Activity-dependent survival and enhanced turnover of calcium in cultured rat cerebellar granule neurons

Neurons survive when their activity is maintained. An influential hypothesis on the cellular mechanism underlying this phenomenon is that there is an appropriate range of intracellular Ca²⁺ concentration ([Ca²⁺]i) for survival. The rat cerebellar granule neuron in culture serves as the most often used model system for the analysis of activity-dependent survival, since it does not survive unless an excitant (KCl or glutamate) is added to the culture medium. Against the above-mentioned hypothesis, we found in our previous examination no difference between steady-state [Ca²⁺]i in granule neurons cultured under high KCl (i.e., survival) and low KCl (i.e., death) conditions. In this report, we present the quantitative background of unchanged [Ca²⁺]i between the two culture conditions. Influx of Ca²⁺ due predominantly to L-type voltage-dependent calcium channels was higher in high KCl cultures than in low KCl cultures. At the same time, efflux of Ca²⁺ due to the activity of Ca²⁺/Na⁺ antiport was also higher in high KCl cultures. Additionally, we found that the endocytotic activity was greater in high KCl cultures than in low KCl cultures, as monitored by the rate of uptake of horseradish peroxidase added to medium. Since the uptake was blocked by an internal Ca²⁺ chelator, the increased endocytotic activity in high KCl cultures might be a consequence of the enhanced Ca²⁺ turnover.     

The effects of an intracellular calcium antagonist HA 1077 on delayed cerebral vasospasm in dogs

Summary The effectiveness of calcium antagonists on a chronic cerebral vasospasm after an SAH is still under debate. Calcium channel blockers such as nimodipine, nefedipine etc. can dilate spastic arteries by intrathecal administration, but not by systemic (iv or po) use. HA 1077 is a novel and potent calcium antagonist vasodilator which is considered to act by employing different mechanisms from the usual calcium channel blockers since it inhibits 1. calcium ionophore A 23187 induced contraction in arterial strips and 2. phenylephrine induced contraction in calcium free media, suggesting that its site of action is in the intracellular space. HA 1077 is water soluble and relatively stable in light.In the present study, the efficacy of HA 1077 was evaluated on dogs by using the spiral arterial strips in vitro and by angiography in vivo. In the arterial strips from the control dogs, a 50% relaxation of KCl (15 mM) induced contraction was obtained by a 10^–6 M HA 1077 for the intracranial basilar and middle cerebral arteries, while a 10^–5 M was needed to obtain the same effect for the extracranial common carotid and vertebral arteries, indicating that HA 1077 is more effective for the intracranial arteries. A vasospasm was produced by the two haemorrhage model of Varsoset al. The average angiographic diameter of the basilar artery was reduced to 60% of the control on SAH day 7. Intravenous infusion of HA 1077 (0.5–3 mg/kg/30 min) significantly dilated the spastic basilar artery (up to 20–30%), for over 2 hours. A fall in the systemic BP remained less than 20% during this time. Such spasmolytic effects by intravenous administration could not have been obtained with the usual calcium channel blockers. HA 1077 may be suitable for the treatment of a vasospasm in humans as well.     

Circulating 1,25-Dihydroxycholecalciferol After Intravenous Injections of l{alpha}-Hydroxycholecalciferol in Patients on Regular Haemodialysis

The formation of 1.25-dihydroxycholecalciferol (1.25-(OH)₂D₃after single intravenous injections of 1-hydroxycholecalciferol(1-OHD₃) was examined in four patients with chronic renal failureon regular haemodialysis. Following 1–3µg 1-OHD₃administered at weekly intervals, 1.25-(OH)₂D₃ appeared in thecirculation within 1 h, and peak concentrations were reachedbetween 2 h and 5 h. By 8 h serum 1.25-(OH)₂D₃ concentrationshad started declining and by 44 h they had returned to baselineafter 1µg 1-OHD₃ but they were still above basal after2 and 3 µg by an average of 30 pmol/l. One week afterinjections, concentrations were back to basal in all patientsstudied. The serum 1,25-(OH)₂D₃ dose response to injected la-OHDwas linear, indicating ample capacity of the liver 25-hydroxylaseto further hydroxylate 1-OHD. However, examination of the individualresponses revealed lower increments in serum 1.25-(OH)₃ concentrationsin the patients with the highest basal serum 25-hydroxyvitaminD concen trations. Intravenous 1-OHD₃ may be useful in the furtherstudy of the interactions between 1.25-(OH)₂₃ calcium and PTHin chronic renal failure, as well as of the hepatic metabolismof vitamin D.     

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 regional distribution of adenosine-regulating enzymes in the left and right ventricle walls of control and hypertrophic heart

Summary The transmural distribution of the adenosine-generating enzyme 5-nucleotidase (5N) and of the adenosine-degrading enzymes adenosine deaminase (ADA), AMP deaminase (AMP-D) and adenosine kinase (Ado-K) were determined across the walls of left and right ventricles of control and hypertrophic rat hearts.The enzyme distribution across the left ventricle wall (but not across the right wall) of normal hearts was not uniform: 5N activity shows its highest levels in the subepicardial and in the subendocardial regions, whereas all the other enzyme activities show their lowest levels. A similar pattern of transmural distribution was also detected in other mammalian species (ox and pig).In the experimental cardiac hypertrophy, caused by two different types of chronic cardiac overload, the levels and the profiles of transmural distribution of 5N and ADA enzyme activities may significantly change across the rat left ventricle wall.     

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.