Stimulation of spontaneous transmitter release at the frog neuromuscular junction by 12-O-tetradecanoylphorbol-13-acetate occurs in the absence of extracellular Ca2+ and is enhanced by depolarization

Summary The effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the release of transmitter at the frog neuromuscular junction has been investigated electrophysiologically. TPA (100 nmol/l) caused a gradual rise in miniature end-plate potential (MEPP) frequency. After 20–30 min MEPP frequency had risen by approximately 40%. This action of the drug was not inhibited by bathing preparations in either Ca²⁺-free medium (0 Ca²⁺-1 mmol/l EGTA) or high Mg²⁺ medium, or by pretreatment with verapamil (5 mol/l). The inactive TPA analogue 4--TPA had no effect on release rate. There was no indication of any positive correlation between resting MEPP frequency and the size of the subsequent response to TPA treatment. Any synergism between [Ca²⁺]_i and TPA treatment is therefore likely to occur at a site other than that which determines spontaneous release rate.The stimulatory effect of TPA was enhanced 2-fold by carrying out the experiments in a partially depolarising saline (10 mmol/l K⁺). When TPA was applied to preparations bathed in Ca²⁺-free depolarising saline, the response to the drug was still significantly greater than that in non-depolarised preparations. It is concluded that responsiveness to TPA is enhanced by depolarisation, but that little, if any, of this enhancement can be attributed to the consequent influx of Ca²⁺.Send offprint requests to S. J. Publicover at the above addressPEL was in receipt of an S.E.R.C. postgraduate awardZYS was in receipt of financial support from Umm Al Qura University, Saudi Arabia     

Improved diastolic function with the calcium antagonist nisoldipine (coat-core) in patients post myocardial infarction: results of the DEFIANT study

A multicentre, randomized, double-blind, placebo-controlled,parallel-group trial was undertaken in 135 patients to determinewhether 4 weeks of treatment with long-acting nisoldipine coat-core(20 mg once a day) could alter diastolic function in patientswith a recent myocardial infarction and with mild left ventriculardysfunction as indicated by a left ventricular ejection fraction50%. The primary endpoint was the change in diastolic fillingparameters assessed by Doppler and two-dimensional echocardiography. The mean time of admission to the study was 20 days (range 7–35)after myocardial infarction. Mean left ventricular ejectionfraction was 41%. The drug increased early diastolic peak velocityat the tips of the mitral leaflet by 0·06 m . s^–1(95% confidence intervals (CI): 0·01, 0·11). Thetime velocity integral was increased by 1·2 cm (95% CI:0·16, 2·27). These findings are indicative ofincreased early diastolic flow across the mitral valve. An importantdeterminant appeared to be a reduced isovolumic relaxation time(by 14·7 ms, 95% CI: -22·5, -6·9). As therewas no change in heart rate, systolic and diastolic blood pressureor cardiac output, after load reduction appeared unlikely asan explanation. Peak workload on exercise was 12 watts higherin the group on nisoldipine (95% CI: 0·8, 23·3).Thus, nisoldipine was shown to improve indices of diastolicventricular function, as well as exercise capacity, in thisgroup of patients. The observed effects of nisoldipine may reflectan anti-ischaemic effect or be due to improved relaxation ofthe myocardium.     

The role of intracellular calcium stores in motilin induced contractions of the longitudinal muscle of the rabbit duodenum

Summary The contraction of longitudinal muscle strips of the rabbit duodenum in response to motilin and acetylcholine was investigated in normal and high K⁺-solutions in the presence and absence of external calcium, in order to demonstrate the existence of pharmaco-mechanical coupling for motilin and to examine whether the peptide mobilizes calcium from an intracellular store. In depolarized smooth muscle (140 mM K⁺), motilin (3.2×10⁹ –1×10^–7 M) and acetylcholine (1×10^–5 M) were still capable of causing a considerable, transient, concentration-dependent contraction in the presence of Ca²⁺. The extra-contraction to motilin was not blocked by tetrodotoxin (1 g/ml) nor by atropine (10^–7 M), but acetylcholine (10^–5 M) was blocked by atropine. Verapamil (10^–7 M) could selectively block the K⁺ contraction without affecting the extra agonist contraction. Nitroprusside was ineffective up to 10^–4 M in high K⁺-solutions, but in normal Hepes-buffer it caused a concentration-dependent rightward shift of the concentration-response curve of motilin and acetylcholine contractions. In a calcium-depleted medium, high K⁺-depolarized muscle strips were still responsive to motilin and acetylcholine, but higher concentrations (10^–6 M) were needed than in the presence of calcium and the contractions reached only 57 +- 11% and 74 +- 9% respectively of the maximal contraction in 1.2 mM Ca²⁺ containing solutions. The response to motilin (10^–6 M) was not only smaller than that to acetylcholine (10^–5 M), it also faded more rapidly with time. The response to one agonist could not be repeated except by using a higher concentration of the same or the other agonist, and the magnitude of this second response depended upon the dose used in the first one. We conclude that pharmaco-mechanical coupling exists for motilin and that this peptide is able to elicit contractions by mobilization of calcium from an intracellular store. This store overlaps with the one used by acetylcholine. Our experiments also reinforce the hypothesis that in the rabbit motilin exerts a direct action upon smooth muscle cells.     

