The relationship between insulin resistance and abnormalities of cellular calcium metabolism and cell membrane abnormalities in patients with essential hypertension

OBJECTIVE: To investigate the relationship between the insulin resistance (IR) and the abnormalities of cellular calcium metabolism and cell membrane in patients with essential hypertension. METHODS: Plasma insulin and glucose before and during OGTT, membrane ATPase activity, cholesterol (C) and phospholipid (P), lipid peroxides (MDA), intracellular total calcium concentration [(Ca2+)i] and calcium binding capacity of inner surface of membranes were determined in 26 normotensive (NT) subjects, 17 hypertensives (HT), and 16 hypertensives associated with IR (HT + IR). RESULTS: The HT + IR group had higher levels of plasma glucose and insulin than that in the NT group and HT group with significant differences. The HT + IR group consistently demonstrated depressed activity of each Na(+)-K(+)-ATPase and Ca(2+)-ATPase studied with significant differences when compared with the NT group, but no difference was found if compared with the HT group. There was a higher [(Ca2+)i] in the HT + IR group than those in the NT group and even in the HT group with significant differences, respectively. Insulin sensitivity in the HT + IR group was significantly lower than that in both groups of HT and NT, respectively. The membrane C/P molar ratio and MDA content in the HT + IR group and HT group were significantly higher than those in the NT group, respectively. CONCLUSIONS: It is likely that there is a causal relation between IR and abnormalities of cellular calcium metabolism and membrane lipids in patients with essential hypertension.     

Calcium solubilization and retention in the gastrointestinal tract in chicks (Gallus domesticus) as a function of gastric acid secretion inhibition and of calcium carbonate particle size

In chicks, immature pullets and laying hens, the inhibition of gastric acid secretion by omeprazole, an H+,K(+)-transporting ATPase (EC 3.6.1.36) inhibitor, greatly increased proventricular and gizzard pH values. Consequently, gizzard soluble Ca concentration deceased and the insoluble Ca fraction increased. Inhibition of acid secretion increased duodenal pH values in immature pullets and laying hens but not in chicks. Duodenal soluble and ionic Ca concentrations were lowered by gastric acid inhibition in chicks and to a larger extent in immature pullets and laying hens. The use of Ca of coarse particle size increased the gizzard insoluble Ca fraction in chicks and pullets. However, it did not influence its soluble Ca fraction in chicks but tended to reinforce the negative effect of omeprazole on soluble Ca in the gizzard and duodenum of chicks and laying hens. Coarse particles of Ca led to an increase in gizzard and duodenal soluble Ca at the end of eggshell calcification in laying hens. An enhancement in the level of Ca in the diet from 10 to 36 g/kg increased gizzard soluble Ca and duodenal soluble and ionic Ca concentrations in immature and adult hens. Intestinal Ca retention and bone mineralization was unaffected by gastric acid inhibition in chicks but were largely diminished by the use of coarse particles of Ca. Gastric acid inhibition was associated in laying hens with decreased Ca retention to a small extent and with reduced eggshell quality. These observations confirm that gastric acid secretion is of importance for CaCO3 solubilization but question its role as a prerequisite for intestinal Ca retention in chicks and even in hens fed on a high Ca diet.     

Developmental features of human striatal tissue transplanted in a rat model of Huntington''s disease

