Agonist-induced intracellular Ca2+ transients in HT29 cells

In the present study we have investigated the mechanism of intracellular Ca2+ activity ([Ca2+]i) changes in HT29 cells induced by adenosine triphosphate (ATP), carbachol (CCH), and neurotensin (NT). [Ca2+]i was measured with the fluorescent Ca2+ indicator fura-2 at the single-cell level or in small cell plaques with high time resolution (1-40Hz). ATP and CCH induced not only a dose-dependent [Ca2+]i peak response, but also changes of the plateau phase. The [Ca2+]i plateau was inversely dependent on the ATP concentration, whereas the CCH-induced [Ca2+]i plateau increased at higher CCH concentrations. NT showed (from 10(-10) to 10(-7) mol/l) in most cases only a [Ca2+]i spike lasting 2-3 min. The [Ca2+]i plateau induced by ATP (10(-6) mol/l) and CCH (10(-5) mol/l) was abolished by reducing the Ca2+ activity in the bath from 10(-3) to 10(-4) mol/l (n = 7). In Ca(2+)-free bathing solution the [Ca2+]i peak value for all three agonists was not altered. Using fura-2 quenching by Mn2+ as an indicator of Ca2+ influx the [Ca2+]i peak was always reached before Mn2+ influx started. Every agonist showed this delayed stimulation of the Ca2+ influx with a lag time of 23 +/- 1.5 s (n = 15) indicating a similar mechanism in each case. Verapamil (10(-6)-10(-4) mol/l) blocked dose dependently both phases (peak and plateau) of the CCH-induced [Ca2+]i increase. Short pre-incubation with verapamil augmented the effect on the [Ca2+]i peak, whereas no further influence on the plateau was observed. Ni2+ (10(-3) mol/l) reduced the plateau value by 70%.     

Differential effects of Ca2+ channel blockers on Ca2+ transients and cell cycle progression in vascular smooth muscle cells

The predominant angiotensin II receptor expressed in the human myometrium is the angiotensin AT2 receptor. This preparation was used for a structure-activity relationship study on angiotensin II analogues modified in positions 1 and 8. The angiotensin AT2 receptor present on human myometrium membranes displayed a high affinity (pKd = 9.18) and was relatively abundant (53-253 fmol/mg of protein). The pharmacological profile was typical of an angiotensin AT2 receptor with the following order of affinities: (angiotensin III > or = angiotensin II > angiotensin I > PD123319 > angiotensin-(1-7) > angiotensin-(1-6) approximately angiotensin IV > Losartan). Modifications of the N-terminal side chain and of the primary amine of angiotensin II were evaluated. Neutralisation of the methylcarboxylate (Asp) to a methylcarboxamide (Asn) or to a hydroxymethyl (Ser) or substitution for a methylsulfonate group (cysteic acid) improved the affinity. Extension from methylcarboxylate (Asp) to ethylcarboxylate (Glu) did not affect the affinity. Introduction of larger side chains such as the bulky p-benzoylphenylalanine (p-Bpa) or the positively charged Lys did not substantially affect the affinity. Complete removal of the side chain (angiotensin III), however, resulted in a significant affinity increase. Removal or acetylation of the primary amine of angiotensin II did not noticeably influence the affinity. Progressive alkylation of the primary amine significantly increased the affinity, betain structures being the most potent. It appears that quite important differences exist between the angiotensin AT1 and AT2 receptors concerning their pharmacological profile towards analogues of angiotensin II modified in position 1. On position 8 of angiotensin II, a structure-activity relationship on the angiotensin AT2 receptor was quite similar to that observed with angiotensin AT1 receptor. Bulky, hydrophobic aromatic residues displayed affinities similar to or even better than [Sarcosine1]angiotensin II. Aliphatic residues, especially those of reduced size, caused a significant decrease in affinity especially [Sarcosine1, Gly8]angiotensin II who showed a 30-fold decrease. Introduction of a positive charge (Lys) at position 8 reduced the affinity even further. Stereoisomers in position 8 (L-->D configuration) also induced lower affinities. The angiotensin AT2 receptor display a structure-activity relationship similar to that observed on the AT1 receptor for the C-terminal position of the peptide hormone. Position 1 structure-activity relationships are however fundamentally different between the angiotensin AT1 and AT2 receptor.     

