L-Type calcium channels mediate water intake induced by angiotensin injection into median preoptic nucleus

Calcium ions are widely accepted as critically important in responses of neurons to a stimulus. We have show previously the central involvement of angiotensin II (ANGII) in water intake. This study determined whether voltage-dependent calcium channels are involved in ANGII-induced behavioral drinking implicating nitrergic mechanism. The antidipsogenic actions of L-type calcium channel antagonists nifedipine, on ANGII-induced drinking behavior were studied when it is injected into the median preoptic nucleus (MnPO). The influence of nitric oxide (NO) on nifedipine antidipsogenic action was also studied by utilizing the N-W-nitro-L-arginine methyl ester (L-NAME) a constitutive nitric oxide synthase inhibitor constitutive (cNOSI) and 7-nitroindazol (7-NIT) a specific neuronal nitric oxide synthase inhibitor (nNOSI) and L-arginine a NO donor. Rats 200-250 g, with cannulae implanted into MnPO, pre-treated into MnPO with either nifedipine, followed by ANGII, drank significantly less water than controls during the first 15 min after injection. However, L-NAME potentiated the dipsogenic effect of ANGII that is blocked by prior injection of nifedipine and L-arginine. 7-NIT injected prior to ANGII into MnPO also potentiated the dipsogenic effect of ANGII but with a less intensity than L-NAME that it is also blocked by prior injection of nifedipine. The results described in this paper provide evidence that calcium channels play important roles in the ANGII-induced behavioral water intake. The structures containing NO in the brain such as MnPO include both endothelial cells and neurons might be responsible for the influence of nifedipine on dipsogenic effect of ANGII. These data shows the correlation between L-type calcium channel and a free radical gas NO produced endogenously from amino acids L-arginine by endothelial and neuronal NO synthase in the control of ANGII-dipsogenic effect. This suggests that an L-type calcium channel participates in both short- and longer-term neuronal actions of ANGII by nitrergic way. (c) 2006 Elsevier B.V. All rights reserved.

Agelaia MP-I: A peptide isolated from the venom of the social wasp, Agelaia pallipes pallipes, enhances insulin secretion in mice pancreatic islets

Peptides isolated from animal venoms have shown the ability to regulate pancreatic beta cell function. Characterization of wasp venoms is important, since some components of these venoms present large molecular variability, and potential interactions with different signal transduction pathways. For example, the well studied mastoparan peptides interact with a diversity of cell types and cellular components and stimulate insulin secretion via the inhibition of ATP dependent K + (K ATP) channels, increasing intracellular Ca 2+ concentration. In this study, the insulin secretion of isolated pancreatic islets from adult Swiss mice was evaluated in the presence of synthetic Agelaia MP-I (AMP-I) peptide, and some mechanisms of action of this peptide on endocrine pancreatic function were characterized. AMP-I was manually synthesized using the Fmoc strategy, purified by RP-HPLC and analyzed using ESI-IT-TOF mass spectrometry. Isolated islets were incubated at increasing glucose concentrations (2.8, 11.1 and 22.2 mM) without (Control group: CTL) or with 10 μM AMP-I (AMP-I group). AMP-I increased insulin release at all tested glucose concentrations, when compared with CTL (P < 0.05). Since molecular analysis showed a potential role of the peptide interaction with ionic channels, insulin secretion was also analyzed in the presence of 250 μM diazoxide, a K ATP channel opener and 10 μM nifedipine, a Ca 2+ channel blocker. These drugs abolished insulin secretion in the CTL group in the presence of 2.8 and 11.1 mM glucose, whereas AMP-I also enhanced insulin secretory capacity, under these glucose conditions, when incubated with diazoxide and nifedipine. In conclusion, AMP-I increased beta cell secretion without interfering in K ATP and L-type Ca 2+ channel function, suggesting a different mechanism for this peptide, possibly by G protein interaction, due to the structural similarity of this peptide with Mastoparan-X, as obtained by modeling. © 2012 Elsevier Ltd.