The actions of the novel calcium (Ca2+) channel antagonist mibefradil (Ro 40-5967), a selective T-type channel blocker in myocardium, were investigated in embryonic rat spinal motoneurones maintained in culture. Whole-cell currents were recorded with the patch-clamp technique. Motoneurones displayed transient, low-voltage-activated (LVA) and, more sustained, high-voltage-activated (HVA) Ca2+ currents. The LVA currents were small and preferentially blocked by amiloride and low doses of nickel. Most of the HVA Ca2+ current flowed through N-type Ca2+ channels, while L-, and P/Q-type channels represented a smaller fraction. Mibefradil caused a rapid and reversible dose-dependent block of inward Ca2+ channel currents. Inhibition was nearly complete at 10 microM, suggesting mibefradil blockade of all subclasses of Ca2+ channels. The IC50 was approximately 1.4 microM on currents measured at 0 mV, from a holding potential of -90 mV. Inhibition of LVA Ca2+ current occurred over the same contraction range. Slow tail currents induced by the dihydropyridine agonist Bay K 8644 were also blocked by mibefradil, although with a slightly lower potency (IC50 = 3.4 microM). These broad inhibitory effects of mibefradil on Ca2+ influx were also supported by the strong inhibition of depolarization-induced intracellular calcium transients, measured from Indo-1 loaded motoneurones imaged with confocal microscopy. We conclude that mibefradil has potent blocking effects on Ca2+ channels in mammalian motoneurones. We hypothesize that therapeutic and pharmacological effects of mibefradil may involve actions on Ca2+ channels other than type T.     

RH不加钙处理对SCM435冷锻钢D类和Ds类夹杂物的影响

SCM435钢的生产流程为80 t BOF-LF-RH-280 mm×325 mm坯连铸。LF终点精炼渣成分为（/%）:45～55CaO,10～15SiO₂,20～30Al₂O₃,∑（FeO+MnO）≤1%。分析了RH加钙（0.0013%Ca）和RH不加钙（0.0002%Ca）对Φ13 mm盘条中D和Ds夹杂物的影响。结果表明,RH不加钙处理工艺夹杂物最大尺寸为7.65μm,Ds≤0.5级合格率为100%;RH加钙处理工艺夹杂物最大尺寸为25.68μm,Ds≤0.5级合格率为95%。在数量控制方面,RH不加钙处理工艺夹杂物指数由RH加钙工艺的0.72降至0.68,D类≤1.0合格率由RH加钙工艺的30%提高至75%;RH不加钙处理工艺夹杂物主要为MgO·Al₂O₃,少量钙铝酸盐夹杂,RH加钙工艺为镁铝尖晶石、钙铝酸盐和CaS多相夹杂。因此,在脆性D类和Ds类夹杂物尺寸、数量和类型控制上,RH不加钙处理工艺改善效果明显     

Arginine vasotocin receptor in the vagina of the oviduct of the hen

The presence of receptor for arginine vasotocin (AVT) in the vagina of the oviduct of the hen was demonstrated by the use of radioligand binding assays on membrane fractions of the tissue. The binding to [125I]AVT was highly competitive with unlabeled AVT. Scatchard analysis revealed that the binding sites are of a single class. The equilibrium dissociation constant (Kd) was 0.48+/-0.05 nM (x+/-SEM; n = 6) in laying hens holding a hard-shelled egg in the uterus (shell gland) and 1.01+/-0.02 nM (n = 6) in nonlaying hens. The maximum binding capacity (Bmax) was 0.41+/-0.04 pmol/mg protein (n = 6) in laying hens and 0.81+/-0.01 pmol/mg protein (n = 6) in nonlaying hens. The Kd value of the laying hens varied from 0.39 to 1.20 nM during an oviposition cycle, showing an increase just prior to oviposition, and the Bmax value also varied from 0.30 to 0.66 pmol/mg protein, showing a gradual increase after 6 h prior to oviposition. In the nonlaying hen, both values were almost constant during a 24-h day. The changes in the binding affinity and capacity of AVT receptor of the vagina may be related to oviposition in the hen.     

Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pHi) and intracellular Ca2+ activity ([Ca2+]i) of HT29 cells was investigated microspectrofluorimetrically using pH- and Ca2+- sensitive fluorescent indicators, [i.e. 2', 7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (Vm) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pKa value. Trimethylamine (20 mmol/l) increased pHi by 0.78 +/- 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pHi and a slow and incomplete recovery: pHi stayed 0.26 +/- 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pHi. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca2+]i transient which reached a peak value within 10-25 s and then slowly declined to a [Ca2+]i plateau. The initial Delta[Ca2+]i induced by trimethylamine (20 mmol/l) was 160 +/- 15 nmol/l (n = 17). The [Ca2+]i peak was independent of the Ca2+ activity in the bath solution, but the [Ca2+]i plateau was significantly lower under Ca2+-free conditions and could be immediately interrupted by application of CO2 (10%; n = 6), a manoeuvre to acidify pHi in HT29 cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca2+ stores completely abolished this [Ca2+]i transient. Tetramethylamine led to higher [Ca2+]i changes than the other amines tested and only this transient could be completely blocked by atropine (10(-6) mol/l). Trimethylamine (20 mmol/l) hyperpolarized Vm by 22.5 +/- 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 +/- 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pHi changes, release Ca2+ from intracellular, inositol-1,4, 5-trisphosphate-sensitive Ca2+ stores and increase Ca2+ influx into HT29 cells. The latter may be related to both the store depletion and the hyperpolarization.     

Q345R 抗酸钢 120 t BOF-LF-RH-260 mm x 2 70 mm CC冶炼工艺实践

分析了"BOF-LF-RH-连铸"生产Q345R抗酸钢的工艺和不同的钙处理方式对钢Ca/S、夹杂物的影响,以及低过热度结合动态轻压下浇铸对铸坯低倍质量的影响。研究表明:采用"LF+RH+钙处理"工艺冶炼,可提高钢中Ca/S,降低钢中A类和B类夹杂物尺寸。RH真空后进行钙处理,成品钢板中出现2.0级的Ds类夹杂,延长钙处理后软吹时间,可减少该类夹杂的尺寸和数量。采用LF/RH双步钙处理工艺,RH钙处理后软吹时间16～20 min,可达到钢板B类、Ds和D类夹杂尺寸控制在≤1.0级,A类和C类夹杂尺寸控制在≤0.5级。利用5～12℃过热度结合动态轻压下技术浇铸,铸坯低倍评级中心偏析达到C类1.0级,各元素偏析度较低。采用该工艺,可实现Q345R抗酸钢成分、夹杂物、低倍质量满足标准要求。     

Modulation of ovarian cytochrome P450 17 alpha-hydroxylase and cytochrome aromatase messenger ribonucleic acid by prolactin in the domestic turkey

The effect of exogenous ovine prolactin (oPRL) on preovulatory follicle P450 17 alpha-hydroxylase (C17) and aromatase (ARO) mRNA abundance was investigated in turkeys. Ovine PRL (124 IU/hen per day) was injected i.m. into four sets (n = 8) of laying turkeys for 2, 4, 8, or 14 days. Vehicle was injected into control hens for 8 days (n = 8). Blood samples were collected and serum was assayed for LH, progesterone (P), testosterone (T), and estradiol (E). Theca layers from the largest (F1) and the third (F3), fifth (F5), and seventh (F7) largest preovulatory follicles and from small white follicles (SWF) were examined for C17 and ARO mRNA contents. The number of atretic follicles increased from 0 (vehicle-injected controls) to 9 (14-day-oPRL-injected hens). Serum E, T, and LH levels decreased, while P levels remained unchanged. There was a transient increase in theca C17 mRNA abundance of 2- and 4-day-oPRL-treated hen follicles. Cytochrome P450 ARO mRNA levels were reduced in SWF and F7 in response to oPRL. Thecal C17 and ARO mRNA content was reduced during follicular maturation in laying hens. ARO mRNA was not detectable in granulosa cells. The progressive decline in C17 and ARO mRNA content associated with follicular maturation as well as the absence of ARO mRNA in granulosa cells is consistent with the secretory activity of P, T, and E in preovulatory follicles. These findings suggest that reduced circulating E may be a consequence of suppressed ARO gene expression whereas the oPRL suppression of T secretion may not be coupled to C17 gene expression.     

