Combined drug triads for synergic neuroprotection in retinal degeneration.

This review focuses on retina degeneration occurring during glaucoma, age-related macular degeneration (AMD), diabetic retinopathy (DR), and retinitis pigmentosa (RP), and on the potential therapeutic use of triads of repositioned medicines, addressed to distinct but complementary targets, to prevent, delay or stop retina cell death. Although myriad pathogenic mechanisms have been implicated in these disorders, common signaling pathways leading to apoptotic cell death to all of them, and to all neurodegenerative diseases are (i) calcium dyshomeostasis/excitotoxicity; (ii) oxidative stress/mitochondrial dysfunction, and (iii) neuroinflammation/P2X7 receptor activation. From a therapeutic point of view, it is relevant to consider the multitarget approach based on the use of combined medicines acting on complementary pathogenic mechanisms that has been highly successful in the treatment of chronic diseases such as cancer, AIDS, pain, hypertension, Parkinson's disease, cardiac failure, depression, or the epilepsies as the basic mechanisms of cell death do not differ between the different CNS degenerative diseases. We suggest the multi-target therapy approach could be more effective compared with single-drug treatments. Used at doses lower than standard, these triads may also be safer and more efficient. After the establishment of a proof-of-concept in animal models of retinal degeneration, potential successful preclinical trials of such combinations may eventually drive to test this concept in clinical trials in patients, first to evaluate the safety and efficacy of the drug combinations in humans and then their therapeutic advantages, if any, seeking the prevention and/or the delay of retina degeneration and blindness.

Blockade by NNC 55-0396, mibefradil, and nickel of calcium and exocytotic signals in chromaffin cells: implications for the regulation of hypoxia-induced secretion at early life.

Adrenal chromaffin cells (CCs) express high-voltage activated calcium channels (high-VACCs) of the L, N and PQ subtypes; in addition, T-type low-VACCs are also expressed during embryo and neonatal life. Effects of the more frequently used T channel blockers NNC 55-0396 (NNC), mibefradil, and Ni2+ on the whole-cell Ba2+ current (IBa), the K+-elicited [Ca2+]c transients and catecholamine secretion have been studied in adult bovine CCs (BCCs) and rat embryo CCs (RECCs). NNC, mibefradil, and Ni2+ blocked BCC IBa with IC50 of 1.8, 4.9 and 70 µM, while IC50 to block IBa in RECCs were 2.1, 4.4 and 41 µM. Pronounced blockade of K+-elicited [Ca2+]c transients and secretion was also elicited by the three agents. However, the hypoxia-induced secretion (HIS) of catecholamine in RECCs was blocked substantially (75%) with thresholds concentrations of NCC (IC20 to block IBa); this was not the case for mibefradil and Ni2+ that required higher concentrations to block the HIS response. Thus, out of the three compounds, NNC seemed to be an adequate pharmacological tool to discern the contribution of T channels to the HIS response, without a contamination with high-VACC blockade.

Hypoxia-elicited catecholamine release is controlled by L-type as well as N/PQ types of calcium channels in rat embryo chromaffin cells.

At early life, the adrenal chromaffin cells respond with a catecholamine surge under hypoxic conditions. This response depends on Ca(2+) entry through voltage-activated calcium channels (VACCs). We have investigated here three unresolved questions that concern this response in rat embryo chromaffin cells (ECCs): 1) the relative contribution of L (α1D, Cav1.3), N (α1B, Cav2.2), and PQ (α1A, Cav2.1) to the whole cell Ca(2+) current (ICa); 2) the relative contribution of L and N/PQ channels to the cytosolic Ca(2+) elevations triggered by hypoxia (Δ[Ca(2+)]c); and 3) the role of L and non-L high-VACCs in the regulation of the catecholamine surge occurring during prolonged (1 min) hypoxia exposure of ECCs. Nimodipine halved peak ICa and blocked 60% the total Ca(2+) entry during a 50-ms depolarizing pulse to 0 mV (QCa). Combined ω-agatoxin IVA plus ω-conotoxin GVIA (Aga/GVIA) blocked 30% of both ICa peak and QCa. This relative proportion of L- and non-L VACCs was corroborated by Western blot that indicated 55, 23, and 25% relative expression of L, N, and PQ VACCs. Exposure of ECCs to hypoxia elicited a mild but sustained Δ[Ca(2+)]c; the area of Δ[Ca(2+)]c was blocked 50% by nifedipine and 10% by Aga/GVIA. Exposure of ECCs to 1-min hypoxia elicited an initial transient burst of amperometric secretory spikes followed by scattered spikes along the time of cell exposure to hypoxia. This bulk response was blocked 85% by nimodipine and 35% by Aga/GVIA. Histograms on secretory spike frequency vs. time indicated a faster initial inactivation when Ca(2+) entry took place through N/PQ channels; more sustained secretion but at a lower rate was associated to Ca(2+) entry through L channels. The results suggest that the HIS response may initially be controlled by L and P/Q channels, but later on, N/PQ channels inactivate and the delayed HIS response is maintained at lower rate by slow-inactivating L channels.