Nitric oxide synthesis in endothelial cytosol: Evidence for a calcium-dependent and a calcium-independent mechanism

Summary Release of nitric oxide (NO) from endothelial cells critically depends on a sustained increase in intracellular free calcium maintained by a transmembrane calcium influx into the cells. Therefore, we studied whether the free cytosolic calcium concentration directly affects the activity of the NO-forming enzyme(s) present in the cytosol from freshly harvested porcine aortic endothelial cells. NO was quantified by activation of a purified soluble guanylate cyclase coincubated with the cytosol. In the presence of 1 mM L-arginine, 0.1 mM NADPH and 0.1 mM EGTA, endothelial cytosol (0.2 mg of cytosolic protein per ml) stimulated the activity of guanylate cyclase 5.0 + 0.5-fold (from 31 + 9 to 153 + 15 nmol cyclic GMP formed per min per mg guanylate cyclase). Calcium chloride increased this stimulation further in a concentration-dependent fashion by up to 136 + 15% (with 2 M free calcium; EC₅₀ 0.3 M). The calcium-dependent and -independent activation of guanylate cyclase was enhanced by superoxide dismutase (0.3 M) and was inhibited by the stereospecifically acting inhibitor of L-arginine-dependent NO formation N^G-nitro-L-arginine (1 mM) and by LY 83583 (1 M), a generator of superoxide anions. Our findings suggest a calcium-dependent and -independent synthesis of NO from L-arginine by native porcine aortic endothelial cells. Send of fprint requests to A. Mülsch, at the above address     

Changes in the inducibility of a hepatic aldehyde dehydrogenase by various effectors

A hepatic soluble aldehyde dehydrogenase (ALDH), inducible by polycyclic aromatic hydrocarbons, was studied in Wistar rats in connection with substances known to affect drug metabolism or aldehyde dehydrogenase activity, such as phenobarbital (PB), disulfiram (DS), -diethylaminoethyl diphenylpropylacetate (SKF 525A) and calcium cyanamide (CC). 3-Methylcholanthrene (MC) was given as a model inducer of ALDH (100 mg/kg, i.p., as a single dose) and the animals were killed after 3 days. Pretreatment with PB (1 g/l drinking water, for 2 weeks) enhanced the inducing effect of MC. On the contrary, pretreatment with DS (100 mg/kg, i.p., daily x4) reduced by 70% the expected increase in ALDH activity. Neither SKF 525A (25 mg/kg, i.p., daily x4), nor CC (5 mg/kg, i.p., daily x4) could affect the action of the inducer. At the above doses, basal ALDH activity was inhibited by DS (30%) and CC (70%), but was not affected at all by PB or SKF 525A. The results were somewhat different when the various effectors tested were administered to animals already treated with MC (20 mg/kg, i.p., daily x6). In this case, DS did not affect the already induced ALDH activity. On the contrary, CC was still an effective inhibitor. Unexpectedly, post-treatment with SKF 525A further enhanced the initial induction brought about by MC. Our findings show that substances affecting microsomal drug metabolism can interfere with the process of ALDH induction by MC. The additive result of PB pretreatment is probably due to the enhanced accumulation of an active metabolite of MC. The opposite effect of DS on drug metabolism could explain the decreased ability of MC to induce ALDH activity. The MC-inducible ALDH isozyme can be effectively inhibited with CC, but not with DS.     

Subsynaptosomal distribution of calcium during aging and 3,4-diaminopyridine treatment

