
pp. H1183-H1187 ◽  
Author(s):  
Kristen M. Park ◽  
Mario Trucillo ◽  
Nicolas Serban ◽  
Richard A. Cohen ◽  
Victoria M. Bolotina
Keyword(s):  
Carotid Arteries ◽  
Cerebral Arteries ◽  
Present Evidence ◽  
Voltage Gated ◽  
Functional Inhibition ◽  
Intracellular Ca ◽  
Dependent Pathway ◽  
Secondary Activation

Store-operated channels (SOC) and store-operated Ca2+ entry are known to play a major role in agonist-induced constriction of smooth muscle cells (SMC) in conduit vessels. In microvessels the role of SOC remains uncertain, in as much as voltage-gated L-type Ca2+ (CaL2+) channels are thought to be fully responsible for agonist-induced Ca2+ influx and vasoconstriction. We present evidence that SOC and their activation via a Ca2+-independent phospholipase A2 (iPLA2)-mediated pathway play a crucial role in agonist-induced constriction of cerebral, mesenteric, and carotid arteries. Intracellular Ca2+ in SMC and intraluminal diameter were measured simultaneously in intact pressurized vessels in vitro. We demonstrated that 1) Ca2+ and contractile responses to phenylephrine (PE) in cerebral and carotid arteries were equally abolished by nimodipine (a CaL2+ inhibitor) and 2-aminoethyl diphenylborinate (an inhibitor of SOC), suggesting that SOC and CaL2+ channels may be involved in agonist-induced constriction of cerebral arteries, and 2) functional inhibition of iPLA2β totally inhibited PE-induced Ca2+ influx and constriction in cerebral, mesenteric, and carotid arteries, whereas K+-induced Ca2+ influx and vasoconstriction mediated by CaL2+ channels were not affected. Thus iPLA2-dependent activation of SOC is crucial for agonist-induced Ca2+ influx and vasoconstriction in cerebral, mesenteric, and carotid arteries. We propose that, on PE-induced depletion of Ca2+ stores, nonselective SOC are activated via an iPLA2-dependent pathway and may produce a depolarization of SMC, which could trigger a secondary activation of CaL2+ channels and lead to Ca2+ entry and vasoconstriction.

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.

TRPC4 forms store-operated Ca2+ channels in mouse mesangial cells

2004 ◽  
Vol 287 (2) ◽  
pp. C357-C364 ◽  
Author(s):  
Xiaoxia Wang ◽  
Jennifer L. Pluznick ◽  
Peilin Wei ◽  
Babu J. Padanilam ◽  
Steven C. Sansom
Keyword(s):  
Transient Receptor Potential ◽  
Mesangial Cells ◽  
Receptor Potential ◽  
Immunocytochemical Staining ◽  
Rt Pcr ◽  
Fura 2 ◽  
Intracellular Ca ◽  
Transient Receptor ◽  
Ca2 Channels

Studies were performed to identify the molecular component responsible for store-operated Ca2+ entry in murine mesangial cells (MMC). Because the canonical transient receptor potential (TRPC) family of proteins was previously shown to comprise Ca2+-selective and -nonselective cation channels in a variety of cells, we screened TRPC1–TRPC7 with the use of molecular methods and the fura 2 method to determine their participation as components of the mesangial store-operated Ca2+ (SOC) channel. Using TRPC-specific primers and RT-PCR, we found that cultured MMC contained mRNA for TRPC1 and TRPC4 but not for TRPC2, TRPC3, TRPC5, TRPC6, and TRPC7. Immunocytochemical staining of MMC revealed predominantly cytoplasmic expression of TRPC1 and plasmalemmal expression of TRPC4. The role of TRPC4 in SOC was determined with TRPC4 antisense and fura 2 ratiometric measurements of intracellular Ca2+ concentration ([Ca2+]i). SOC was measured as the increase in [Ca2+]i after extracellular Ca2+ was increased from <10 nM to 1 mM in the continued presence of thapsigargin. We found that TRPC4 antisense, which reduced plasmalemmal expression of TRPC4, inhibited SOC by 83%. Incubation with scrambled TRPC4 oligonucleotides did not affect SOC. Immunohistochemical staining identified expressed TRPC4 in the glomeruli of mouse renal sections. The results of RT-PCR performed to distinguish between TRPC4-α and TRPC4-β were consistent with expression of both isoforms in brain but with only TRPC4-α expression in MMC. These studies show that TRPC4-α may form the homotetrameric SOC in mouse mesangial cells.

