Visualization of NMDA receptor-induced mitochondrial calcium accumulation in striatal neurons.

Ca2+ influx through NMDA receptor-gated channels and the subsequent rise in intracellular Ca2+ concentration ([Ca2+]i) have been implicated in cytotoxic processes that lead to irreversible neuronal injury. While many studies have focused on cytosolic Ca2+ homeostasis, much less is known about Ca2+ fluxes in subcellular organelles, such as mitochondria. The mitochondria play an important role in Ca2+ homeostasis by sequestering cytosolic Ca2+ loads. However, mitochondrial Ca2+ overload can impair ATP synthesis, induce free radical formation, and lead to lipid peroxidation. Thus, it is also important to understand the mitochondrial Ca2+ fluxes induced by NMDA. In this study, changes in mitochondrial Ca2+ concentration ([Ca2+]m) in cultured striatal neurons were monitored with a Ca(2+)-binding fluorescent probe, rhod-2, and laser scanning confocal microscopy. The rhod-2 fluorescence signal was highly localized in mitochondrial areas of confocal images. A rapid increase of [Ca2+]m was observed when neurons were treated with 100 microM NMDA. The increased [Ca2+]m induced by NMDA could not be observed in the presence of ruthenium red, an inhibitor of the mitochondrial Ca2+ uniporter, or CCCP, a protonophore that breaks down the mitochondrial membrane potential necessary for Ca2+ uptake. The magnitude and reversibility of changes in [Ca2+]m induced by NMDA were variable. In neurons receiving multiple pulses of NMDA, [Ca2+]m did not return to baseline. The elevated [Ca2+]m may persist indefinitely and may rise further after successive NMDA exposures. These data demonstrate that Ca2+ accumulates in mitochondria in response to NMDA receptor activation. This Ca2+ accumulation may play a role in the excitotoxic mitochondrial dysfunction induced by NMDA.

Comparison of the effects of cimetidine and ranitidine on the pharmacokinetics of disopyramide in man.

The widely prescribed antiulcer agents cimetidine and ranitidine have the potential to affect the absorption, metabolism or renal excretion of disopyramide. This study investigated the effect of a single oral dose of cimetidine or ranitidine on the pharmacokinetics of disopyramide and mono-N-dealkyldisopyramide in six healthy volunteers. The treatment was conducted in a randomized cross-over design. Serum levels and urinary recoveries of disopyramide and mono-N-dealkyldisopyramide were assayed by HPLC. Cimetidine significantly elevated the maximum plasma concentration of disopyramide, the area under the plasma concentration-time curve and the total amount of disopyramide excreted unchanged in the urine, but the serum profile of mono-N-dealkyldisopyramide was not significantly affected. The effects of ranitidine on the pharmacokinetics of disopyramide and mono-N-dealkyldisopyramide were not significant. The interaction between cimetidine and disopyramide occurred mainly at the site of absorption. The results indicate that cimetidine, but not ranitidine, significantly increased the absorption of orally administered disopyramide.

Histamine induces oscillations of mitochondrial free Ca2+ concentration in single cultured rat brain astrocytes.

1. The free Ca2+ concentration of mitochondria ([Ca2+]m) in cultured rat brain astrocytes was measured with a fluorescent Ca2+ indicator, rhod-2, and laser confocal microscopy. 2. Confocal images revealed a rhod-2 distribution that matched mitochondrial localization. 3. Using a Ca2+ ionophore, ionomycin, to clamp the [Ca2+]m from 0 to 100 microM in order to obtain the minimal and maximal fluorescence of rhod-2 in situ, a 3.5 +/- 0.4-fold increase in fluorescence intensity was observed, suggesting that the fluorescence of intramitochondrial rhod-2 was responding in a Ca(2+)-sensitive manner, thereby allowing measurements of [Ca2+]m in single astrocytes. 4. Exposure of fura-2-loaded astrocytes to 100 microM histamine produced a rapid and transient increase in cytosolic Ca2+ concentration ([Ca2+]c) that lasted for several tens of seconds. The spike in [Ca2+]c was frequently followed by variable numbers of repetitive oscillations of Ca2+, which appeared to dampen in amplitude with time. 5. This pattern of histamine-induced [Ca2+]c oscillations was also observed in rhod-2-loaded cells suggesting that [Ca2+]m fluctuated with a similar frequency. 6. The oscillations of [Ca2+]m, but not of [Ca2+]c, were abolished by a proton ionophore, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), and by Ruthenium Red, a mitochondrial Ca(2+)-uniporter inhibitor. 7. These results suggest that the mitochondrial Ca2+ transport systems in cultured rat brain astrocytes are able to relay receptor-mediated [Ca2+]m oscillations into mitochondria.

Differences in ligand binding and phosphoinositide turnover between M1 muscarinic receptor gene transfected cells and mouse and rat brain membranes.

