Role of l-Ca(2+) channels in intestinal pacing in wild-type and W/W(V) mice

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-Ca(2+) channels are believed to be primarily responsible for the influx of calcium generating a contraction in smooth muscle. However, l-Ca(2+) 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/W(V)), we found that l-Ca(2+) channel currents are required for pacing at normal frequencies to occur. Application of 1 muM 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-Ca(2+) channels with BAY K 8644 neither removed nor recovered the pacing gradient. W/W(V) mice, which lack ICC-MP, also exhibited a pacing gradient in LM. Application of nicardipine to LM segments of W/W(V) 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/W(V) tissue. In conclusion, we found that l-Ca(2+) 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/W(V) mouse intestine.     

Hyperlipidemia in mast cell-deficient W/WV mice

Approximately 70% of the W/WV mice lacking mast cells due to a genetic defect showed hypertriglyceridemia combined with hypercholesterolemia. Increases of various magnitudes in chylomicrons, very-low-density lipoprotein, and intermediate-density lipoprotein were observed in the plasma of W/WV mice compared to those in the plasma of congenic normal mice. The increase in these lipoproteins was seen even in normolipidemic W/WV mice. Activities of both lipoprotein lipase and hepatic triacylglycerol lipase in the plasma after heparin injection were markedly lower in the W/WV mice than in the congenic normal mice, although activities of both lipoprotein lipase in the heart and adipose tissue and hepatic triacylglycerol lipase in the liver were not decreased. These results suggest that the W/WV mice have genetic defects in one or more of the following: secretion of both lipases from their synthesising cells, transport to the endothelium, and anchoring to the endothelial surface. Heparin deficiency in these mice may be responsible for the impairment and, thereby, may partially contribute to the hyperlipidemia.     

Uterine histamine content in mast-cell deficient (W/WV) mice during experimentally induced deciduoma formation

This study examines the changes in uterine histamine content in mast-cell normal and deficient mice during deciduoma formation. Surgical trauma (e.g., sutures) and intraluminal injection of oil produce deciduoma formation in ovariectomized mast-cell normal (+/+) and mast-cell deficient (W/W^V) pseudopregnant mice that had previously received a single control ovary transplanted under the kidney capsule. Mast-cell normal (+/+) mice exhibit significant elevation of total uterine histamine content during both trauma and oil induced deciduoma formation. Mast-cell deficient (W/W^V) mice under conditions of similar deciduoma formation did not show any change in total uterine histamine content. These results indicate that the change in histamine observed in mast-cell normal mice during deciduoma formation is most likely of mast cell origin. The lack of any measurable change in uterine histamine in mast-cell deficient mice indicates that an increase of non-mast cell histamine is not a prerequisite for deciduoma formation.     

Effect of age, vitamin D, and calcium on the regulation of rat intestinal epithelial calcium channels

Transepithelial transport of calcium involves uptake at the apical membrane, movement across the cell, and extrusion at the basolateral membrane. Active vitamin D metabolites regulate the latter two processes by induction of calbindin D and the plasma membrane ATPase (calcium pump), respectively. The expression of calbindin D and the calcium pump declines with age in parallel with transepithelial calcium transport. The apical uptake of calcium is thought to be mediated by the recently cloned calcium channels-CaT1 (or ECaC2, TRPV6) and CaT2 (or ECaC1, TRPV5). The purpose of these studies was to determine whether there were age-related changes in intestinal calcium channel regulation and to identify the dietary factors responsible for their regulation. Young (2 months) and adult (12 months) rats were fed either a high calcium or low calcium diet for 4 weeks. The low calcium diet significantly increased duodenal CaT1 and CaT2 mRNA levels in both age groups, but the levels in the adult were less than half that of the young. The changes in calcium channel expression with age and diet were significantly correlated with duodenal calcium transport and with calbindin D levels. To elucidate the relative roles of serum 1,25(OH)2D3 and calcium in the regulation of calcium channel expression, young rats were fed diets containing varying amounts of calcium and vitamin D. Dietary vitamin D or exogenous 1,25(OH)2D3 more than doubled CaT1 mRNA levels, and this regulation was independent of dietary or serum calcium. These findings suggest that the apical calcium channels, along with calbindin and the calcium pump, may play a role in intestinal calcium transport and its modulation by age, dietary calcium, and 1,25(OH)2D3.     