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 l-Ca2+ channels in intestinal pacing in wild-type and W/WV mice

2005 ◽  
Vol 288 (3) ◽  
pp. G439-G446 ◽  
Author(s):  
Geoffrey Boddy ◽  
E. E. Daniel
Keyword(s):  
Smooth Muscle ◽  
Bay K 8644 ◽  
Wild Type ◽  
Uniform Contraction ◽  
Mouse Intestine ◽  
Cells Of Cajal ◽  
Channel Currents ◽  
Ca2 Channels ◽  
Frequency Gradient

Rhythmic contractions generating transit in the digestive tract are paced by a network of cells called interstitial cells of Cajal (ICC) found in the myenteric plexus (MP). ICC generate cyclic depolarizations termed “slow waves” that are passively transmitted to the smooth muscle to initiate contractions. The opening of l-Ca2+ channels are believed to be primarily responsible for the influx of calcium generating a contraction in smooth muscle. However, l-Ca2+ channels are not thought to be important in generating the pacing current found in ICC. Using intact segments of circular (CM) and longitudinal (LM) muscle from wild-type mice and mice lacking c-kit kinase (W/WV), we found that l-Ca2+ channel currents are required for pacing at normal frequencies to occur. Application of 1 μM nicardipine caused a significant decrease in contraction amplitude and frequency in LM and CM that was successfully blocked with BAY K 8644. Nicardipine also abolished the pacing gradient found throughout the intestines, resulting in a uniform contraction frequency of 30–40/minute. Stimulating l-Ca2+ channels with BAY K 8644 neither removed nor recovered the pacing gradient. W/WV mice, which lack ICC-MP, also exhibited a pacing gradient in LM. Application of nicardipine to LM segments of W/WV mouse intestine did not reduce pacing frequency, and in jejunum, resulted in a slight increase. BAY K 8644 did not affect pacing frequency in W/WV tissue. In conclusion, we found that l-Ca2+ channel activity was required for normal pacing frequencies and to maintain the pacing frequency gradient found throughout the intestines in wild-type but not in W/WV mouse intestine.

Role of Ca2+ channels in NE-induced increase in [Ca2+]iand tension in fetal and adult cerebral arteries

1999 ◽  
Vol 277 (1) ◽  
pp. R286-R294 ◽  
Author(s):  
Wen Long ◽  
Yu Zhao ◽  
Lubo Zhang ◽  
Lawrence D. Longo
Keyword(s):  
Plasma Membrane ◽  
Cerebral Arteries ◽  
Channel Blockers ◽  
Developmental Age ◽  
Intracellular Ca ◽  
Lanthanum Ion ◽  
Near Term ◽  
Dose Dependent Decrease ◽  
Nonpregnant Adult

