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.     

Differential potentiation by calcium antagonists of neuromuscular blockade induced by pancuronium and succinylcholine in cats in vivo

Summary The effects of several calcium antagonists (verapamil, nicardipine and two diltiazem isomers, d-cis and l-cis diltiazem) alone and associated to non-depolarizing (pancuronium) and depolarizing (succinylcholine) neuromuscular blockers, were evaluated on sciatic nerve-tibialis anterior muscle preparations from cats in vivo. The calcium antagonists used (at 0.1 and 0.5mg/kg iv) did not modify the height of muscular twitches elicited indirectly. However, these agents potentiated in a dose-dependent way the neuromuscular blockade induced by iv pancuronium (2–40g/kg) and succinylcholine (6–200g/kg). The order of potency in increasing the effects of pancuronium was nicardipine d-cis diltiazem verapamil, whereas the order of potency in enhancing succinylcholine effects was d-cis diltiazem verapamil nicardipine. The effects of diltiazem were stereoselective, thus the potentiation induced by d-cis diltiazem was significantly greater in all cases than that induced by l-cis diltiazem, which suggests that calcium channel blockade plays a role in these interactions. However, other mechanisms such as calcium antagonists-induced nicotinic receptor desensitization may also be involved.     

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.     

Baclofen: reduction of presynaptic calcium influx in the cat spinal cord in vivo

 In the ventral horn of the lumbar spinal cord of cats anaesthetised with pentobarbitone sodium, microelectrophoretically administered (–)-baclofen, but not (+)-baclofen, reversibly reduced the duration of the orthodromic action potential of muscle group Ia afferent terminations, but not those of muscle group I afferent myelinated fibres. The presumably submicromolar concentrations are already known to reversibly reduce excitatory transmitter release from muscle group Ia afferent terminations. Action potential durations were estimated from threshold recovery curves after an orthodromic impulse using an extracellular microstimulation technique. Both of these presynaptic effects of (–)-baclofen were blocked by baclofen antagonists, and neither appeared to be reduced by the potassium channel blocking agents tetraethylammonium and 4-aminopyridine. Tetraethylammonium and 4-aminopyridine also did not significantly modify the reduction by (–)-baclofen of monosynaptic field potentials in the lumbar cord of rats anaesthetised with pentobarbitone sodium. In the cat the maximum reduction by (–)-baclofen of termination action potentials was considerably less than that produced by cadmium ions, which, unlike (–)-baclofen, also reduced the action potential duration of group I myelinated fibres. These findings are consistent with a reduction by (–)-baclofen of the influx of calcium through voltage-activated channels in the membrane of group Ia terminations, a proposal which also accounts for the reduction by (–)-baclofen of the release of GABA at axo-axonic depolarizing synapses on these terminations. The results are discussed in relation to the mode of action of (–)-baclofen and the different sensitivities of transmitter release at various central synapses. Received: 30 May 1996 / Accepted: 11 September 1996     

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.     

海藻酸钙微胶珠的载药性能优化

利用高压静电法制备了海藻酸钙微胶珠，以牛血红蛋白作为模型药物，考察了牛血红蛋白（Hb）的稳定性、制备条件对微胶珠载药的影响。结果表明：Hb在10℃下能稳定存在，1．8％（w／v）Na-Alg与4．5％（w／v）CaCl2制备的微胶珠载药量较大，真空干燥的微胶殊彼此粘连破坏严重，冷冻干燥则表面出现明显的内陷，乙醇梯度洗脱-真空干燥球形度较好。水凝胶态的微胶珠载药后球形度完好，是理想的载药方式，24h时载药量达28．4％，为干燥后的2～3倍。