Calcium, calcium channels, and calcium channel antagonists

Voltage-dependent Ca2+ channels are an important pathway for Ca2+ influx in excitable cells. They also represent an important site of action for a therapeutic group of agents, the Ca2+ channel antagonists. These drugs enjoy considerable use in the cardiovascular area including angina, some arrhythmias, hypertension, and peripheral vascular disorders. The voltage-dependent Ca2+ channels exist in a number of subclasses characterized by electrophysiologic, permeation, and pharmacologic criteria. The Ca2+ channel antagonists, including verapamil, nifedipine, and diltiazem, serve to characterize the L channel class. This channel class has been characterized as a pharmacologic receptor, since it possesses specific drug-binding sites for both antagonists and activators and it is regulated by homologous and heterologous influences. The Ca2+ channels of both voltage- and ligand-regulated classes are likely to continue to be major research targets for new drug design and action.     

Differential expression of T-type calcium channels in P/Q-type calcium channel mutant mice with ataxia and absence epilepsy

Mutations in P/Q-type calcium channels generate common phenotypes in mice and humans, which are characterized by ataxia, paroxysmal dyskinesia, and absence seizures. Subsequent functional changes of T-type calcium channels in thalamus are observed in P/Q-type calcium channel mutant mice and these changes play important roles in generation of absence seizures. However, the changes in T-type calcium channel function and/or expression in the cerebellum, which may be related to movement disorders, are still unknown. The leaner mouse exhibits severe ataxia, paroxysmal dyskinesia, and absence epilepsy due to a P/Q-type calcium channel mutation. We investigated changes in T-type calcium channel expression in the leaner mouse thalamus and cerebellum using quantitative real-time polymerase chain reaction (qRT-PCR) and quantitative in situ hybridization histochemistry (ISHH). qRT-PCR analysis showed no change in T-type calcium channel alpha 1G subunit (Cav3.1) expression in the leaner thalamus, but a significant decrease in alpha 1G expression in the whole leaner mouse cerebellum. Interestingly, quantitative ISHH revealed differential changes in alpha 1G expression in the leaner cerebellum, where the granule cell layer showed decreased alpha 1G expression while Purkinje cells showed increased alpha 1G expression. To confirm these observations, the granule cell layer and the Purkinje cell layer were laser capture microdissected separately, then analyzed with qRT-PCR. Similar to the observation obtained by ISHH, the leaner granule cell layer showed decreased alpha 1G expression and the leaner Purkinje cell layer showed increased alpha 1G expression. These results suggest that differential expression of T-type calcium channels in the leaner cerebellum may be involved in the observed movement disorders.     

Cell proliferation, calcium influx and calcium channels

Both increases in the basal cytosolic calcium concentration ([Ca²⁺]_cyt) and [Ca²⁺]_cyt transients play major roles in cell cycle progression, cell proliferation and division. Calcium transients are observed at various stages of cell cycle and more specifically during late G₁ phase, before and during mitosis. These calcium transients are mainly due to calcium release and reuptake by the endoplasmic reticulum (ER) and are observed over periods of hours in oocytes and mammalian cells. Calcium entry sustains the ER Ca²⁺ load and thereby helps to maintain these calcium transients for such a long period. Calcium influx also controls cell growth and proliferation in several cell types. Various calcium channels are involved in this process and the tight relation between the expression and activity of cyclins and calcium channels also suggests that calcium entry may be needed only at particular stages of the cell cycle. Consistent with this idea, the expression of l-type and T-type calcium channels and SOCE amplitude fluctuate along the cell cycle. But, as calcium influx regulates several other transduction pathways, the presence of a specific connection to trigger activation of proliferation and cell division in mammalian cells will be discussed in this review.     

Evidence for a sodium/calcium exchanger and voltage-dependent calcium channels in adipocytes

The objective of these studies is to identify and characterize Ca2+-transport systems that may be of potential importance in the action of Ca2+-mobilizing hormones in the adipocyte. Using the Ca2+-sensitive photoprotein, aequorin, [Ca2+]i was estimated to be 0.15 microM, assuming an intracellular [Mg2+] of 1 mM. Substitution of Na+ with choline+ caused a transient increase in [Ca2+]i which was inversely related to extracellular [Na+], consistent with operation of a mediated Na+-Ca2+ exchange system. The stoichiometry was 3Na+:Ca2+. Elevation of extracellular K+ caused an increase in [Ca2+]i that was blocked by the Ca2+ channel antagonist, diltiazem, by omitting extracellular Ca2+, or by substituting Sr2+ for Ca2+. These findings indicate the presence of an Na+-Ca2+ exchanger and voltage-sensitive Ca2+ channels in adipocytes.     