Role of intracellular Ca2+ stores in smooth muscle of human penile erectile tissue

The image of plastic surgery as portrayed by the media is of concern to all plastic surgeons. In order to assess knowledge about the specialty, a questionnaire was devised and given to five groups of participants: general practitioners, medical students, nurses, plastic surgical out-patient attendees, and the general public. The results revealed that general practitioners, nurses and medical students in the Cambridge area are, on the whole, knowledgeable about the role of plastic surgery. However, the general public are not so well educated and 23.7% of them could not think of five conditions treated by plastic surgeons, and felt that burns and cosmetic problems were the commonest conditions dealt with. Improved liaison with general practitioners, other specialties and more teaching of undergraduates, coupled with more effective promotion of the skills on offer might permit better use to be made of the specialty.     

Dissociation of intracellular Ca2+ release and Ca2+ entry response to 5-hydroxytryptamine in cultured canine tracheal smooth muscle cells

The relationship between the agonist-sensitive Ca2+ pool and those discharged by the Ca2+ -ATPase inhibitor thapsigargin (TG) were investigated in canine tracheal smooth muscle cells (TSMCs). In fura-2-loaded TSMCs, 5-hydroxytryptamine (5-HT) stimulated a rapid increase in intracellular Ca2+ ([Ca2+]i), followed by a sustained plateau phase that was dependent on extracellular Ca2+. In such cells, TG produced a concentration-dependent increase in [Ca2+]i, which remained elevated over basal level for several minutes and was substantially attenuated in the absence of extracellular Ca2+. Application of 5-HT after TG demonstrated that the TG-sensitive compartment partly overlapped the 5-HT-sensitive stores. Pre-treatment of TSMCs with TG significantly inhibited the increase in [Ca2+]i induced by 5-HT in a time-dependent manner. Similar results were obtained with two other Ca2+ -ATPase inhibitors, cyclopiazonic acid and 2,5-di-t-butylhydroquinone. Although these inhibitors had no effect on phosphoinositide hydrolysis, Ca2+ -influx was stimulated by these agents. These results suggest that depletion of the agonist-sensitive Ca2+ stores is sufficient for activation of Ca2+ influx. Some characteristics of the Ca2+ -influx activated by depletion of internal Ca2+ stores were compared with those of the agonist-activated pathway. 5-HT-stimulated Ca2+ influx was inhibited by La3+, membrane depolarisation, and the novel Ca2+ -influx blocker 1-?beta-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl?-1H-imidazole hydrochloride (SKF96365). Likewise, activation of Ca2+ influx by TG also was blocked by La3+, membrane depolarisation, and SKF96365. These results suggest that (1) in the absence of PI hydrolysis, depletion of the agonist-sensitive internal Ca2+ stores in TSMCs is sufficient for activation of Ca2+ influx, and (2) the agonist-activated Ca2+ influx pathway and the influx pathway activated by depletion of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool are indistinguishable.     

Voltage-dependent Ca2+ channels in arterial smooth muscle cells

The past years have seen some significant advances in our understanding of the functional and molecular properties of voltage-dependent Ca2+ channels in arterial smooth muscle. Molecular cloning and expression studies together with experiments on native voltage-dependent Ca2+ channels revealed that these channels are built upon a molecular structure with properties appropriate to function as the main source for Ca2+ entry into arterial smooth muscle cells. This Ca2+ entry regulates intracellular free Ca2+, and thereby arterial tone. We summarize several avenues of recent research that should provide significant insights into the functioning of voltage-dependent Ca2+ channels under conditions that occur in arterial smooth muscle. These experiments have identified important features of voltage-dependent Ca2+ channels, including the steep steady-state voltage-dependence of the channel open probability at steady physiological membrane potentials between -60 and -30 mV, and a relatively high permeation rate at physiological Ca2+ concentrations, being about one million Ca2+ ions/s at -50 mV. This calcium permeation rate seems to be a feature of the pore-forming Ca2+ channel alpha1 subunit, since it was identical for native channels and the expressed alpha1 subunit alone. The channel activity is regulated by dihydropyridines, vasoactive hormones and intracellular signaling pathways. While the membrane potential of smooth muscle cells primarily regulates arterial muscle tone through alterations in Ca2+ influx through dihydropyridine-sensitive voltage-dependent ('L-type') Ca2+ channels, the role of these channels in the differentiation and proliferation of vascular smooth muscle cells is less clear. We discuss recent findings suggesting that other Ca2+ permeable ion channels might be important for the control of Ca2+ influx in dedifferentiated vascular smooth muscle cells.     