Diltiazem como alternativa ao betabloqueador na angiotomografia de artérias coronárias

FUNDAMENTO: A redução da frequência cardíaca (FC) na angiografia por tomografia das artérias coronarianas (ATCCor) é fundamental para a qualidade de imagem. A eficácia dos bloqueadores de cálcio como alternativas para pacientes com contraindicações aos betabloqueadores não foi definida. OBJETIVOS: Comparar a eficácia na redução da FC e variabilidade RR do metoprolol e diltiazem na ATCCor. MÉTODOS: Estudo prospectivo, randomizado, aberto, incluiu pacientes com indicação clínica de ATCCor, em ritmo sinusal, com FC>70bpm e sem uso de agentes que interferissem com a FC. Cinquenta pacientes foram randomizados para grupos: metoprolol IV 5-15 mg ou até FC≤60 bpm(M), e diltiazem IV 0,25-0,60mg/kg ou até FC≤60 bpm (D). Pressão arterial (PA) e FC foram aferidas na condição basal, 1min, 3min e 5min após agentes, na aquisição e após ATCCor. RESULTADOS: A redução da FC em valores absolutos foi maior no grupo M que no grupo D (1, 3, 5min, aquisição e pós-exame). A redução percentual da FC foi significativamente maior no grupo M apenas no 1 min e 3 min após início dos agentes. Não houve diferença no 5 min, durante a aquisição e após exame. A variabilidade RR percentual do grupo D foi estatisticamente menor do que a do grupo M durante a aquisição (variabilidade RR/ FC média da aquisição). Um único caso de BAV, 2:1 Mobitz I, revertido espontaneamente ocorreu (grupo D). CONCLUSÃO: Concluímos que o diltiazem é uma alternativa eficaz e segura aos betabloqueadores na redução da FC na realização de angiografia por tomografia computadorizada das artérias coronarianas. (Arq Bras Cardiol. 2012; [online].ahead print, PP.0-0)

Effects of MK-801 and amphetamine treatments on allergic lung inflammatory response in mice

Glutamate acts as a neurotransmitter within the Central Nervous System (CNS) and modifies immune cell activity. In lymphocytes, NMDA glutamate receptors regulate intracellular calcium, the production of reactive oxygen species and cytokine synthesis. MK-801, a NMDA receptor open-channel blocker, inhibits calcium entry into mast cells, thereby preventing mast cell degranulation. Several lines of evidence have shown the involvement of NMDA glutamate receptors in amphetamine (AMPH)-induced effects. AMPH treatment has been reported to modify allergic lung inflammation. This study evaluated the effects of MK-801 (0.25mg/kg) and AMPH (2.0mg/kg), given alone or in combination, on allergic lung inflammation in mice and the possible involvement of NMDA receptors in this process. In OVA-sensitized and challenged mice, AMPH and MK-801 given alone decreased cellular migration into the lung, reduced IL-13 and IL10 levels in BAL supernatant, reduced ICAM-1 and L-selectin expression in granulocytes in the BAL and decreased mast cell degranulation. AMPH treatment also decreased IL-5 levels. When both drugs were administered, treatment with MK-801 reversed the decrease in the number of eosinophils and neutrophils induced by AMPH in the BAL of OVA-sensitized and challenged mice as well as the effects on the expression of L-selectin and ICAM-1 in granulocytes, the IL-10, IL-5 and IL-13 levels in BAL supernatants and increased mast cell degranulation. At the same time, treatment with MK-801, AMPH or with MK-801+AMPH increased corticosterone serum levels in allergic mice. These results are discussed in light of possible indirect effects of AMPH and MK-801 via endocrine outflow from the CNS (i.e., HPA-axis activity) to the periphery and/or as a consequence of the direct action of these drugs on immune cell activity, with emphasis given to mast cell participation in the allergic lung response of mice.

Aktivitätsabhängige Kontrolle der Apoptose in neonatalen kortikalen Neuronen und Etablierung eines Biosensors zur Echtzeitanalyse der Caspase-3-Aktivierung