Endogenous photoproteins,calcium channels and calcium transients during metamorphosis in hydrozoans

Summary There are species of hydrozoans, Eutonina victoria, Mitrocomella polydiademata, and Phialidium gregarium whose eggs contain calcium-specific photoproteins. These cytoplasmic photoproteins are synthesized during oogenesis. During the cleavage stages of embryogenesis they are distributed to all of the cells of the developing planula larva. The amount of photoprotein slowly declines during the development of the planula larva, and markedly declines when the planula undergoes metamorphosis to become a polyp.Oocytes, unfertilized eggs, and fertilized eggs prior to the first cleavage do not produce light when treated with KCl. The ability to respond to KCl appears about the time of first cleavage, and is correlated with the appearance of active membrane responses. Both the KCl response and the action potentials will occur in sodium-free sea water, and both are inhibited by calcium channel blockers. These and other experiments suggest that voltage sensitive calcium channels first become active at about the time of first cleavage. These channels also appear on the same schedule in both unfertilized eggs and in enucleated egg fragments, which have been artificially activated with A23187.Developing planulae produce few or no spontaneous light responses before gastrulation. Later the frequency and magnitude of spontaneous light production increases presumably due to an increasing frequency and magnitude of calcium transients. Both the natural trigger of metamorphosis (bacteria) and an artificial trigger (CsCl) cause a conspicuous series of calcium transients. When these transients are inhibited by calcium channel blockers, metamorphosis is also inhibited.     

Decreased neutrophils and megakaryocytes in anemic mice of genotype W/W

The concentration of neutrophils and megakaryocytes was determined in the marrow of anemic mice of genotype W/W^v and their normal (+/+) litter mates. In all groups studied, the humerus of W/W^v mice contained significantly less neutrophils and megakaryocytes than did normal animals. Blood neutrophil concentration was less in all groups of W/W^v mice but in only one group which was the youngest group studied, did this value differ significantly from normal. The blood and marrow neutrophil response to endotoxin was similar in W/W^v and “+/+” animals. This suggests that the neutrophilic system of W/W^v mice responds to this stimulus in a relatively normal manner, much as their erythroid system responds to hypoxia, and androgens.     

Proteinuria of nonautoimmune origin in wild-type FVB/NJ mice

FVB/NJ mice frequently are used as transgenic hosts, but the suitability of this genetic background for transgenic and congenic models of systemic autoimmunity have not been reported. In this study, FVB/NJ mice were evaluated for the presence of serum autoantibodies and autoimmune kidney pathology. Previously unreported albuminuria was observed in aged female FVB/NJ mice; however, serum autoantibody testing, light microscopic evaluation of differentially stained renal sections, and evaluation of renal sections for immunoglobulin deposits revealed that the albuminuria was not of autoimmune etiology. Anecdotally, multiple characteristics of the FVB/NJ strain, including albuminuria, cholesterolemia, mild podocyte foot process effacement in aged female FVB/NJ kidneys and predisposition to enhanced Th2 immune responses, is reminiscent of human minimal change nephrotic syndrome (MCNS). We propose that mapping of genetic polymorphisms that are responsible for these traits in FVB/NJ mice may lead to increased understanding of mild nephrotic syndromes including MCNS and other proteinurias.     

Cysteine strings,calcium channels and synaptic transmission

Multidisciplinary studies have led to the discovery and characterization of cysteine string proteins (csps) in both Drosophila and Torpedo. Phenotypic analysis of csp mutants in Drosophila demonstrates a crucial role for csp in synaptic transmission. Expression studies of Torpedo csp (Tcsp) in Xenopus oocytes suggests that the protein has some role in the function of presynaptic Ca²⁺ channels. However, biochemical purification of Tcsp indicates that is associated with synaptic vesicles rather than with the plasma membrane of presynaptic terminals where Ca²⁺ channels reside. These results suggest a model in which csps serve as a link by which docked synaptic vesicles could modulate the activity of presynaptic Ca²⁺ channels.     

Single-channel gating and regulation of human L-type calcium channels in cardiomyocytes of transgenic mice

Overexpression of human cardiac L-type Ca(2+) channel pores (hCa(v)1.2) in mice causes heart failure. Earlier studies showed Ca(v)1.2-mRNA increase by 2.8-fold, but whole-cell current density enhancement by 相似文献   

Cations affect the rate of gating charge recovery in wild-type and W434F Shaker channels through a variety of mechanisms