Ca2+ signalling by endothelin receptors in rat and human cultured airway smooth muscle cells

1. The aim of the current study was to characterize the ET receptor subtypes in cultured airway smooth muscle cells derived from rat trachea and human bronchus using radioligand binding techniques and to investigate the coupling of ET receptors to intracellular calcium signalling mechanisms using endothelin receptor-selective agonists (sarafotoxin S6c) and antagonists (BQ-123, BQ-788) and digital image fluorescence microscopy. 2. Confluent rat airway smooth muscle cells in culture possessed a mixed ET receptor population (30% ETA : 70% ETB), with a density of approximately 3400+/-280 ETA and 8000+/-610 ETB receptors/cell (n = 3 experiments). The density of ETB, but not ETA receptors increased substantially in serum-containing medium. However, a 2-day period of serum deprivation, which inhibited cellular growth, substantially reduced ETB receptor density such that the ET receptor subtype proportions were approximately equal (55% ETA; 45% ETB) and similar to those previously observed in intact rat tracheal smooth muscle. 3. Challenge of rat airway smooth muscle cells in culture with endothelin- 1 elicited a concentration-dependent biphasic increase in [Ca2+]i (EC50: 16 nM), that comprised an initial transient peak [Ca2+]i increase (typically 350 nM) followed by a modest sustained component. The endothelin-1-induced biphasic [Ca2+]i increase was primarily due to ETA receptor activation, although a modest and inconsistent ETB response was observed. The ETA-mediated [Ca2+]i increase was due primarily to the mobilization of IP3-sensitive and to a lesser extent ryanodine-sensitive intracellular calcium stores. In contrast, ETB receptor activation was exclusively coupled to extracellular calcium influx. 4. Somewhat surprisingly, human airway smooth muscle cells in culture contained a homogeneous population of ETA receptors at a density of 6100+/-800 receptors cell(-1) (n = 3 experiments). Serum deprivation was without effect on either ET receptor subtype proportion or ETA receptor density. Challenge of human airway smooth muscle cells with endothelin-1 provoked a concentration-dependent increase in [Ca2+]i (EC50: 15 nM), with a peak [Ca2+]i increase to greater than 700 nM. Furthermore, the ETA-mediated calcium response in these human airway smooth muscle cells in culture was entirely dependent upon the mobilization of calcium from intracellular stores. 5. In summary, rat cultured tracheal airway smooth muscle cells contained both ETA and ETB receptors. ETA receptors, the numbers of which remained constant during cell growth, were linked to the release of Ca2+ from intracellular stores and a strong rise in [Ca2+]i in the majority of airway smooth muscle cells. In stark contrast, the numbers of ETB receptors increased significantly during cell growth, an effect that was diminished substantially by incubation in serum-free medium. Moreover, despite the greater number of ETB receptors, their activation in a small number of airway smooth muscle cells produced only a weak rise in [Ca2+]i, which appeared to be attributable to the influx of extracellular Ca2+. In contrast, the populations of ET receptors and their linkage to [Ca2+]i were markedly different in the human cultured airway smooth muscle cells used in the current study compared to that previously observed in intact human isolated bronchial smooth muscle.     