高速铁路车轴用25CrMoVNi超低氧钢RH精炼过程非金属夹杂物的行为

25CrMoVNi钢由120 t EAF-LF-RH脱气-φ600 mm圆坯连铸工艺生产,EAF出钢时加Al预脱气使[Al]s≥0.030%,并加入石灰造渣预精炼,LF精炼时炉渣表面加Al粒扩散脱氧,LF精炼渣的组成为（/%）:53~57CaO,10~13SiO₂,27~28Al₂O₃,6~9MgO,0.09~0.10MnO。RH脱气精炼结果表明,RH后T[O]由脱气前0.001 3%~0.001 5%降至0.000 5%;钢中TCa由0.001 9%降至0.000 9%~0.001 7%;夹杂物发生MgO·Al₂O₃→（MgO）z（CaO）x（Al₂O₃）y→（CaO）x（Al₂O₃）y的转变;最后以尖晶石类固相夹杂物数量迅速减少,以钙铝酸盐类的液相夹杂物数量呈现出先增加后减少,钢中夹杂物由6.7个/mm²下降至2.7个/mm²     

Genomic organization, sequence, and transcriptional regulation of the human eotaxin gene

Our previous studies showed that early, stage I preneoplastic cells (sup+ I) are highly susceptible to apoptosis, whereas the later, stage II preneoplastic cells (sup- II) are relatively resistant. To examine possible mechanisms that might explain these differences in the regulation of apoptosis, Ca2+ homeostasis was analyzed and comparisons were made between these two Syrian hamster embryo cell lines. The Ca2+ indicator, fura-2, and fluorescent microscopy were used to measure intracellular free calcium concentrations, [Ca2+]i. The results indicated that the [Ca2+]i level in logarithmically growing sup+ I cells (approximately 100 nM) was considerably lower than that observed in sup- II cells (approximately 260 nM). Serum removal resulted in a reduction of [Ca2+]i in the sup+ I cells (approximately 82 nM), whereas the [Ca2+]i level in sup- II cells did not change. Endoplasmic reticulum (ER) calcium levels were determined by measuring thapsigargin-releasable Ca2+. Reduced ER calcium was consistently observed in cells induced to undergo apoptosis. Specifically, thapsigargin-releasable Ca2+ was greatly reduced in sup+ I cells (45 nM) as compared to sup- II cells (190 nNM) after 4 h in low serum. When sup- II cells were placed under conditions that resulted in apoptosis (thapsigargin or okadaic acid), decreased ER calcium was observed. To determine whether reduced ER calcium had a causative effect in apoptosis, ER calcium levels were exogenously increased in sup+ I cells by raising extracellular Ca2+ to 3 mM; ER calcium levels were maintained, and apoptosis was blocked. Studies were performed to determined whether the decrease in ER calcium could be attributed to reduced Ca2+ influx at the plasma membrane. To measure directly whether Ca2+ entry was decreased in sup+ I cells in 0.2% serum, Mn2+ uptake was used to monitor Ca2+ influx. The data show that in low serum, the rate of thapsigargin-induced Mn2+ entry in sup+ I cells was approximately 50% lower than that of sup- II cells, demonstrating that capacitative entry is reduced in sup+ I cells. In further support of this hypothesis, thapsigargin-treated sup+ I cells (0.2% serum) showed decreased Ca2+ entry upon raising extracellular Ca2+ from 0 to 2 mM. We report the novel finding that early preneoplastic cells, which exhibit a high propensity to undergo apoptosis, have decreased calcium entry at the plasma membrane, resulting in decreased ER calcium pools. This study provides new insight into mechanisms that can be involved in the regulation/dysregulation of apoptosis during neoplastic progression. Furthermore, the data imply that preneoplastic cells, which have developed a mechanism to maintain ER calcium, would be less susceptible to apoptosis and would thus have an increased potential for becoming transformed.     