In this study we examine the effects of ionic conditions on the gating charge movement in the fast inactivation-removed wild-type Shaker channel and its W434F mutant. Our results show that various ionic conditions influence the rate at which gating charge returns during repolarization following a depolarizing pulse. These effects are realized through different mechanisms, which include the regulation of channel closing by occupying the cavity, the modulation of transitions into inactivated states, and effects on transitions between closed states via a direct interaction with the channel's gating charges. In generating these effects the cations act from the different binding sites within the pore. Ionic conditions, in which conducting wild-type channels close at different rates, do not significantly affect the rate of charge recovery upon repolarization. In these conditions, channel closing is fast enough not to be rate-limiting in the charge recovery process. In the permanently P-inactivated mutant channel, however, channel closing becomes the rate-limiting step, presumably due to weakened ion-ion interactions inside the pore and a slower intrinsic rate of gate closure. Thus, variations in closing rate induced by different ions are reflected as variations in the rate of charge recovery. In 115 mM internal Tris(+) and external K(+), Cs(+), or Rb(+), low inward permeation of these ions can be observed through the mutant channel. In these instances, channel closing becomes slower than in Tris(+)(O)//Tris(+)(I) solutions showing resemblance to the wild-type channel, where higher inward ionic fluxes also retard channel closing. Our data indicate that cations regulate the transition into the inactivated states from the external lock-in site and possibly the deep site. The direct action of barium on charge movement is probably exerted from the deep site, but this effect is not very significant for monovalent cations.     

Regulation of megakaryocytes in W/Wv mice

W/Wv mice were injected with antiplatelet serum to produce thrombocytopenia or with platelet transfusions to induce thrombocytosis. The responses of their platelets and megakaryocytes were followed to determine if proliferative abnormalities of the megakaryocytic system would be detected. W/Wv mice responded normally to the stimulation from thrombocytopenia with rebound thrombocytosis, macromegakaryocytosis, and macrothrombocytosis. The megakaryocytes of these mice became smaller than normal in response to post-thrombocytopenic rebound thrombocytosis but not to transfusion-induced thrombocytosis. Thus, endogenous thrombocytosis appeared to be a more potent suppressor of megakaryocyte growth than exogenous. These results failed to reveal an effective abnormality of the thrombocytopoietic regulatory system of W/Wv mice in spite of their intrinsically reduced numbers of megakaryocytes and the well known defect of stem cell proliferation. Thrombocytopoietic regulation appeared, therefore, to occur mainly at the committed, rather then pluripotential, stem cell level, and normal responses of the platelet system were observed in spite of severe abnormalities at the pluripotential stem cell level.     

Pain perception in mice lacking the beta3 subunit of voltage-activated calcium channels

The importance of voltage-activated calcium channels in pain processing has been suggested by the spinal antinociceptive action of blockers of N- and P/Q-type calcium channels as well as by gene targeting of the alpha1B subunit (N-type). The accessory beta3 subunits of calcium channels are preferentially associated with the alpha1B subunit in neurones. Here we show that deletion of the beta3 subunit by gene targeting affects strongly the pain processing of mutant mice. We pinpoint this defect in the pain-related behavior and ascending pain pathways of the spinal cord in vivo and at the level of calcium channel currents and proteins in single dorsal root ganglion neurones in vitro. The pain induced by chemical inflammation is preferentially damped by deletion of beta3 subunits, whereas responses to acute thermal and mechanical harmful stimuli are reduced moderately or not at all, respectively. The defect results in a weak wind-up of spinal cord activity during intense afferent nerve stimulation. The molecular mechanism responsible for the phenotype was traced to low expression of N-type calcium channels (alpha1B) and functional alterations of calcium channel currents in neurones projecting to the spinal cord.     

Differential expression of T‐type calcium channels in P/Q‐type calcium channel mutant mice with ataxia and absence epilepsy

Mutations in P/Q‐type calcium channels generate common phenotypes in mice and humans, which are characterized by ataxia, paroxysmal dyskinesia, and absence seizures. Subsequent functional changes of T‐type calcium channels in thalamus are observed in P/Q‐type calcium channel mutant mice and these changes play important roles in generation of absence seizures. However, the changes in T‐type calcium channel function and/or expression in the cerebellum, which may be related to movement disorders, are still unknown. The leaner mouse exhibits severe ataxia, paroxysmal dyskinesia, and absence epilepsy due to a P/Q‐type calcium channel mutation. We investigated changes in T‐type calcium channel expression in the leaner mouse thalamus and cerebellum using quantitative real‐time polymerase chain reaction (qRT‐PCR) and quantitative in situ hybridization histochemistry (ISHH). qRT‐PCR analysis showed no change in T‐type calcium channel α1G subunit (Cav3.1) expression in the leaner thalamus, but a significant decrease in α1G expression in the whole leaner mouse cerebellum. Interestingly, quantitative ISHH revealed differential changes in α1G expression in the leaner cerebellum, where the granule cell layer showed decreased α1G expression while Purkinje cells showed increased α1G expression. To confirm these observations, the granule cell layer and the Purkinje cell layer were laser capture microdissected separately, then analyzed with qRT‐PCR. Similar to the observation obtained by ISHH, the leaner granule cell layer showed decreased α1G expression and the leaner Purkinje cell layer showed increased α1G expression. These results suggest that differential expression of T‐type calcium channels in the leaner cerebellum may be involved in the observed movement disorders. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005