Protein kinase C inhibits the Ca(2+)-activated K+ channel of cultured porcine coronary artery smooth muscle cells

The effect of protein kinase C (C-kinase) on the Ca(2+)-activated K+ channel (KCa-channel) was studied in cultured smooth muscle cells from porcine coronary artery by the patch-clamp technique. In cell-attached patches, bath application of phorbol 12-myristate 13-acetate (PMA, 1 microM), a C-kinase activator, significantly decreased the open probability of the activated KCa-channel in the presence of the calcium ionophore A23187 (20 microM), which increases intracellular Ca2+. This decrease in the open probability was reversed by subsequent application of staurosporine (1 nM), a C-kinase inhibitor. Application of 1-oleoyl-2-acetylglycerol (OAG, 30 microM) or 1,2-dioctanoylglycerol (DG8; 30 microM), activators of C-kinase, also inhibited KCa-channel activation by A23187, and these inhibitions were also reversed by staurosporine. PMA (1 microM) also inhibited KCa-channel activation by dibutylyl cyclic AMP (db-cAMP, 2 mM) or caffeine (30 mM). In inside-out patches, bath application of the C-kinase fraction from rat brain in the presence of ATP (1 mM) and PMA (1 microM) markedly inhibited the KCa-channel. These results indicate that activation of C-kinase inhibits the KCa-channel and may cause membrane depolarization and vascular contraction.     

Farnesol inhibits L-type Ca2+ channels in vascular smooth muscle cells

Earlier experiments with animal and human arteries have shown that farnesol, a natural 15-carbon (C15) isoprenoid, is an inhibitor of vasoconstriction (Roullet, J.-B., Xue, H., Chapman, J., McDougal, P., Roullet, C. M., and McCarron, D. A. (1996) J. Clin. Invest. 97, 2384-2390). We report here that farnesol reduced KCl- and norepinephrine-dependent cytosolic Ca2+ transients in fura-2-loaded intact arteries. An effect on Ca2+ signaling was also observed in cultured aortic smooth muscle cells (A10 cells). In these cells, farnesol reduced KCl-induced [Ca2+]i transients and mimicked the inhibitory effect of Ca2+-free medium on the [Ca2+]i response to both 12,13-phorbol myristate acetate, a protein kinase C activator, and thapsigargin, a specific endoplasmic reticulum ATPase inhibitor. Perforated patch-clamp experiments further showed in two vascular smooth muscle cell lines (A10 and A7r5), a reversible, dose-dependent inhibitory effect of farnesol on L-type Ca2+ currents (IC50 = 2.2 microM). Shorter (C10, geraniol) and longer (C20, geranylgeraniol) isoprenols were inactive. L-type Ca2+ channel blockade also occurred under tight (gigaohm) seal configuration using cell-attached, single-channel analysis, thus suggesting a possible action of farnesol from within the intracellular space. We finally demonstrated that farnesol did not affect Ca2+-sensitive pathways implicated in smooth muscle contraction, as tested with alpha-toxin permeabilized arteries. Altogether, our results indicate that farnesol is an inhibitor of vascular smooth muscle Ca2+ signaling with plasma membrane Ca2+ channel blocker properties. The data have implications for the endogenous and pharmacological regulation of vascular tone by farnesol or farnesol analogues.     

Spatial heterogeneity of caffeine- and inositol 1,4,5-trisphosphate-induced Ca2+ transients in isolated snail neurons

Inositol 1,4,5-trisphosphate- and caffeine-induced Ca2+ release was examined in neurons isolated from the mollusc Helix pomatia using Ca2+ indicator fura-2 and fluorescent digital-imaging microscopy technique. Extracellular application of caffeine caused a fast and pronounced augmentation of [Ca2+]i whose amplitude and kinetics differ in the centre of the cell and near its membrane. Mean values of caffeine-induced increase of [Ca2+]i were 0.97 +/- 0.11 microM at the periphery and 0.53 +/- 0.13 microM in the centre. The rates of rise and relaxation of caffeine-evoked [Ca2+]i transients were faster near the membrane. Pressure injection of inositol, 1,4,5-trisphosphate into the same neurons produced an abrupt and significant increase of [Ca2+]i in the centre (mean value of inositol 1,4,5-trisphosphate-induced elevation = 0.55 +/- 0.11 microM) while the response was smaller or even absent near the cellular membrane. Inositol 1,4,5-trisphosphate- and caffeine-induced Ca2+ transients did not affect each other. The data obtained indicate that in snail neurons these two calcium pools are not overlapping and at least some part of the caffeine-sensitive store is located close to the cellular membrane and that the inositol 1,4,5-trisphosphate-sensitive one is located in the centre of the cell.     

