CSF omeprazole concentration and albumin quotient following high dose intravenous omeprazole in dogs.

Clinical signs of syringomyelia and hydrocephalus occur secondary to cerebrospinal fluid (CSF) accumulation within the central nervous system. Omeprazole is recommended to treat these conditions despite little evidence of its capacity to decrease CSF production in the dog. Studies into new treatments are hampered by difficulties in measuring CSF production. The albumin quotient (QAlb), the ratio between CSF and serum albumin concentrations, may reflect CSF production and any decrease in CSF production should be associated with an increase in QAlb. The primary objective of this study was to determine CSF omeprazole concentration after administration of a high intravenous dose of omeprazole and to evaluate its impact on QAlb in the dog. The second aim was to validate QAlb as a surrogate marker of CSF production. Eighteen dogs were included in this prospective crossover placebo-controlled study. Each dog received omeprazole (10 mg/kg), acetazolamide (50 mg/kg) combined with furosemide (1 mg/kg) and saline. Blood and CSF samples were obtained on day 0 and then every 7 days, one hour after drug administration. Omeprazole concentrations (2.0 ±â€¯0.4 µmol/L) reached in CSF after high dose omeprazole were lower than the concentrations previously described as decreasing CSF production in dogs. There was no significant increase in QAlb following administration of acetazolamide/furosemide, prohibiting validation of QAlb as a surrogate marker for CSF production. Several dogs presented transient mild side effects after injection of acetazolamide/furosemide. High dose omeprazole was well tolerated in all dogs.

New insights on culture and calcium signalling in neurons and astrocytes from epileptic patients.

Primary brain cell cultures are a useful tool for understanding the physiopathology of epilepsy and for searching new potential antiepileptic drugs. These cell types are usually prepared from murine species and few human models have been described. The main goal of this study is the establishment of experimental conditions to isolate and culture neurons and astrocytes from human brain and to test its functionality. The tissues came from antiepileptic drug-resistant epileptic patients undergoing surgery. Human neurons and astrocytes were isolated following an enzymatic and mechanical dissociation protocol. Cultures were viable for 3-6 weeks. Cytological characterization was performed by immunocytochemistry using specific antibodies against both neuron (anti-NeuN) and astrocyte (anti-GFAP) protein markers. In order to test their viability and functionality, cells were loaded with the fluorescent calcium probe fura-2 and variations in cytosolic calcium concentrations ([Ca2+]c) were measured by cell imaging. [Ca2+]c increases were evoked upon cell stimulation with high K+ (KCl 75 mM), glutamate (500 µM) or bicuculline (100 µM). Interestingly, spontaneous [Ca2+]c transients were also observed in some neuron-like cells. A novel unreported finding in this study has been the incorporation of human serum that was critical for cell functionality. The setting of these human cultures open the opportunity to new insights on culture and calcium signalling studies on the mechanism(s) of cell resistance to antiepileptic drugs, as well as to studies on plasticity, maturation and possible neurite emission for graft studies.

Calcium-dependent inhibition of L, N, and P/Q Ca2+ channels in chromaffin cells: role of mitochondria.

The hypothesis that the buffering of Ca(2+) by mitochondria could affect the Ca(2+)-dependent inhibition of voltage-activated Ca(2+) channels, (I(Ca)), was tested in voltage-clamped bovine adrenal chromaffin cells. The protonophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), the blocker of the Ca(2+) uniporter ruthenium red (RR), and a combination of oligomycin plus rotenone were used to interfere with mitochondrial Ca(2+) buffering. In cells dialyzed with an EGTA-free solution, peak I(Ca) generated by 20 msec pulses to 0 or +10 mV, applied at 15 sec intervals, from a holding potential of -80 mV, decayed rapidly after superfusion of cells with 2 microm CCCP (tau = 16.7 +/- 3 sec; n = 8). In cells dialyzed with 14 mm EGTA, CCCP did not provoke I(Ca) loss. Cell dialysis with 4 microm ruthenium red or cell superfusion with oligomycin (3 microm) plus rotenone (4 microm) also accelerated the decay of I(Ca). After treatment with CCCP, decay of N- and P/Q-type Ca(2+) channel currents occurred faster than that of L-type Ca(2+) channel currents. These data are compatible with the idea that the elevation of the bulk cytosolic Ca(2+) concentration, [Ca(2+)](c), causes the inhibition of L- and N- as well as P/Q-type Ca(2+) channels expressed by bovine chromaffin cells. This [Ca(2+)](c) signal appears to be tightly regulated by rapid Ca(2+) uptake into mitochondria. Thus, it is plausible that mitochondria might efficiently regulate the activity of L, N, and P/Q Ca(2+) channels under physiological stimulation conditions of the cell.

Blockade by agmatine of catecholamine release from chromaffin cells is unrelated to imidazoline receptors.