RH-喂线钙处理的管线钢X80非金属夹杂物变性效果分析

在钙处理对夹杂物变性作用进行分析的基础上,结合钢厂生产X80管线钢(%:≤0.08C、≤1.85Mn、≤0.060Als)的工业性试验,利用冶金热力学原理,分析计算了Al₂O₃变性为低熔点钙铝酸盐所需钙含量的范围和避免单相CaS析出硫含量的范围,同时对≤0.002%S和0.025%～0.035%Al的RH处理钢水按出站[Ca]_Tot=(40～50)×10-6计算喂Ca-Si线进行钙处理,并对中间包钢水和铸坯中的夹杂物进行了检测。结果表明,X80管线钢试验炉次平均[Ca]_Tot为41×10^-6,[S]为23×10^-6,均在理论计算范围内;同时经钙处理后,钢中绝大部分夹杂物CaO-CaS-Al₂O₃复合夹杂,钙处理效果良好。     

Expression of functional P2-purinergic receptors in primary cultures of human colorectal carcinoma cells

Primary cell cultures of human colorectal carcinomas were established and characterized immunocytochemically. In the isolated cancer cells intracellular Ca2+ concentrations ([Ca2+]i) were measured by the fura-2 method. Stimulation with either extracellular ATP or UTP caused a biphasic rise of [Ca2+]i in a dose-dependent manner and cross-desensitization between both nucleotides was observed. The rank order of potency was ATP >== UTP > ATP-gamma-S > ADP > adenosine which is characteristic for a P2U-receptor subtype. Selective agonists of P1-, or P2X- purinoceptors had no effect on [Ca2+]i. The initial rise in [Ca2+]i was independent of extracellular calcium [Ca2+]e, whereas the second phase was not observed under [Ca2+]e-free conditions suggesting a capacitative Ca2+-entry-mechanism. Intracellular Ca2+ mobilization was proven by use of the Ca2+-ATPase inhibitor thapsigargin. P2U-specific mRNA could be detected by RT-PCR in both colorectal tumor tissues and in the human colorectal cancer cell line HT 29. In HT 29 cells, the hydrolysis-resistant ATP analog ATP-gamma-S inhibited cell proliferation and, also, induced apoptosis in a dose-dependent manner. Thus, human colorectal cancer cells express functional P2U-receptors which may play a role in the regulation of cell proliferation and apoptosis.     

Altered glycine transport by cerebral tissue and decreased Na+ and Ca++ pump activities during organophosphorus-ester-induced delayed neurotoxicity development period

Uptake of [U-14C] glycine during the organophosphorus-ester-induced delayed neurotoxicity (OPIDN) development period was studied. Diisopropyl fluorophosphate (DFP), a delayed neurotoxic organophosphorus ester was administered to adult rats and hens. Results showed a decreased accumulation of glycine in hen cerebral cortex slices during the delayed neurotoxicity development period. An altered sensitivity toward transport inhibitors 2,4-dinitrophenol and ouabain was observed in DFP-treated hens. An altered neuronal membrane function during the OPIDN development period is reported in the present work. Brain Na+, K(+)-ATPase and Ca(++)-ATPase activities decreased during the neurotoxicity development period. The decrease in Ca(++)-ATPase activity persisted in hens until the complete development of neurotoxic symptoms. Decreased Ca++ pump activity is correlated with altered membrane function during OPIDN.     

Role of reactive oxygen species in the signalling cascade of cyclosporine A-mediated up-regulation of eNOS in vascular endothelial cells

1. The action of mibefradil was studied on wild type class A calcium (Ca2+) channels and various class A/L-type channel chimaeras expressed in Xenopus oocytes. The mechanism of Ca2+ channel block by mibefradil was evaluated with two microelectrode voltage clamp. 2. Resting-state dependent block (or initial block) of barium currents (IBa) through class A Ca2+ channels was concentration dependent with an IC50 value of 208+/-23 microM. 3. Mibefradil (50 microM) did not significantly affect the midpoint voltage of the steady-state inactivation curve suggesting that inactivation does not promote Ca2+ channel block. Chimaeric class A/L-type Ca2+ channels inactivating with faster or slower kinetics than wild type class A channels were equally well inhibited by mibefradil as wild type class A channels. 4. Frequent Ca2+ channel activation facilitated IBa inhibition by mibefradil (use-dependent block). Recovery from use-dependent block was voltage-dependent, being slower at depolarized membrane potentials (tau = 75+/-15 s at -70 mV, (n=6) vs tau = 20+/-2 s at -100 mV, (n=6), P<0.05). 5. We suggest that use-dependent block of class A Ca2+ channels by mibefradil occurs because of slow recovery from open channel block (SROB) and not because of drug binding to inactivated channels. 6. Voltage-dependent slow recovery from open state-dependent block provides a molecular basis for understanding the cardiovascular profile of mibefradil such as selectivity for vasculature and relative lack of negative inotropic effects.     