Acetaldehyde depresses shortening and intracellular Ca2+ transients in adult rat ventricular myocytes

Numerous studies have shown that the developing tip of a neurite, the growth cone, can respond to environmental cues with behaviors such as guidance or collapse. To assess whether a given cell type can use more than one second-messenger pathway for a single behavior, we compared the influence of two well-characterized guidance cues on growth cones of chick temporal retinal ganglion cells. The first cue was the repulsive activity derived from the posterior optic tectum (p-membranes), and the second was the collapse-inducing activity derived from oligodendrocytes known as NI35/NI250. p-Membranes caused permanent growth cone collapse with no recovery after several hours, while NI35 caused transient collapse followed by recovery after about 10 min. The p-membrane-induced collapse was found to be Ca2+ independent, as shown using the Ca2+-sensitive dye Fura-2 and by the persistence of collapse in Ca2+-free medium. Dantrolene, a blocker of the ryanodine receptor, had only a minor effect on the collapse frequency caused by p-membranes. In contrast, the NI35-induced collapse was clearly Ca2+ dependent. [Ca2+]i increased sevenfold preceding collapse, and both dantrolene and antibodies against NI35 significantly reduced both the Ca2+ increase and the collapse frequency. Thus, even in a single cell type, growth cone collapse induced by two different signals can be mediated by two different second-messenger systems.     

Stochastic simulation of a single inositol 1,4,5-trisphosphate-sensitive Ca2+ channel reveals repetitive openings during 'blip-like' Ca2+ transients

Confocal microscope studies with fluorescent dyes of inositol 1,4,5-trisphosphate (InsP3)-induced intracellular Ca2+ mobilization recently established the existence of 'elementary' events, dependent on the activity of individual InsP3-sensitive Ca2+ channels. In the present work, we try by theoretical stochastic simulation to explain the smallest signals observed in those studies, which were referred to as Ca2+ 'blips' [Parker I., Yao Y. Ca2+ transients associated with openings of inositol trisphosphate-gated channels in Xenopus oocytes. J Physiol Lond 1996; 491: 663-668]. For this purpose, we assumed a simple molecular model for the InsP3-sensitive Ca2+ channel and defined a set of parameter values accounting for the results obtained in electrophysiological bilayer experiments [Bezprozvanny I., Watras J., Ehrlich B.E. Bell-shaped calcium-response curves of Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature 1991; 351: 751-754; Bezprozvanny I., Ehrlich B.E. Inositol (1,4,5)-trisphosphate (InsP3)-gated Ca channels from cerebellum: conduction properties for divalent cations and regulation by intraluminal calcium. J Gen Physiol 1994; 104: 821-856]. With a stochastic procedure which considered cytosolic Ca2+ diffusion explicitly, we then simulated the behaviour of a single channel, placed in a realistic physiological environment. An attractive result was that the simulated channel exhibited bursts of activity, arising from repetitive channel openings, which were responsible for transient rises in Ca2+ concentration and were reminiscent of the relatively long-duration experimental Ca2+ blips. The influence of the values chosen for the various parameters (affinity and diffusion coefficient of the buffers, luminal Ca2+ concentration) on the kinetic characteristics of these theoretical blips is analyzed.     

Characterization of action potential-triggered [Ca2+]i transients in single smooth muscle cells of guinea-pig ileum

Cytology can be a rewarding diagnostic technique in equine practice. The respiratory tract readily lends itself to sampling for cytologic evaluation from the upper to lower regions of the system. This article discusses preservation and staining techniques that will allow the practitioner to present satisfactory samples to the laboratory. General considerations for cytologic analysis are discussed as well as the specific findings for individual disorders of the respiratory tract. The proper use of cytologic findings in conjunction with other diagnostic techniques for the respiratory tract are also discussed.     

