Aron Jurkiewicz

Mitochondrial calcium, oxidative stress and apoptosis in a neurodegenerative disease model induced by 3‐nitropropionic acid

Intracellular calcium homeostasis is important for cell survival. However, increase in mitochondrial calcium (Ca2+m) induces opening of permeability transition pore (PTP), mitochondrial dysfunction and apoptosis. Since alterations of intracellular Ca2+ and reactive oxygen species (ROS) generation are involved in cell death, they might be involved in neurodegenerative processes such as Huntingtons disease (HD). HD is characterized by the inhibition of complex II of respiratory chain and increase in ROS production. In this report, we studied the correlation between the inhibitor of the complex II, 3‐nitropropionic acid (3NP), Ca2+ metabolism, apoptosis and behavioural alterations. We showed that 3NP (1 mm) is able to release Ca2+m, as neither Thapsigargin (TAP, 2 µm) nor free‐calcium medium affected its effect. PTP inhibitors and antioxidants inhibited this process, suggesting an increase in ROS generation and PTP opening. In addition, 3NP (0.1 mm) also induces apoptotic cell death. Behavioural changes in animals treated with 3NP (20 mg/kg/day for 4 days) were also attenuated by pre‐ and co‐treatment with vitamin E (VE, 20 mg/kg/day). Taken together, our results show that complex II inhibition could involve Ca2+m release, oxidative stress and cell death that may precede motor alterations in neurodegenerative processes such as HD.

Evidence for participation of nitric oxide in excitatory neurotransmission in rat vas deferens

A depression of the fast, non-adrenergic, and also of the slow, adrenergic, components of muscle contraction in response to intramural nerve stimulation was induced by the blocker of nitric oxide synthase NG-nitro-L-arginine methyl ester (L-NAME), in rat vas deferens. Effects of exogenous noradrenaline or ATP were not reduced by L-NAME. However, L-arginine also caused an inhibition of electrically induced effects in most of the preparations, contrary to the expectations for a precursor of nitric oxide synthesis. In spite of these difficulties L-arginine antagonized the action of L-NAME. These results indicate that nitric oxide is involved in excitatory nerve-muscle transmission in vas deferens.

Effect of full agonists following calcium deprivation in rat vas deferens

The contractile effects of maximum doses of adrenaline, noradrenaline, methacholine, acetylcholine, serotonin and barium chloride were studied following substitution of a medium without calcium for the normal nutrient solution. Except for the last agonist, the effects fall to about 10% within the first 3 min with prompt return to normal value upon reintroduction of regular fluid. This recovery is, however, slower if the previous incubation in Ca-free solution is prolonged. When barium chloride is used in a calcium-free medium, the maximum height of contractions falls exponentially at a t1/2 of about 180 min. This decay can be accelerated by giving successive 5-min doses of the agonist or by using EDTA. It is hypothesised that excitation--contraction coupling in rat vas deferens depends on at least two different calcium sources: a deep site associated with the effects of barium, and a superficial one, related to the other agonists. To explain the slow recovery after prolonged calcium lack, a third compartment in series with the latter is suggested. No indication is found that the biphasic effects of barium depend on two different calcium pools.

Functional properties of agmatine in rat vas deferens

Experiments were performed with rat vas deferens to verify whether agmatine, an endogenous ligand for adrenoceptors and imidazoline receptors, can influence sympathetic neurotransmission, with respect to contractions induced by transmural nerve stimulation, contractions induced by exogenous noradrenaline, and overflow of endogenous noradrenaline. It was shown that agmatine (a) caused a dose-dependent potentiation of electrically induced twitches, up to about 70% in relation to controls, (b) shifted to the right the inhibitory concentration-response curves for clonidine on electrically induced twitches, indicating competitive antagonism at presynaptic alpha-adrenoceptors, with a pA2 value of 4.12 +/- 0.10, (c) shifted to the right the concentration-response curves for noradrenaline-induced contractions, indicating competitive antagonism at postsynaptic alpha-adrenoceptors as well, with a pA2 value of 4.03 +/- 0.10, and (d) caused a dose-dependent increase of KCI-induced overflow of noradrenaline, up to about 90% in relation to controls. It is concluded that agmatine has multiple effects on sympathetic neurotransmission in rat vas deferens.