The blockade of exocytosis induced by the putative endogenous ligand for imidazoline receptors, agmatine, was studied by using on-line measurement of catecholamine release in bovine adrenal medullary chromaffin cells. Agmatine inhibited the acetylcholine-evoked release of catecholamines in a concentration-dependent manner (IC(50)=366 microM); the K(+)-evoked release of catecholamines was unaffected. Clonidine (100 microM) and moxonidine (100 microM) also inhibited by 75% and 50%, respectively, the acetylcholine-evoked response. In cells voltage-clamped at -80 mV, the intermittent application of acetylcholine pulses elicited whole-cell inward currents (I(ACh)) that were blocked 63% by 1 mM agmatine. The onset of blockade was very fast (tau(on) = 31 ms); the recovery of the current after washout of agmatine also occurred very rapidly (tau(off = 39 ms). Efaroxan (10 microM) did not affect the inhibition of I(ACh) elicited by 1 mM agmatine. I(ACh) was blocked 90% by 100 microM clonidine and 50% by 100 microM moxonidine. The concentration-response curve for acetylcholine to elicit inward currents was shifted to the right in a non-parallel manner by 300 microM agmatine. The blockade of I(ACh) caused by agmatine (100 microM) was similar at various holding potentials, around 50%. When intracellularly applied, agmatine did not block I(ACh). At 1 mM, agmatine blocked I(Na) by 23%, I(Ba) by 14%, I(K(Ca)) by 16%, and I(K(VD)) by 18%. In conclusion, agmatine blocks exocytosis in chromaffin cells by blocking nicotinic acetylcholine receptor currents. In contrast to previous views, these effects seem to be unrelated to imidazoline receptors.

Mechanisms of blockade by the novel migraine prophylactic agent, dotarizine, of various brain and peripheral vessel contractility.

The novel antimigraineur, dotarizine, inhibited 5-HT (5 hydroxytryptamine)-evoked contractions of rabbit vertebral, aorta, femoral and mesenteric arteries, with IC(50)s of 1.35, 1.40, 0.52 and 1.09 microM, respectively. Flunarizine had little effect on these contractions, while ketanserin was more potent (IC(50)s of 0.17 microM for vertebral, 0.22 microM for aorta, 0.05 microM for femoral and 0.03 microM for mesenteric arteries). At 10 microM, dotarizine caused 40% blockade of K(+)-evoked contractions of rabbit aorta, and 70% inhibition of 5-HT-evoked responses; these values were 30% and 20% for 10 microM flunarizine. Contractions of rabbit aorta elicited by noradrenaline, angiotensin II or prostaglandin F(2alpha) were not affected by 10 microM dotarizine or flunarizine. Ketanserin shifted to the right, in parallel, the concentration-response curves for 5-HT in rabbit aorta; however, dotarizine caused a non-competitive type of blockade, increasing the maximum 5-HT contraction at 30 nM and decreasing it at 3 and 30 microM. K(+)-evoked contractions of rabbit aorta were halved by 3 microM dotarizine in a voltage-independent manner; flunarizine caused a delayed-type, non-reversible post-drug blockade, and exhibited some voltage-dependence. Blockade by nifedipine was voltage-dependent and fully reversible. Ca(2+)-evoked contractions of depolarised bovine middle cerebral arteries were blocked by 1--3 microM dotarizine in a non-surmountable manner. Contraction of these vessels evoked by electrical stimulation was blocked 50% and 70% by 1 and 3 microM dotarizine, respectively. Dotarizine (1--3 microM) also inhibited to a similar extent the K(+)-evoked [(3)H]noradrenaline release from cultured rat sympathetic neurones. These data suggest that the mechanism of blockade by dotarizine of cerebral vessels contractility has three components: (i) presynaptic inhibition of noradrenaline release; (ii) blockade of postsynaptic vascular 5-HT receptors; (iii) blockade of Ca(2+)entry into the vascular smooth muscle cell cytosol. The compound does not affect the vascular receptors for noradrenaline, angiotensin II or prostaglandin F(2alpha).

PF9404C, a new slow NO donor with beta receptor blocking properties.

1. PF9404C is the S-S diesteroisomer of a novel blocker of beta adrenergic receptors with vasodilatory properties. It causes a concentration-dependent relaxation of rat aorta helical strips pre-contracted with 10(-6) M noradrenaline (NA; IC(50) 33 nM). It was equipotent to nitroglycerin (NTG; IC(50) 49 nM), but much more potent than isosorbide dinitrate (ISD; IC(50) 15,000 nM). 2. Oxyhaemoglobin (10 microM) shifted to the right the concentration-response curve for the relaxation induced by PF9404C (IC(50) 530 nM) or NTG (IC(50) 61 nM). 3. Either methylene blue (MB) or ODQ (1 microM each) largely prevented the vasorelaxing responses to increasing concentrations of PF9404C or NTG. 4. In rat aorta smooth muscle cells, PF9404C increased the formation of cyclic GMP from 3 pmol mg(-1) protein in basal conditions, to 53 pmol mg(-1) protein in 10 microM PF9404C. Neither metoprolol nor carvedilol enhanced cyclic GMP. 5. In the electrically driven guinea-pig left atrium, PF9404C blocked the inotropic effects of isoprenaline in a concentration-dependent manner. Its IC(50) (30 nM) was similar to that for S-propranolol (22.4 nM) and lower than the IC(50)s for metoprolol (120 nM) and atenolol (192 nM). The beta-adrenergic ligand (-)-[(3)H]-CGP12177 (0.2 nM) was displaced from its binding to rat brain membranes with K(i) of 7 nM, 17 nM, 170 nM and 1.2 microM respectively for PF9404C, S-(-)propranolol, metoprolol, and atenolol. 6. The data are consistent with the idea that the S-S diesteroisomer PF9404C, is a potent vasorelaxing agent, as well as a blocker of cardiac beta adrenergic receptors. The mechanism of its vasorelaxing effects involves the slow generation of NO. This molecule can, therefore, exhibit antihypertensive and cardioprotective actions through a double mechanism, NO donation and beta blockade.