Evidence for Mg2, ATP-dependent accumulation of Ca2+ by the intracellular non-mitochondrial calcium store in uterine smooth muscle cells

Using carbachol contracture as the experimental model for testing the properties of the intracellular calcium store in intact tissue and 45Ca2+ accumulation in the chemically skinned by digitonin smooth muscle cells isolated from oestrogen-dominated rat uterus the evidence for the presence of Mg2+, ATP-dependent Ca2+ pump in the non-mitochondrial store has been found which is supposed to play a key role in the process of refilling' of the store on the cytoplasmic level. The experiments performed on intact muscle showed that the functional activity of the carbachol-releasable Ca2+ store is critically dependent on Ca2+ entry. It is found that Ca2+ entry via voltage operated Ca2+ channels or on the Na(+)-Ca2+ exchange was needed to refill the store in this tissue. However, when Ca2+ extrusion systems located in the plasma membrane were inhibited by La3+, the store retained its ability to discharge and reaccumulate Ca2+ released on the regular basis suggesting the presence of the energy-dependent Ca2+ accumulating system in the store. The process of the store refilling was totally inhibited by cyclopiazonic acid. Chemically skinned uterine smooth muscle cells demonstrated the presence of Mg2+, ATP-dependent accumulation of Ca2+ in the non-mitochondrial (ruthenium red insensitive) intracellular store(s) potentiated by Ca(2+)-precipitating anions (potassium oxalate and phosphate), in a time- and concentration dependent way which was inhibited by Ca(2+)-ionophore A 23187 (5 microM) and cyclopiazonic acid with Ki = 0.4 microM. It is suggested that in the uterine smooth muscle of the oestrogen-dominated rats, nonmitochondrial receptor-operated intracellular calcium store is represented by endoplasmic reticulum.     

Heparin inhibits human coronary artery smooth muscle cell migration

Heparin, an anticoagulant, has been shown to reduce neointimal proliferation and restenosis following vascular injury in experimental studies, but the clinical trials of heparin in coronary balloon angioplasty have been negative. The current study, therefore, examined the effect of heparin on basal or stimulated migration by serum and platelet-derived growth factor (PDGF)-BB in cultured human coronary artery smooth muscle cells (SMCs) by Boyden's chamber method. In addition, the reversibility of the heparin effect on human coronary artery SMC migration was examined. Fetal calf serum (FCS) and PDGF-BB stimulated SMC migration in a concentration-dependent manner. Heparin in moderate to high concentration (10 to 100 U/mL) exhibited concentration-related inhibition of FCS- and PDGF-BB-stimulated SMC migration; however, a low concentration (1 U/mL) of heparin had no inhibitory effects. Heparin also had weak inhibitory effects on nonstimulated SMC migration. The SMCs that were exposed to a high concentration (100 U/mL) of heparin for 6 hours were capable of migrating after a short lag period of removal of heparin from the culture medium. These SMCs also showed recovery of responses to FCS and PDGF-BB by migrating significantly greater than the nonstimulated level. Furthermore, heparin-containing medium did not contain detached cells. These results indicate that heparin inhibits human coronary artery SMC migration, especially when stimulated by FCS or PDGF-BB, and that this inhibitory effect of heparin is reversible and not simply a function of killing cells.     

Adrenomedullin increases intracellular Ca2+ and inositol 1,4,5-trisphosphate in human oligodendroglial cell line KG-1C

The effects of adrenomedullin (AM), a hypotensive peptide, were investigated in cultured human oligodendroglial cell line KG-1C. Human AM increased the intracellular Ca2+ concentration ([Ca2+]i) at concentrations greater than 10(-7) M. Human calcitonin gene-related peptide (CGRP), a peptide structurally related to AM, also increased [Ca2+]i with a potency similar to that of AM. AM increased [Ca2+]i in the absence of extracellular Ca2+. Further, AM increased inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) level in a concentration-dependent manner similar to that of AM-induced [Ca2+]i, suggesting that AM-induced elevation of [Ca2+]i is due to Ca2+ release from Ins(1,4,5)P3-sensitive stores. AM (10(-9) to 10(-6) M) increased cAMP in a concentration-dependent manner. Forskolin also increased cAMP, but did not mimic the [Ca2+]i-raising effect of AM. These findings suggest that functional AM receptors are present in oligodendroglial KG-1C cells and that AM increases [Ca2+]i through a mechanism independent of cAMP.     