Die Apoptose spielt eine entscheidende Rolle während der normalen Entwicklung des zentralen Nervensystems. Elektrische Aktivität und die Versorgung mit trophischen Faktoren sind ausschlaggebend für das Überleben von Neuronen. Um zu untersuchen, welche zellulären Prozesse die aktivitätsabhängige Apoptose in organotypischen Schnittkulturen des neugeborenen Neokortex beeinflussen, wurde in der vorliegenden Arbeit immunzytochemisch das Auftreten aktivierter Caspase-3, nach pharmakologischer Beeinflussung von Ionenkanälen und membranständigen Rezeptoren analysiert. Die Unterdrückung neuronaler Aktivität durch den Natriumionenkanalblocker TTX führte zu einem signifikanten Verlust kortikaler Neuronen. Ein ähnlicher Anstieg der Zahl apoptotischer Neurone konnte durch Applikation von Antagonisten ionotroper Glutamatrezeptoren, GABAA-Rezeptoren oder neuronaler Gap Junctions induziert werden. Jedoch konnte bei einigen Antagonisten die apoptosefördernde Wirkung erst nach längerer Einwirkung beobachtet werden. Im Weiteren wurde eine Methode etabliert, mit deren Hilfe eine Echtzeitanalyse der Apoptose kortikaler Neurone unter dem Entzug trophischer Faktoren in Gegenwart unterschiedlicher extrazellulärer Kaliumkonzentrationen ermöglicht wurde. Dazu wurden dissoziierte kortikale Kulturen mit dem pCaspase3-sensor Vektor transfiziert. Das durch dieses Plasmid codierte fluoreszente Protein wird Caspase-3 abhängig gespalten. In der vorliegenden Arbeit konnte gezeigt werden, dass der Caspase3-sensor spezifisch für die Aktivierung der Caspase-3 ist, und dass die Überlebensfähigkeit der transfizierten Neurone durch das Transfektionsprotokoll nicht beeinflusst wird.

Trpv6 mediates intestinal calcium absorption during calcium restriction and contributes to bone homeostasis

Energy-dependent intestinal calcium absorption is important for the maintenance of calcium and bone homeostasis, especially when dietary calcium supply is restricted. The active form of vitamin D, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is a crucial regulator of this process and increases the expression of the transient receptor potential vanilloid 6 (Trpv6) calcium channel that mediates calcium transfer across the intestinal apical membrane. Genetic inactivation of Trpv6 in mice (Trpv6(-/-)) showed, however, that TRPV6 is redundant for intestinal calcium absorption when dietary calcium content is normal/high and passive diffusion likely contributes to maintain normal serum calcium levels. On the other hand, Trpv6 inactivation impaired the increase in intestinal calcium transport following calcium restriction, however without resulting in hypocalcemia. A possible explanation is that normocalcemia is maintained at the expense of bone homeostasis, a hypothesis investigated in this study. In this study, we thoroughly analyzed the bone phenotype of Trpv6(-/-) mice receiving a normal (approximately 1%) or low (approximately 0.02%) calcium diet from weaning onwards using micro-computed tomography, histomorphometry and serum parameters. When dietary supply of calcium is normal, Trpv6 inactivation did not affect growth plate morphology, bone mass and remodeling parameters in young adult or aging mice. Restricting dietary calcium had no effect on serum calcium levels and resulted in a comparable reduction in bone mass accrual in Trpv6(+/+) and Trpv6(-/-) mice (-35% and 45% respectively). This decrease in bone mass was associated with a similar increase in bone resorption, whereas serum osteocalcin levels and the amount of unmineralized bone matrix were only significantly increased in Trpv6(-/-) mice. Taken together, our findings indicate that TRPV6 contributes to intestinal calcium transport when dietary calcium supply is limited and in this condition indirectly regulates bone formation and/or mineralization.

Mechanism of Ca(v)1.2 channel modulation by the amino terminus of cardiac beta2-subunits

L-type calcium channels are composed of a pore, alpha1c (Ca(V)1.2), and accessory beta- and alpha2delta-subunits. The beta-subunit core structure was recently resolved at high resolution, providing important information on many functional aspects of channel modulation. In this study we reveal differential novel effects of five beta2-subunits isoforms expressed in human heart (beta(2a-e)) on the single L-type calcium channel current. These splice variants differ only by amino-terminal length and amino acid composition. Single-channel modulation by beta2-subunit isoforms was investigated in HEK293 cells expressing the recombinant L-type ion conducting pore. All beta2-subunits increased open probability, availability, and peak current with a highly consistent rank order (beta2a approximately = beta2b > beta2e approximately = beta2c > beta2d). We show graded modulation of some transition rates within and between deep-closed and inactivated states. The extent of modulation correlates strongly with the length of amino-terminal domains. Two mutant beta2-subunits that imitate the natural span related to length confirm this conclusion. The data show that the length of amino termini is a relevant physiological mechanism for channel closure and inactivation, and that natural alternative splicing exploits this principle for modulation of the gating properties of calcium channels.