The present study describes some unexpected receptor mediated effects of N-methylcarbamylcholine on mouse M1 muscarinic receptor gene transfected cell line (M1Y1) that were not evident from biochemical studies with mouse and rat brain tissue where N-methylcarbamylcholine exhibited only nicotinic properties. Although N-methylcarbamycholine was devoid of muscarinic properties in mouse and rat brain preparations, as determined by phosphoinositide turnover and inhibition of [3H]QNB binding, it exhibited significant muscarinic characteristics in the transfected M1Y1 cell line. At a concentration of 10(-6) M or greater, N-methylcarbamycholine caused a transient increase in intracellular Ca2+ of 50 s duration that was reversible by atropine or pirezepine. The Ca(2+)-transient was not elicited by other nicotinic agents such as nicotine and N,N-dimethylcarbamylcholine, a close analogue of N-methylcarbamylcholine, with comparable affinity for nicotinic receptors and devoid of muscarinic activity. N-Methylcarbamylcholine also stimulated phosphoinositide turnover in M1Y1 cells with an estimated EC50 value 10 times greater than that of carbachol, and the effect was blocked by atropine. Both carbachol and N-methylcarbamycholine inhibited [3H]QNB binding in a concentration-dependent manner; however, the IC50 for carbachol was over two orders of magnitude greater than that observed in mouse and rat brain membranes. In considering possible explanations for the differential characteristics of N-methylcarbamylcholine in mouse and rat brain as compared to the transfected M1Y1 cells, it was concluded that the difference may be attributable to differences in the receptor-transduction coupling efficiency and the microenvironment of the muscarinic receptors.

Mitochondrial free Ca2+ concentration in living cells.

Evidence has accrued during the past two decades that mitochondrial Ca2+ plays an important role in the regulation of numerous cell functions such as energy metabolism. This implies that mitochondrial Ca2+ transport systems might be able to relay the changes of cytosolic Ca2+ concentration ([Ca2+]c) into mitochondrial matrix for regulating biochemical activities. To substantiate this idea, measurements of intramitochondrial free Ca2+ concentration ([Ca2+]m) become essential. In this article, we review the results from recent studies attempting to measure [Ca2+]m in living cells. In addition, the significance of each study is discussed.

Localization of the stomach-projecting neurons in the dorsal motor nucleus of the vagus nerve in the guinea pig.

The medullary origin of cells of the cervical vagus nerve and the vagal innervation of the stomach in the guinea pig were studied using the retrograde transport of horseradish peroxidase. The horseradish peroxidase was injected into the cervical portion of the vagus nerve, and also into the greater or lesser curvature of the stomach. The animals were perfused with fixative two days after the injection. The medulla oblongata containing the dorsal motor nucleus of the vagus nerve was sectioned and processed histochemically with the tetramethyl benzidine method. The injection of horseradish peroxidase in the cervical vagus nerve resulted in heavy retrograde labelling of neurons in the ipsilateral dorsal motor nucleus and in the nucleus ambiguous. Following the injection of horseradish peroxidase into the greater curvature of the stomach, the stomach-projecting neurons which were bilaterally labelled were localized in the dorsal and dorsolateral part of the dorsal motor nucleus. Although also bilaterally labelled in the dorsal and dorsolateral part of the dorsal motor nucleus, the neurons projecting to the lesser curvature of the stomach were predominantly (approx. 70%) located in the left dorsal motor nucleus. Our study suggests that the parasympathetic preganglionic neurons innervating the greater and lesser curvatures of the stomach are organized viscerotopically in the dorsal motor nucleus in the guinea pig.

Pre- and postsynaptic effects of nicotine on the mouse phrenic nerve-diaphragm preparation.

Nicotine at less than or equal to 33 microM enhanced the single twitch response to indirect stimulation but potentiated the blocking effect of tubocurarine. Failure of tetanic contraction (tetanic fade) occurred on stimulation at 100 Hz. At 76 microM, nicotine induced a first phase rapid (10 min) inhibition of twitch response followed later (60-90 min) by a second phase complete block. Neostigmine partially restored the response at either phase of block whereas diaminopyridine completely antagonized the blockade. The end-plate was depolarized maximally by only 10-15 mV within 30 min with 43 microM nicotine. The depolarization was maintained but was antagonized by tubocurarine. The twitch response induced by direct stimulation was unchanged indicating no depolarization block ensued. The amplitudes of both EPP (0.7 Hz) and MEPP were markedly depressed in parallel indicating a curare-like postsynaptic inhibition without an effect on the release of transmitter. It is concluded that nicotine blocks the neuromuscular transmission by a dual mechanism by its partial agonist action. At higher frequencies of transmission, nicotine (greater than or equal to 22 microM) also produced a remarkable run-down of EPP just like other receptor antagonists suggesting that the nerve terminal acetylcholine receptors are not particularly sensitive to nicotine as those on the autonomic ganglia.