Creatine supplementation improves intracellular Ca2+ handling and survival in mdx skeletal muscle cells

Rapid review, digital recording, on-line quantification, and three-dimensional reconstruction are all essential in the evaluation of intracoronary ultrasound images during coronary interventions. We describe a low-cost method that offers all these necessary features. The proposed method uses the QuickTime compatible video digitizers of standard multimedia Apple Macintosh or PowerPC desktop computers and the freeware software Object Image 1.60.     

Cd2+ and Co2+ at micromolar concentrations mobilize intracellular Ca2+ via the generation of inositol 1,4,5-triphosphate in bovine chromaffin cells

To understand the mechanisms underlying the Cd2+- and Co2+-induced intracellular Ca2+ mobilization, we measured the levels of inositol phosphates using bovine chromaffin cells. Studies using HPLC indicated that Cd2+, Co2+ and methacholine significantly increased the generation of 1,4,5-IP3. The results suggest that Cd2+ and Co2+ mobilize Ca2+ from IP3-sensitive Ca2+ stores, possibly through the presumptive Cd2+ receptor.     

Influence of cellular incorporation of n-3 eicosapentaenoic acid on intracellular Ca2+ concentration and membrane potential in vascular smooth muscle cells

Recombinant antibody fragments expressed in the cytoplasm of cells have considerable practical potential. However in the reducing environment of the cytoplasm, the intradomain disulphide bonds are not formed and the fragments are unstable and expressed in low yields. Here we attempted to overcome these limitations. We first isolated an antibody single chain Fv fragment that binds and activates an inactive mutant beta-galactosidase. We then subjected the gene encoding the scFv fragment to random mutation in vitro by error-prone polymerase chain reaction, and co-expressed the mutant beta-galactosidase and mutant antibody fragments in lac- bacteria. By plating on limiting lactose, we selected for antibody mutants with improved expression, and after four successive rounds of mutation and selection, isolated an antibody fragment that is expressed in the bacterial cytoplasm with yields of 0.5 g/l in a shaker flask (A600 nm of 5.5) and 3.1 g/l (A600 nm=33) in a fermentor. Analysis of the mutant antibody fragments revealed that the disulphide bonds are reduced in the cytoplasm, and that the fragments could be denatured and renatured efficiently under reducing conditions in vitro. This shows that with a suitable method of screening or selection, it is possible to make folded and functional antibody fragments in excellent yield in the cytoplasm.     

Alterations in contractility and intracellular Ca2+ transients in isolated bundles of skeletal muscle fibers from rats with chronic heart failure

To determine if chronic heart failure (CHF) leads to functional or structural alterations of skeletal muscle, we compared intracellular Ca2+ signaling, contractility, and the rate of fatigue development, together with electron microscopy (EM), in skeletal muscle preparations from rats with myocardial infarction-induced CHF versus sham-operated control rats. Bundles of 100 to 200 cells were dissected from the extensor digitorum longus (EDL) muscle of control (n = 13) and CHF (n = 19) rats and were either loaded with aequorin or fixed for EM. Muscles from CHF rats exhibited depressed tension development compared with control muscles during twitches (1.4 +/- 0.2 versus 2.8 +/- 0.7 g/mm2, P < .05) and maximal tetani (5.3 +/- 1.4 versus 10.7 +/- 2.4 g/mm2, P < .05). Depressed tension in CHF was accompanied by reduced quantitative [Ca2+]i release during twitches (0.7 +/- 0.1 versus 0.4 +/- 0.1 microM, P < .05) and during maximal tetani (1.8 +/- 0.3 versus 0.9 +/- 0.2 microM, P < .05). Skeletal muscle from CHF rats also demonstrated prolonged intracellular Ca2+ transients during twitches and tetani and accelerated fatigue development. EM revealed a lack of cellular atrophy in the CHF rats. In conclusion, EDL skeletal muscle from rats with CHF had intrinsic abnormalities in excitation-contraction coupling unrelated to cellular atrophy. These findings indicate that CHF is a condition accompanied by EDL skeletal muscle dysfunction.