[Treatment of hypertension in type 2 diabetes mellitus--2002 update]

Arterial hypertension and diabetes are potent independent risk factors for cardiovascular, cerebral, renal and peripheral (atherosclerotic) vascular disease. The prevalence of hypertension in diabetic individuals is approximately twice that in the non-diabetic population. Diabetic individuals with hypertension have a greater risk of macrovascular and microvascular disease than normotensive diabetic individuals. Hypertension is a major contributor to morbidity and mortality in diabetes, and should be recognized and treated early. Type 2 diabetes and hypertension share certain risk factors such as overweight, visceral obesity, and possibly insulin resistance. Life-style modifications (weight reduction, exercise, limitation of daily alcohol intake, stop smoking) are the foundation of hypertension and diabetes management as the definitive treatment or adjunctive to pharmacological therapy. Additional pharmacological therapy should be initiated when life-style modifications are unsuccessful or hypertension is too severe at the time of diagnosis. All classes of antihypertensive drugs are effective in controlling blood pressure in diabetic patients. For single-agent therapy, ACE-inhibitors, angiotensin receptor blocker, beta-blockers, and diuretics can be recommended. Because of concerns about the lower effectiveness of calcium channel blockers in decreasing coronary events and heart failure and in reducing progression of renal disease in diabetes, it is recommended to use these agents as second-line drugs for patients who cannot tolerate the other preferred classes or who require additional agents to achieve the target blood pressure. The choice depends on the patients specific treatment indications since each of these drugs have potential advantages and disadvantages. In patients with microalbuminuria or clinical nephropathy, both ACE-inhibitors and angiotensin receptor blockers are considered first line therapy for the prevention of and progression of nephropathy. Since treatment is usually life-long, cost effectiveness should be included in treatment evaluation.

Active intestinal calcium transport in the absence of transient receptor potential vanilloid type 6 and calbindin-D9k

To study the role of the epithelial calcium channel transient receptor potential vanilloid type 6 (TRPV6) and the calcium-binding protein calbindin-D9k in intestinal calcium absorption, TRPV6 knockout (KO), calbindin-D9k KO, and TRPV6/calbindin-D(9k) double-KO (DKO) mice were generated. TRPV6 KO, calbindin-D9k KO, and TRPV6/calbindin-D9k DKO mice have serum calcium levels similar to those of wild-type (WT) mice ( approximately 10 mg Ca2+/dl). In the TRPV6 KO and the DKO mice, however, there is a 1.8-fold increase in serum PTH levels (P < 0.05 compared with WT). Active intestinal calcium transport was measured using the everted gut sac method. Under low dietary calcium conditions there was a 4.1-, 2.9-, and 3.9-fold increase in calcium transport in the duodenum of WT, TRPV6 KO, and calbindin-D9k KO mice, respectively (n = 8-22 per group; P > 0.1, WT vs. calbindin-D9k KO, and P < 0.05, WT vs. TRPV6 KO on the low-calcium diet). Duodenal calcium transport was increased 2.1-fold in the TRPV6/calbindin-D9k DKO mice fed the low-calcium diet (P < 0.05, WT vs. DKO). Active calcium transport was not stimulated by low dietary calcium in the ileum of the WT or KO mice. 1,25-Dihydroxyvitamin D3 administration to vitamin D-deficient null mutant and WT mice also resulted in a significant increase in duodenal calcium transport (1.4- to 2.0-fold, P < 0.05 compared with vitamin D-deficient mice). This study provides evidence for the first time using null mutant mice that significant active intestinal calcium transport occurs in the absence of TRPV6 and calbindin-D9k, thus challenging the dogma that TRPV6 and calbindin-D9k are essential for vitamin D-induced active intestinal calcium transport.

The characterization of an epithelial chloride channel and purification of a channel component