Modulation of calcium responses by altered peptide ligands in a human T cell clone

To determine whether altered peptide ligands (APL) affect calcium signaling events, we investigated changes in intracellular calcium concentration ([Ca2+]i) in human T cell clone stimulated with either the fully agonistic peptide M12p54-68, the partially agonistic analogue E63V or the simple antagonistic analogue E58M. Both E63V and E58M stimulated a Ca2+ response in approximately 40% of T cells, whereas M12p54-68 did so in approximately 70% of T cells. The most predominant pattern of a Ca2+ increase induced by M12p54-68 was a small sinusoidal peak followed by a sustained high response. The most frequent pattern of calcium response induced by E63V was a continuous high response without a preceding sinusoidal peak, whereas that induced by E58M was large with frequent oscillations. Genistein, an inhibitor of the protein tyrosine kinases (PTK), markedly inhibited the wild-type peptide-induced increase in [Ca2+]i, whereas it marginally inhibited the response induced by E63V or E58M. In contrast, GF109203X, a protein kinase C (PKC)-specific inhibitor, markedly inhibited the E63V- or E58M-induced Ca2+ response, whereas it marginally affected the wild peptide-induced Ca2+ response. Furthermore, in nominal Ca2+-free medium, the E58M-induced Ca2+ response was almost completely blocked, while the M12p54-68- or E63V-induced responses were only partially inhibited. Our results suggest that the Ca2+ response induced by the fully agonistic peptide depends on activation of the genistein-sensitive signaling pathway, including PTK, whereas the Ca2+ response to a simple antagonistic APL completely depends on extracellular Ca2+ and activation of the GF109203X-sensitive signaling pathway, including PKC. These differences in the CA2+i response in recognition of different APL may parallel the unique T cell activation patterns induced by APL in human T cells.     

Treponema denticola outer membrane inhibits calcium flux in gingival fibroblasts

Treponema denticola is a cultivable oral spirochete which perturbs the cytoskeleton in cultured cells of oral origin, but intracellular signalling pathways by which it affects actin assembly are largely unknown. As the outer membrane (OM) of Treponema denticola disrupts actin-dependent processes that normally require precise control of intracellular calcium, we studied the effects of an OM extract on internal calcium release, ligand-gated and calcium release-activated calcium channels, and related mechanosensitive cation fluxes in human gingival fibroblasts (HGF). Single-cell ratio fluorimetry demonstrated that in resting cells loaded with Fura-2, baseline intracellular Ca2+ concentration ([Ca2+]i) was not affected by treatment with OM extract, but normal spontaneous [Ca2+]i oscillations were dramatically increased in frequency for 20 to 30 min followed by complete blockade. OM extract inhibited ATP-induced and thapsigargin-induced release of calcium from intracellular stores by 40 and 30%, respectively. Addition of Ca2+ to the extracellular pool following depletion of intracellular Ca2+ by thapsigargin and extracellular Ca2+ by EGTA yielded 59% less replenishment of [Ca2+]i in OM extract-treated than in control HGF. In cells loaded with collagen-coated ferric oxide beads to stimulate integrin-dependent calcium release, baseline [Ca2+]i was nearly doubled but was not significantly different in control and OM extract-treated cells. Magnetically generated tensile forces on the beads induced >300% increases of [Ca2+]i above baseline. Cells preincubated with OM extract exhibited dose-dependent and time-dependent reductions in stretch-induced [Ca2+]i transients, which were due to neither loss of beads from the cells nor cell death. The T. denticola OM inhibitory activity was eliminated by heating the OM extract to 60 degrees C and by boiling but not by phenylmethylsulfonyl fluoride treatment. Thus nonlipopolysaccharide, nonchymotrypsin, heat-sensitive protein(s) in T. denticola OM can evidently inhibit both release of calcium from internal stores and uptake of calcium through the plasma membrane, possibly by interference with calcium release-activated channels.     