In vitro denervation of the rat vas deferens through hypothermic storage

1 The rat vas deferens was excised, stored at 4–6°C and tested after 24, 48, 72 or 96 h for its contractile activity and for the presence of innervation. 2 The maximal contractile capacity of the vas, tested through cumulative concentrations of barium chloride (3 × 10−2 m) was progressively reduced from about 110 mm to about 63 mm after 72 h, without further decay after 96 h. Spontaneous rhythmic contractions were practically absent. 3 A loss of endogenous pools of catecholamines was indicated by four parameters: (a) a decline of about 80% after 24 h and of more than 95% after 48 h of the contractile effect of the indirect sympathomimetic agonist tyramine; (b) a fall of about 20%, 50% and 85% on the concentration of noradrenaline, respectively after 24, 48 and 72 h; (c) a fall of about 25% and 90% after respectively 24 and 48 h, of the activity of dopamine‐β‐hydroxylase (DBH); (d) a decline of noradrenaline‐induced histofluorescence on cross sections of the vas. 4 A loss of neuronal uptake capacity was indicated by: (a) a progressive variation of the apparent affinity for adrenaline, expressed as pD2 values, that increased by about 1.5 log units (corresponding to a 30 fold potentiation) after 72 h, and (b) a reduction of the ability of cocaine to potentiate the contractile effects of adrenaline. 5 The pD2 values for barium chloride, 5‐hydroxytryptamine (5‐HT) and histamine were not significantly changed, while the corresponding value for acetylcholine was slightly but significantly reduced by about 0.8 log units. 6 The maximal heights of concentration‐response curves for noradrenaline, acetylcholine, histamine and 5‐HT were reduced by 42–66% in relation to controls. However, when this reduction was measured in relation to the corresponding barium effect, by means of the relative responsiveness ratio (p), a small though significant increase was observed for noradrenaline, and a fall for the other drugs. 7 It is concluded that: (1) the values for the various biochemical and pharmacological parameters decline at different rates, though revealing altogether that denervation is completed by at least 85% after 72 h of hypothermic storage; (2) two of the results, i.e., the lack of spontaneous rhythmic contractions and the lack of increased contractile effects for acetylcholine, 5‐HT and histamine, indicate that in these conditions the vas is devoid of the so‐called nonspecific signs of denervation.

Low dihydropyridine receptor density in vasa deferentia of castrated rats.

Radioligand binding studies in crude membrane preparations of vasa deferentia of normal rats, with the 1,4‐dihydropyridine (+)‐[3H]‐PN200–110 (isradipine) showed typical saturation isotherms. The binding exhibited a KD of 259 ± 60 pm and Bmax of 144 ± 20 fmol mg−1 protein. The low KD and the stereoselective displacement of (+)‐[3H]‐PN200–110 binding by (+)‐ and (−)‐PN200–110 and by nifedipine suggests that these tissues contain dihydropyridine receptors probably coupled to voltage‐sensitive, L‐type calcium channels. In membrane preparations from vasa deferentia from rats castrated 30 days previously the maximum specific binding was 25 ± 10 fmol mg−1 protein, representing only 11% of total binding; thus, the calculation of reliable KD values was not feasible. These findings suggest that a testicular hormone, possibly testosterone, plays an important role in the regulation of dihydropyridine‐sensitive, voltage‐dependent calcium channels in the rat vas deferens.

Role of the Endoplasmic Reticulum and Mitochondria on Quantal Catecholamine Release from Chromaffin Cells of Control and Hypertensive Rats

Here, we present the first study on the effects of compounds that interfere with calcium (Ca2+) handling by the endoplasmic reticulum (ER) and mitochondria on amperometrically measured quantal catecholamine release from single adrenal chromaffin cells of control and spontaneously hypertensive rats (SHRs). Acetylcholine (ACh) or K+ pulses triggered spike bursts of secretion by Ca2+ entry through Ca2+ channels. ER Ca2+ release triggered by a mixture of caffeine, ryanodine, and thapsigargin (CRT) or carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (a mitochondrial protonophore) also caused bursts of secretory spikes. The spike bursts generated by ACh, K+, CRT, and FCCP were 3 to 4 times longer in SHRs compared with control cells; furthermore, the individual spikes were faster and had 3-fold greater quantal size. In additional experiments, a 90-s treatment was made with CRT or FCCP to block Ca2+ handling by the ER and mitochondria. In these conditions, the integrated spike burst responses elicited by ACh and K+ were potentiated 2- to 3-fold in control and SHR cells. This suggests that variations in Ca2+ entry and its subsequent redistribution into the ER and mitochondria are not responsible for the greater secretion seen in SHRs compared with control cells; rather, such differences seem to be due to greater quantal content of spike bursts and to greater quantal size of individual amperometric events.