A membrane fraction (M$\sb{\rm PS}$), enriched in Cl$\sp-$ channels, has been isolated from bovine tracheal epithelia and renal cortex homogenates by hydrophobic chromatography. The tracheal fraction shows a 37 fold enrichment of Cl$\sp-$ channels over crude tracheal homogenates by net Cl$\sp-$ measurements in membrane vesicles. Alkaline phosphatase and (Na$\sp+$ + K$\sp+$)-ATPase are not found in these membranes, suggesting that they are not apical or basolateral plasma membranes. The M$\sb{\rm PS}$ fraction exhibits a protein profile unlike that of other membrane fractions with major proteins of 200 kDa and 42 kDa, proteins of 30 to 35 kDa, and lesser amounts of other proteins. Reconstitution of M$\sb{\rm PS}$ fractions from both trachea and kidney into planar lipid bilayers demonstrates the presence of a single type of anion channel. The current-voltage relationship of this channel is linear with a slope conductance of 84 pS in symmetrical 400 mM KCl, and is identical to that of the predominant anion channel observed in tracheal apical membranes under similar conditions (Valdivia, Dubinsky, and Coronado. Science, 1988). In addition, the voltage dependence, selectivity sequence of Cl$\sp- >$ Br$\sp- \ge$ I$\sp-$, and inhibition by low concentrations of the Cl$\sp-$ channel blocker, DIDS, correspond to those of the predominant apical membrane channel. Thus, although the M$\sb{\rm PS}$ fraction appears to be of subcellular origin, it may be functionally related to an apical membrane Cl$\sp-$ permeability. When renal M$\sb{\rm PS}$ membranes were treated with the detergent octyl-glucoside (OG, 2%) and centrifuged, the supernatant, sM$\sb{\rm PS}$, showed a 2 to 7-fold enrichment in specific Cl$\sp-$ flux activity compared with the detergent treated M$\sb{\rm PS}$. These solubilized proteins were then size fractionated on a Superose 12 HPLC gel filtration column, followed by fractionation on a Mono Q HPLC anion exchange column. Fractions that eluted in high salt consistently exhibited significant Cl$\sp-$ flux activity. These fractions had protein profiles consisting of a major band at 34 kDa, a band at 66 kDa, and variable faint bands. Fractions eluting in lower salt had protein profiles consisting of a single band at 34 kDa, and often had little or no Cl$\sp-$ flux activity. However, co-reconstitution of the low salt, solely 34 kDa protein-containing Mono Q fractions with sM$\sb{\rm PS}$ resulted in an enhancement of flux activity compared to that of sM$\sb{\rm PS}$ reconstituted alone. Flux assays of active Mono Q fractions showed that the channel retained its DIDS sensitivity. Applying sM$\sb{\rm PS}$ to a DIDS-affinity column and eluting with salt resulted in fractions with protein profiles again consisting of at least one major band at 34 kDa, a band at 66 kDa, and variable faint bands. Co-reconstitution with sM$\sb{\rm PS}$ again resulted in an enhancement of activity. Thus, the 34 kDa protein appears to be a component of the M$\sb{\rm PS}$ Cl$\sp-$ channel. ^

A heteromeric potassium channel involved in the modulation of the plasma membrane potential is essential for the survival of African trypanosomes.

Discovery of novel drug targets may lead to improved treatment of trypanosomiasis. We characterize here 2 gene products of Trypanosoma brucei that are essential for the growth of bloodstream form (BSF) parasites, as shown by RNA interference (RNAi)-mediated down-regulation of the individual mRNAs. The primary sequences of the 2 proteins--protein encoded by gene Tb927.1.4450 (TbK1) and protein encoded by gene Tb927.9.4820 (TbK2)--indicate that both belong to the family of putative, Ca(2+)-activated potassium channels. The proteins were expressed in Xenopus laevis oocytes and their functions investigated by use of electrophysiological techniques. Only combined expression of TbK1 and TbK2 results in the formation of sizeable currents, indicating that these proteins probably assemble into a heteromeric ion channel. The current mediated by this channel shows little time and voltage dependence and displays a permeability ratio of K(+)/Na(+) of >20. The known potassium channel blocker barium inhibits this channel with a half-maximal inhibitory concentration (IC50) of 98 ± 15 μM. The membrane potential of trypanosomes was measured with a fluorescent dye. Individual RNAi-mediated down-regulation of TbK1 or TbK2 eliminates a potassium conductance in the plasma membrane of BSF. Thus, this heteromeric potassium channel is involved in the modulation of the plasma membrane potential and represents a novel drug target in T. brucei.

Modulation of high voltage -activated calcium channels by phosphorylation

High voltage-activated (HVA) calcium channels from rat brain and rabbit heart are expressed in Xenopus laevis oocytes and their modulation by protein kinases studied. A subtype of the HVA calcium current expressed by rat brain RNA is potentiated by the phospholipid- and calcium-dependent protein kinase (PKC). The calcium channel clone $\alpha\sb{\rm1C}$ from rabbit heart is modulated by the cAMP-dependent protein kinase (PKA), and another factor present in the cytoplasm.^ The HVA calcium channels from rat brain do not belong to the L-type subclass since they are insensensitive to dihydropyridine (DHP) agonists and antagonists. The expressed currents do contain a N-type fraction which is identified by inactivation at depolarized potentials, and a P-type fraction as defined by blockade by the venom of the funnel web spider Agelenopsis Aperta. A non N-type fraction of this current is potentiated, by using phorbol esters to activate PKC. This residual fraction of current resembles the newly described Q-type channel from cerebellar granule cells in its biophysical properties, and potentiation by activation of PKC.^ The $\alpha\sb{\rm1C}$ clone from rabbit heart is expressed in oocytes and single-channel currents are measured using the cell-attached and cell-excised patch clamp technique. The single-channel current runs down within two minutes after patch excision into normal saline bath solution. The catalytic subunit of PKA + MgATP is capable of reversing this rundown for over 15 minutes. There also appears to be an additional factor present in the cytoplasm necessary for channel activity as revealed in experiments where PKA failed to prevent rundown.^ These data are important in that these types of channels are involved in synaptic transmission at many different types of synapses. The mammalian synapse is not accessible for these types of studies, however, the oocyte expression system allows access to HVA calcium channels for the study of their modulation by phosphorylation. ^