In vascular smooth muscle, elevation of agonist-induced intracellular Ca2+ concentration ([Ca2+]i) occurs via both Ca2+ release from intracellular stores and Ca2+influx across the plasma membrane. In the cerebral vasculature of the fetus and adult the relative roles of these mechanisms have not been defined. To test the hypothesis that plasma membrane L-type and receptor-operated Ca2+ channels play a key role in NE-induced vasoconstriction via alterations in plasma membrane Ca2+ flux and that this may change with developmental age, we performed the following study. In main branch middle cerebral arteries (MCA) from near-term fetal (∼140 days) and nonpregnant adult sheep, we quantified NE-induced responses of vascular tension and [Ca2+]i(by use of fura 2) under standard conditions in response to several Ca2+ channel blockers and in response to zero extracellular Ca2+. In fetal and adult MCA, maximal NE-induced tensions (g) were 0.91 ± 0.12 ( n = 10) and 1.61 ± 0.13 ( n = 12), respectively. The pD2 values for NE-induced tension were both 6.0 ± 0.1, whereas the fetal and adult maximum responses (%Kmax) were 107 ± 16 and 119 ± 7, respectively. The fetal and adult pD2 values for NE-induced increase of [Ca2+]iwere 6.2 ± 0.1 and 6.4 ± 0.1, respectively, whereas maximum [Ca2+]iresponses were 81 ± 9 and 103 ± 15% of Kmax, respectively. After 10−5 M NE-induced contraction, nifedipine resulted in dose-dependent decrease in vessel tone and [Ca2+]iwith pIC50 values for fetal and adult tensions of 7.3 ± 0.1 and 6.6 ± 0.1, respectively ( P < 0.01; n = 4 each), whereas pIC50 for [Ca2+]iresponses were 7.2 ± 0.1 and 6.9 ± 0.1, respectively. The pIC50 values for tension for diltiazem and verapamil were somewhat lower but showed a similar relationship. The receptor-operated Ca2+ channel blocker 2-nitro-4 carboxyphenyl- N,N-diphenyl carbamate showed little effect on NE-induced vessel contractility or [Ca2+]i. In the absence of extracellular Ca2+ for 2 min, 10−5 M NE resulted in markedly attenuated responses of adult MCA tension and [Ca2+]ito 39 ± 7 and 73 ± 8% of control values ( n = 4). For fetal MCA, exposure to extracellular Ca2+concentration resulted in essentially no contractile or [Ca2+]iresponse ( n = 4). Similar blunting of NE-induced tension and [Ca2+]iwas seen in response to 10−3M lanthanum ion. These findings provide evidence to suggest that especially in fetal, but also in adult, ovine MCA, Ca2+ flux via L-type calcium channels plays a key role in NE-induced contraction. In contrast, Ca2+ flux via receptor-operated Ca2+ channels is of less importance. This developmental difference in the role of cerebrovascular plasma membrane Ca2+ channels may be an important association with increased Ca2+sensitivity of the fetal vessels.

Effect of acidosis on tension and [Ca2+]iin rat cerebral arteries: is there a role for membrane potential?

1998 ◽  
Vol 274 (2) ◽  
pp. H655-H662 ◽  
Author(s):  
Hong-Li Peng ◽  
Peter E. Jensen ◽  
Holger Nilsson ◽  
Christian Aalkjær
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Isometric Force ◽  
Cerebral Arteries ◽  
Bathing Solution ◽  
Hypercapnic Acidosis ◽  
Small Arteries ◽  
Intracellular Ca ◽  
Intracellular Microelectrodes

The cellular mechanism responsible for the reduction of tension in cerebral small arteries to acidosis is not known. In this study the role of smooth muscle intracellular Ca2+ concentration ([Ca2+]i) and membrane potential for the relaxation to acidosis was investigated in isolated rat cerebral small arteries. Isometric force was measured simultaneously with [Ca2+]i(fura 2) or with membrane potential (intracellular microelectrodes), and acidosis was induced by increasing[Formula: see text] or reducing[Formula: see text] of the bathing solution. Both hypercapnic and normocapnic acidosis were associated with a reduction of intracellular pH [measured with 2′,7′-bis-(carboxyethyl)-5 (and -6)-carboxyfluorescein], caused relaxation, and reduced [Ca2+]i. However, whereas hypercapnic acidosis caused hyperpolarization, normocapnic acidosis was associated with depolarization. It is concluded that a reduction of [Ca2+]iis in part responsible for the direct effect of the acidosis on the vascular smooth muscle both during normo- and hypercapnia. The mechanism responsible for the reduction of [Ca2+]idiffers between the hypercapnic and normocapnic acidosis, being partly explained by hyperpolarization during hypercapnic acidosis, whereas it is seen despite depolarization during normocapnic acidosis.

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.

ROLE OF ENDOTHELIUM AND VASOCONSTRICTOR PROSTANOIDS IN NOREPINEPHRINE-INDUCED VASOCONSTRICTION IN ISOLATED RAT COMMON CAROTID ARTERIES

2000 ◽  
Vol 22 (5) ◽  
pp. 543-554 ◽  
Author(s):  
Naoki Furuhashi ◽  
Seiji Miyazaki ◽  
Masanori Kamura ◽  
Shoichi Emura ◽  
Keigo Yasuda
Keyword(s):  
Carotid Arteries ◽  
Common Carotid Arteries ◽  
Isolated Rat
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