ω-Conotoxins block neurotransmission in the rat vas deferens by binding to different presynaptic sites on the N-type Ca2+ channel

Electrically-induced twitch responses of the prostatic segment of vas deferens (0.1 Hz, 65 V, 1 ms) are mainly due to the transient presynaptic release of ATP, which acts postsynaptically on non-adrenergic receptors to contract smooth muscle cells. These responses were fully blocked by nanomolar concentrations of the omega-conotoxins GVIA, MVIIA, and MVIIC, most likely by inhibiting Ca2+ entry through presynaptic N-type Ca2+ channels controlling the release of ATP. Repeated washout of the toxins allowed the recovery of contractions, except for omega-conotoxin GVIA, whose inhibitory effects remained unchanged for at least 60 min. In addition, micromolar concentrations of omega-conotoxin MVIIC were unable to protect against the irreversible inhibition of twitch contractions induced by nanomolar concentrations of omega-conotoxin GVIA. At low extracellular Ca2+ concentrations (1.5 mM), 20 nM of omega-conotoxin GVIA or MVIIA inhibited completely the twitch contractions in about 10 min. In 5 mM Ca2+ the blockade of twitch contractions after 10 min was 70% for both toxins. In 1.5 mM Ca2+ omega-conotoxin MVIIC (1 microM) inhibited completely the twitch contraction after 10 min. In 5 mM Ca2+ blockade developed very slowly and was very poor after 30 min, omega-conotoxin MVIIC depressed the response by only 20%. These results are compatible with the idea that the three omega-conotoxins block the purinergic neurotransmission of the vas deferens by acting on presynaptic N-type voltage-dependent Ca2+ channels. However, omega-conotoxin MVIIC seems to bind to sites different from those recognised by omega-conotoxin GVIA and MVIIA, which are markedly differentiated by their Ca2+ requirements for binding to their receptors.

Ca2+ release-activated channels in rat stomach smooth muscle cells.

In rat stomach fundus, contractions induced by Ca2+ (1.8 mM) were strikingly potentiated by thapsigargin. This potentiation was partially inhibited by the blockers of Ca2+ release activated channels (CRACs), miconazole and SK&F96365 ([1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole, HCL]) and slightly blocked by the antagonist of calcium voltage-operated channels (VOCs), isradipine. In dissociated cells in a 0Ca solution, thapsigargin potentiated the increase in intracellular calcium after reintroduction of Ca2+. This potentiation was partially reduced by the CRAC blockers, but not by the VOC blockers. This data suggests that calcium influx increased due to the depletion of intracellular calcium by thapsigargin and that this influx occurs predominantly through CRACs.

Single-Vesicle Catecholamine Release Has Greater Quantal Content and Faster Kinetics in Chromaffin Cells from Hypertensive, as Compared with Normotensive, Rats

In a previous study performed in the intact adrenal gland (Lim et al., 2002), stimulation with acetylcholine (ACh) or high K+ concentrations (K+) produced greater catecholamine release in spontaneously hypertensive rats (SHR), as compared with normotensive animals. In this study, the time course of secretion was in the range of minutes. Hence, we do not know whether enhanced release is due to greater quantal content and/or distinct kinetics in SHRs and control animals. To get insight into the mechanism involved in such enhanced catecholamine secretory responses, we performed a single-vesicle release study in primary cultures of adrenal chromaffin cells, recorded with amperometry. Cells were stimulated with 2-s pulses of 1 mM ACh or 70 mM K+. The secretory responses to ACh or K+ pulses in SHR cells as compared with control cells had the following characteristics: 1) double number of secretory events, 2) 4-fold augmentation of total secretion, 3) cumulative secretion that saturated slowly, 4) 3-fold higher complex events with two to four superimposed spikes that may be explained by faster spike kinetics, 5) about 2- to 3-fold higher event frequency at earlier post stimulation periods, and 6) 2- to 5-fold higher quantal content of simple spikes. We conclude that SHR cells have faster and larger catecholamine release responses, explained by more vesicles ready to undergo exocytosis and greater quantal content of vesicles. This could have relevance to further understand the pathogenic mechanisms involved in the development of high blood pressure, as well as in the identification of new drug targets to treat hypertension.