Since previous studies showed that calcium uptake by synaptosomes from rodents declines with aging [30], the subsynaptosomal distribution of calcium was determined with the disruption method of Scott et al. [37]. Calcium uptake by the mitochondrial (digitonin-resistant) and non-mitochondrial (digitonin-labile) compartments, as well as total uptake, were determined at 2, 5 and 10 min. After a 10 min incubation under resting conditions (5 mM-KCl), total calcium uptake decreased at 10 months (−14.6%) and 30 months (−33.0%) of age; mitochondrial calcium uptake increased by 10 months (+11.2%) but declined by 30 months (−17.5%); the nonmitochondrial calcium compartment declined at 10 (−34.7%) and 30 (−43.4%) months when compared to the 3 month old control. With potassium depolarization (31 mM-KCl), total calcium uptake declined from 100% (3 months) to 73.8% (10 months) or 53.0% (30 months); mitochondrial calcium uptake declined from 100% (3 months) to 85.6% (10 months) or 68.4% (30 months); non-mitochondrial calcium uptake decreased at 10 (−34.3%) and 30 (−57.7%) months of age when compared to 3 months (100%). The deficits in calcium homeostasis are not due to changes in synaptosomal volumes or to diminished membrane potentials, as assessed by tetraphenylphosphonium ion accumulation. 3,4-Diaminopyridine partially reversed the alterations in total, mitochondrial and non-mitochondrial calcium uptake by synaptosomes from aged mice.     

Evaluation of the osmoregulatory function of taurine in brain cells

Summary Homogenous primary cultures of mouse astrocytes and cortical neurons were used to clarify the role of taurine in ion and osmoregulation in the CNS. This study indicates that both neurons and glial cells have uptake systems for taurine. The cell water content does not change during loading of cells with taurine. Chemical analysis indicates that part of the accumulated taurine is metabolized and that the product(s) are stored in the cells. Extracellular taurine (1 mM) has no effect on K⁺, Na⁺, Cl^-, or Ca²⁺ movements in astrocytes. However, astrocytes loaded to a taurine content which corresponds a concentration of 60 mM (corresponds to normal mouse cortex levels) show a 50% reduction in their K⁺ accumulation by carriers and a 100% increase in Ca²⁺ turnover rates. Movements of Ca²⁺ and K⁺ are involved in neurotransmission. It appears that taurine stored in glial cells, has an important effect on ion homeostasis in the CNS and may act indirectly on neuronal excitability.     

Evolution of Vertebroplasty: A Biomechanical Perspective

This paper is a collection of computational, finite element studies on vertebroplasty performed in our laboratory, which attempts to provide new biomechanical evidence and a fresh perspective into how the procedure can be implemented more effectively toward the goal of preventing osteoporosis-related fractures. The percutaneous application of a bone cement to vertebral defects associated with osteoporotic vertebral compression fracture has proven clinical successful in alleviating back pain. When the biomechanical efficacy of the procedure was examined, however, vertebroplasty was found to be limited in its ability to provide sufficient augmentation to prevent further fractures without risking complications arising from cement extravasations. The procedure may instead be more efficient biomechanically as a prophylactic treatment, to mechanically reinforce osteoporotic vertebrae at risk for fracture. Patient selection for such intervention may be reliably achieved with the more accurate fracture risk assessments based on vertebral strength, predicted using geometrically detailed, specimen-specific finite element models, rather than on bone density alone. Optimal cement volume, placement, and material properties were also recommended. The future of vertebroplasty involving biodegradable augmentation material laced with osteogenic agents that upon release will stimulate new bone growth and increase bone mass was proposed.     

多孔碳酸化羟基磷灰石骨水泥的生物相容性研究

目的探讨一种新型的代骨材料--多孔碳酸化羟基磷灰石骨水泥的组织相容性.方法合成碳酸化羟基磷灰石骨水泥,添加成孔剂,制备能原位固化形成多孔结构的碳酸化羟基磷灰石代骨材料,并通过细胞毒性实验和肌内埋植实验检测其组织相容性.结果多孔碳酸化羟基磷灰石骨水泥的浸提液对骨髓基质细胞的生长无影响.细胞于材料表面的生长速度、形态与空白对照组无差别.肌内植入实验发现,材料周围纤维组织包膜的最大厚度为22.5μm,未发现炎性细胞反应.结论多孔碳酸化羟基磷灰石骨水泥具有良好的组织相容性,材料测试结果符合和标准.     

Lysophosphatidic acid induces human natural killer cell chemotaxis and intracellular calcium mobilization

We have investigated the effects of the bioactive lipid lysophosphatidic acid (LPA) on several functions of activated human natural killer (NK) cells. Flow cytometric and immunoblot analyses show that these cells express LPA(1, )LPA(2) and LPA(3). LPA but not its precursor phosphatidic acid (PA) induces the chemotaxis of NK cells, an activity that is inhibited by prior treatment of the cells with pertussis toxin (PTX). In addition, LPA induces the mobilization of intracellular calcium, an effect that is markedly inhibited by PTX, but is not inhibited by the addition of EGTA. PA also induces calcium flux in NK cells, but with much lower efficacy than LPA. Cross-desensitization experiments demonstrate that LPA and PA utilize different receptors. Moreover, LPA or PA but not sphingosine 1-phosphate, enhances IFN-gamma secretion by activated NK cells. Our results may shed some light on the findings that activated NK cells are found at the sites of tumor growth.