Calcium cell culture

Miller, A. L., et al. (1991). Imaging free calcium in cultured Aplysia bag cell neurons. Biol. Bull. 181 325. 

This finding suggests that the fast calcium events we have recently identified are not a peculiarity of astrocytes in cell culture but may correspond to events taking place in astrocytes of the living brain. Establishing whether fast calcium events in vivo are associated to transmitter release via SLMV exocytosis, becomes, therefore, of the outmost importance in order to define the type of modulatory influence exerted by astrocytes on neighboring neuronal circuits. 

Figure 4.7 Changes in intraceiiuiar calcium in cultured rat ventricular myocytes exposed to oxidant stress. Calcium was measured using the fluorescent probe Fura>2. The ratio of the Fura-2 fluorescence measured at 340 and 380 nm excitation is shown and this is proportional to the intracellular calcium concentration. The fast-speed traces shown (note the 3.5 s time-scale) were recorded after various durations of oxidant stress. Myocytes under control conditions (before t = 0) show spontaneous calcium transients. These transients decreased in frequency with oxidant stress until cells failed to show spontaneous activity but continued to maintain a low intracellular calcium.

During ischaemia, NOS is activated by calcium influx or by cytokines like tumour necrosis factor (TNF) or by lipopolysaccharide (LPS) and NO is produced in excess. It has been proposed that the excitotoxic effect of glutamate, which contributes to ischaemia-induced neuronal damage, is mediated by increased production of NO via a chain of events that includes increases in intracellular calcium (via glutamate activation of NMDA receptors), calcium activation of NOS, production of NO and peroxynitrite, and induction of lipid peroxidation. In fact, N-nitro-L-atginine, a selective inhibitor of NOS, has been shown to prevent glutamate-induced neurotoxicity in cortical cell cultures (Dawson rf /., 1991). 

Artursson P, C Magnusson. (1990). Epithelial transport of drugs in cell culture. II. Effect of extracellular calcium concentration on the paracellular transport of drugs of different lipophilicities across monolayers of intestinal epithelial (Caco-2) cells. J Pharm Sci 79 595-600. 

Calcium antagonists are able to affect nitric oxide production and suppress the peroxyni-trite-induced damage. Thus, nifedipine enhanced the bioavailability of endothelial NO in porcine endothelial cell cultures supposedly through an antioxidative mechanism [288], Pretreatment with nisoldipine, a vascular-selective calcium blocker of dihydropyridine-type, of confluent bovine aortic endothelial cells suppressed the peroxynitrite-induced GSH loss and increased cell survival [289]. 

In regards to necrosis, it is clear that the old adage an ounce of prevention is worth a pound of cure applies. Agents that stabilize ion homeostasis have proved to be effective in preventing necrosis in cell culture studies. For example, drugs that activate plasma membrane potassium ion channels or chloride ion channels can prevent membrane depolarization and so inhibit sodium and calcium ion influx. Agents that prevent large sustained increases in intracellular free calcium levels can also prevent neuronal... 

Cell cultures of Catharanthus roseus entrapped in calcium alginate have been employed by Takemoto and Achiwa to deracemize pyridyl alcohols such as 15 and 16 [17] (Scheme 8). 

While most appfications were performed in suspended cell cultures some authors showed that the application of NADH-dependent fluorescence monitoring is also possible in immobifized cell systems. Here the growth of Clostridium acetobutylicum and the Saccharomyces cerevisiae immobilized in different calcium alginate structures was studied. However, calibration of the culture fluorescence signal with the biomass concentration was not possible but qualitatively an increasing biomass also led to an increase in the fluorescence signals. 

Johnson ME et al Effect of local anesthetic on neuronal cytoplasmic calcium and plasma membrane lysis (necrosis) in a cell culture model. Anesthesiology 2002 97 1466. [PMID 12459673]... 

Macdonald, R. L., Skerritt, J. H., Werz, M. A. Adenosine agonists reduce voltage-dependent calcium conductance of mouse sensory neurones in cell culture, J. Physiol. 1986, 370, 75-90. 

Goldberg M. P. and Choi D. W. (1993). Combined oxygen and glucose deprivation in cortical cell culture Calcium-dependent and calcium-independent mechanisms of neuronal injury. J. Neurosci. 13 3510-3524. 

Marian, M.J., Mukhopadhyay, P., Borchman, D., Tang, D., Paterson, C.A., 2007, Regulation of sarco/endoplasmic and plasma membrane calcium ATPase gene expression by calcium in cultured human lens epithelial cells. Cell Calcium 41, 87—95. 

Cannabinoids may also cause effects via mechanisms distinct from the cannabinoid receptor pathways. The most extensively investigated compound is (+)-HU 211, a synthetic cannabinoid with a stereochemistry opposite to that present in the naturally occurring compounds. It does not produce THC-type effects in animals and shows insignificant binding to the CB, receptor. However, HU 211 blocks A-methyl-n-aspartate (NMDA) receptors and calcium uptake through the NMDA-receptor-ion channel in primary cell cultures. HU 211 is a potent blocker of NMDA-induced tremor, seizures, and lethality in mice. It may therefore prove useful as a nonpsychoactive drug that protects against NMDA-receptor-mediated neurotoxicity. This is supported by the potent attenuation of NMDA-receptor-mediator neurotoxicity in cell cultures by HU 211. 

Exposure of rat primary mixed hippocampal cell cultures to either sodium nitroprusside (SNP 100 xM) or 3-morpholinosydnonimine resulted in both a decrease in cell survival and an increase in free-radical accumulation. These SNP-induced events were blocked by either EGb 761 (10 to 100 pg/ml) or its flavonoid fraction CP 205 (25 pg/ml), as well as by inhibitors of protein kinase C (PKC chelerythrine) and 1-type calcium channels (nitrendipine). In contrast, the terpenoid constituents of EGb 761, known as bilobalide and ginkgolide B, as well as inhibitors of phospholipases A [3-[4-octadecyl)benzoyl]acrylic acid (OBAA)] and C (U-73122), failed to display any significant effects. Moreover, EGb 761 (50 pg/ml), CP 205 (25 pg/ml), and chelerythrine were also able to rescue hippocampal cells preexposed to SNP (up to 1 mM). Finally, EGb 761 (100 g/ml) was shown to block the activation of PKC induced by SNP (100 xM). These data suggest that the protective and rescuing abilities of EGb 761 are not only attributable to the antioxidant properties of its flavonoid constituents but also by their ability to inhibit NO-stimulated PKC activity (Figure 36.1). 

In cell culture preparations, diphenylhydantoin, carbamazepine and valproate have been shown to reduce membrane excitability at therapeutically relevant concentrations. This membrane-stabilizing effect is probably due to a block in the sodium channels. High concentrations of diazepam also have similar effects, and the membrane-stabilizing action correlates with the action of these anticonvulsants in inhibiting maximal electroshock seizures. Intracellular studies have shown that, in synaptosomes, most anticonvulsants inhibit calcium-dependent calmodulin protein kinase, an effect which would contribute to a reduction in neurotransmitter release. This action of anticonvulsants would appear to correlate with the potency of the drugs in inhibiting electroshock seizures. The result of all these disparate actions of anticonvulsants would be to diminish synaptic efficacy and thereby reduce seizure spread from an epileptic focus. 

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