Calcium-mediated intracellular messengers modulate the serotonergic effects on axonal excitability

We carried out experiments to investigate the mechanisms of serotonin-induced axonal excitability changes using isolated dorsal columns from young (seven to 11-day-old) Long-Evan's hooded rats. Conducting action potentials were activated by submaximal (50%) and supramaximal constant current electrical stimuli and recorded with glass micropipette electrodes. In experiment 1, to study Ca(2+)-mediated mechanisms, we superfused the preparations with Ringer solutions containing varying Ca2+ concentrations. Following superfusion with Ca(2+)-free Ringer solution for 4 h, we tested initial responses to serotonin agonists. Studies then were repeated after preparations had been washed for 1 h with Ringer solution containing 1.5 mM Ca2+ and 1.5 mM Mg2+. After 4 h superfusion of Ca(2+)-free Ringer solution, quipazine (a serotonin2A agonist, 100 microM) did not induce significant axonal excitability changes (amplitude change of 1.4 +/- 1.3%, percentage of predrug control level, +/-S.D., n = 6). A 100 microM concentration of 8-hydroxy-dipropylaminotetralin (a serotonin1A agonist) reduced response amplitudes by 36.3 +/- 4.2% (+/-S.D., P < 0.0005, n = 7) and prolonged latencies by 22.3 +/- 4.3% (+/-S.D., P < 0.0005, n = 7). Application of serotonin (100 microM) decreased amplitudes by 6.6 +/- 5.0% (+/-S.D., P < 0.05, n = 6). Extracellular calcium concentration ([Ca2+]e) was measured at various depths in the dorsal column with ion-selective microelectrodes. Four hours' superfusion with Ca(2+)-free Ringer solution reduced [Ca2+]e to less than 0.1 mM in dorsal columns. In 1.5 mM Ca2+ Ringer solution, quipazine increased the amplitudes by 38.3 +/- 5.8% (P < 0.0005, n = 6). Likewise, serotonin increased the amplitudes by 13.8 +/- 4.9% (P < 0.005, n = 6). In contrast however, 8-hydroxy-dipropylaminotetralin still reduced amplitudes by 35.0 +/- 6.4% (P < 0.0005, n = 7) and prolonged latencies by 24.1 +/- 4.5% (P < 0.0005, n = 7). In experiment 2, we investigated calcium-dependent and cAMP-mediated protein kinase signalling pathways to evaluate their role as intracellular messengers for serotonin2A receptor activation. Two protein kinase inhibitors, 50 microM H7 (an inhibitor of protein kinase C and c-AMP dependent protein kinase) and 100 microM D-sphingosine (an inhibitor of protein kinase A and C) effectively eliminated the excitatory effects of the serotonin2A agonist. 100 microM cadmium (a Ca2+ channel blocker) also blocked the effects of quipazine. Neither these protein kinase inhibitors nor cadmium alone affected action potential amplitudes. These results suggest that replacing Ca2+ with Mg2+ blocks the excitatory effects of quipazine but does not prevent the inhibitory effects of 8-hydroxy-dipropylaminotetralin, and calcium-mediated protein kinase mechanisms modulate axonal excitability changes induced by serotonin and its agonist.     

Intracellular calcium responses of circadian pacemaker neurons measured with fura-2