A release mechanism for stored ATP in ocular ciliary epithelial cells

Purines can modify ciliary epithelial secretion of aqueous humor into the eye. The source of the purinergic agonists acting in the ciliary epithelium, as in many epithelial tissues, is unknown. We found that the fluorescent ATP marker quinacrine stained rabbit and bovine ciliary epithelia but not the nerve fibers in the ciliary bodies. Cultured bovine pigmented and nonpigmented ciliary epithelial cells also stained intensely when incubated with quinacrine. Hypotonic stimulation of cultured epithelial cells increased the extracellular ATP concentration by 3-fold; this measurement underestimates actual release as the cells also displayed ecto-ATPase activity. The hypotonically triggered increase in ATP was inhibited by the Cl−-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) in both cell types. In contrast, the P-glycoprotein inhibitors tamoxifen and verapamil and the cystic fibrosis transmembrane conductance regulator (CFTR) blockers glybenclamide and diphenylamine-2-carboxylate did not affect ATP release from either cell type. This pharmacological profile suggests that ATP release is not restricted to P-glycoprotein or the cystic fibrosis transmembrane conductance regulator, but can proceed through a route sensitive to NPPB. ATP release also was triggered by ionomycin through a different NPPB-insensitive mechanism, inhibitable by the calcium/calmodulin-activated kinase II inhibitor KN-62. Thus, both layers of the ciliary epithelium store and release ATP, and purines likely modulate aqueous humor flow by paracrine and/or autocrine mechanisms within the two cell layers of this epithelium.

Antisense oligonucleotides against rat brain α1E DNA and its atrial homologue decrease T-type calcium current in atrial myocytes

Low voltage-activated, or T-type, calcium currents are important regulators of neuronal and muscle excitability, secretion, and possibly cell growth and differentiation. The gene (or genes) coding for the pore-forming subunit of low voltage-activated channel proteins has not been unequivocally identified. We have used reverse transcription–PCR to identify partial clones from rat atrial myocytes that share high homology with a member of the E class of calcium channel genes. Antisense oligonucleotides targeting one of these partial clones (raE1) specifically block the increase in T-current density that normally results when atrial myocytes are treated with insulin-like growth factor 1 (IGF-1). Antisense oligonucleotides targeting portions of the neuronal rat α1E sequence, which are not part of the clones detected in atrial tissue, also block the IGF-1-induced increase in T-current, suggesting that the high homology to α1E seen in the partial clone may be present in the complete atrial sequence. The basal T-current expressed in these cells is also blocked by antisense oligonucleotides, which is consistent with the notion that IGF-1 up-regulates the same gene that encodes the basal current. These results support the hypothesis that a member of the E class of calcium channel genes encodes a low voltage-activated calcium channel in atrial myocytes.

Calcium-dependent Clustering of Inositol 1,4,5-Trisphosphate Receptors

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP3), which operates as an InsP3-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor (FcεR1) leads to activation of phospholipase C γ isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways, release of InsP3-sensitive intracellular Ca2+ stores, and a sustained phase of Ca2+ influx. These events are accompanied by a redistribution of type II InsP3 receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP3 receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP3 receptor clustering occurs within 5–10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 μM, and maximal clustering responses are dependent on the presence of extracellular calcium. InsP3 receptor aggregation may be a characteristic cellular response to Ca2+-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4–2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36M3R cells. Stimulation of these three cell types leads to a reduction in InsP3 receptor levels only in AR4–2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP3 receptors may contribute to the previously observed changes in affinity for InsP3 in the presence of elevated Ca2+ and/or may establish discrete regions within refilled stores with varying capacity to release Ca2+ when a subsequent stimulus results in production of InsP3.