The circadian pacemaker in the eye of the mollusk Bulla gouldiana is located within basal retinal neurons (BRNs) that express a circadian rhythm in cell culture. Light and other depolarizing stimuli shift the phase of the pacemaker in the eye through a process that requires extracellular calcium and is blocked by Ni2+. To test directly if an influx of Ca2+ is present throughout depolarizing treatments that produce phase shifts, dissociated BRNs in cell culture were loaded with a membrane-permeable form of the calcium-sensitive dye fura-2, and then depolarized with elevated levels of extracellular K+. Calcium levels in the BRNs remained elevated during treatments with 50 mM K+ lasting 1 h, a sufficient duration to phase shift the circadian pacemaker. Lowering extracellular free Ca2+ (approx. 1.7 x 10(-7) M) during depolarization blocked the rise in intracellular Ca2+, verifying that a Ca2+ influx is required. The sustained Ca2+ elevation during depolarization was also prevented with 50 mM Ni2+, which blocks phase shifts of the rhythm to depolarization, but not with 5 mM Ni2+, which does not block phase shifts. The initial rise in [Ca2+]i in response to 50 mM K+ was largest on average during the subjective night. The results show that a critical portion of the entrainment pathway persists in pacemaker neurons during cell culture, and that the phase-shifting stimulus may depend on a prolonged Ca2+ signal.     

Correlation of EGTA and calcium-blocking agents on the response of the bladder to in vitro ischemia

The effects of repetitive field stimulation (model of hyperrelexia) on the responses of isolated strips of rabbit urinary bladder to FS and carbachol were evaluated under a variety of incubation conditions. Compared to control conditions, 2 h of repetitive FS in normal, oxygenated Tyrode's solution followed by incubation for 1 h with no stimulation resulted in a 50% decrease in contractile response to FS and a 30% decrease in the response to carbachol. Incubation in the absence of O2 and glucose was used as an in vitro model for ischemia. Repetitive stimulation during in vitro ischemia resulted in a significantly greater decrease in the contractile responses to FS and carbachol than did in vitro ischemia without repetitive stimulation. The magnitude of contractile dysfunctions in response to both stimuli were significantly reduced in the presence of EGTA (calcium chelator), diltiazem (calcium channel blocker) or pincidil (potassium channel opener). Incubation with thapsigargin (SR calcium uptake inhibitor) + ryanodine (SR calcium storage inhibitor) had no effect. The results of these studies indicate that inhibition of Ca2+ entry reduces the contractile dysfunctions induced by repetitive stimulation in the presence of in vitro ischemia. Inhibition of Ca2+i storage and release had no significant effect on the magnitude of contractile dysfunctions induced by repetitive stimulation an in vitro ischemia.     

In vivo treatment with calcitriol (1,25(OH)2D3) reverses age-dependent alterations of intestinal calcium uptake in rat enterocytes

The vitamin D endocrine system has been involved in the impairment of intestinal calcium absorption during aging. Alterations in the nongenomic mechanism of calcitriol (1,25-dihydroxy-vitamin D3; [1, 25(OH)2D3] have been recently evidenced. In enterocytes isolated from aged rats, 1,25(OH)2D3 stimulation of Ca2+ channels through the cAMP/PKA pathway is blunted. We have now investigated whether in vivo administration of calcitriol to senescent rats reverses the absence of hormonal effects in isolated intestinal cells. In enterocytes from 20-24-month-old rats given 1,25(OH)2D3 for 3 days (30 ng/100 g bw/day), calcitriol (10(-10) M, 3-5 minutes) stimulated Ca2&plus uptake and intracellular cAMP to the same degree and protein quinase A (PKA) activity to a lesser degree than in enterocytes from young animals. Significantly higher basal levels of cAMP and PKA detected in enterocytes from old rats were not affected by prior injection of animals with 1,25(OH)2D3. When the aged rats were injected with 25(OH)D3, similar Ca2+ influx, cAMP, and PKA responses to in vitro stimulation with calcitriol were obtained. 1, 25(OH)2D3-dependent changes in Ca2+ uptake by enterocytes from both young and old rats treated with calcitriol were totally suppressed by the cAMP antagonist Rp-cAMPS, whereas the response to the agonist Sp-cAMPS was markedly depressed in aged animals. These results suggest that intestinal resistance to nongenomic 1,25(OH)2D3 stimulation of duodenal cell Ca2+ uptake develops in rats upon aging and show that in vivo administration of 1,25(OH)2D3 or its precursor to senescent rats restores the ability of the hormone to stimulate duodenal cell calcium influx through the cAMP messenger system.