Novel features on the regulation by mitochondria of calcium and secretion transients in chromaffin cells challenged with acetylcholine at 37°C.

From experiments performed at room temperature, we know that the buffering of Ca(2+) by mitochondria contributes to the shaping of the bulk cytosolic calcium transient ([Ca(2+)]c) and secretion transients of chromaffin cells stimulated with depolarizing pulses. We also know that the mitochondrial Ca(2+) transporters and the release of catecholamine are faster at 37°C with respect to room temperature. Therefore, we planned this investigation to gain further insight into the contribution of mitochondrial Ca(2+) buffering to the shaping of [Ca(2+)]c and catecholamine release transients, using some novel experimental conditions that have not been yet explored namely: (1) perifusion of bovine chromaffin cells (BCCs) with saline at 37°C and their repeated challenging with the physiological neurotransmitter acetylcholine (ACh); (2) separate blockade of mitochondrial Ca(2+) uniporter (mCUP) with Ru360 or the mitochondrial Na(+)/Ca(2+) exchanger (mNCX) with CGP37157; (3) full blockade of the mitochondrial Ca(2+) cycling (mCC) by the simultaneous inhibition of the mCUP and the mNCX. Ru360 caused a pronounced delay of [Ca(2+)]c clearance and augmented secretion. In contrast, CGP37157 only caused a tiny delay of [Ca(2+)]c clearance and a mild decrease in secretion. The mCC resulting in continued Ca(2+) uptake and its release back into the cytosol was interrupted by combined Ru360 + CGP37157 (Ru/CGP), the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, or combined oligomycin + rotenone (O/R); these three treatments caused a mild but sustained elevation of basal [Ca(2+)]c that, however, was not accompanied by a parallel increase in basal secretion. Nevertheless, all treatments caused a pronounced augmentation of ACh-induced secretion, with minor changes of the ACh-induced [Ca(2+)]c transients. Combined Ru/CGP did not alter the resting membrane potential in current-clamped cells. Additionally, Ru/CGP did not increase basal [Ca(2+)]c near subplasmalemmal sites and caused a mild decrease in the size of the readily releasable vesicle pool. Our results provide new functional features in support of the view that in BCCs there are two subpopulations of mitochondria, M1 underneath the plasmalemma nearby exocytotic sites and M2 at the core cell nearby vesicle transport sites. While M1 serves to shape the ACh-elicited exocytotic response through its efficient Ca(2+) removal by the mCUP, M2 shapes the lower [Ca(2+)]c elevations required for new vesicle supply to the exocytotic machinery, from the large reserve vesicle pool at the cell core. The mCUP of the M1 pool seems to play a more prominent role in controlling the ACh responses, in comparison with the mNCX.

Bcl2 mitigates Ca2+ entry and mitochondrial Ca2+ overload through downregulation of L-type Ca2+ channels in PC12 cells.

Altered calcium homeostasis and increased cytosolic calcium concentrations ([Ca2+]c) are linked to neuronal apoptosis in epilepsy and in cerebral ischemia, respectively. Apoptotic programmed cell death is regulated by the antiapoptotic Bcl2 family of proteins. Here, we investigated the role of Bcl2 on calcium (Ca2+) homeostasis in PC12 cells, focusing on L-type voltage-dependent calcium channels (VDCC). Cytosolic Ca2+ transients ([Ca2+]c) and changes of mitochondrial Ca2+ concentrations ([Ca2+]m) were monitored using cytosolic and mitochondrially targeted aequorins of control PC12 cells and PC12 cells stably overexpressing Bcl2. We found that: (i) the [Ca2+]c and [Ca2+]m elevations elicited by K+ pulses were markedly depressed in Bcl2 cells, with respect to control cells; (ii) such depression of [Ca2+]m was not seen either in digitonin-permeabilized cells or in intact cells treated with ionomycin; (iii) the [Ca2+]c transient depression seen in Bcl2 cells was reversed by shRNA transfection, as well as by the Bcl2 inhibitor HA14-1; (iv) the L-type Ca2+ channel agonist Bay K 8644 enhanced K(+)-evoked [Ca2+]m peak fourfold in Bcl2, and twofold in control cells; (v) in current-clamped cells the depolarization evoked by K+ generated a more hyperpolarized voltage step in Bcl2, as compared to control cells. Taken together, our experiments suggest that the reduction of the [Ca2+]c and [Ca2+]m transients elicited by K+, in PC12 cells overexpressing Bcl2, is related to the reduction of Ca2+ entry through L-type Ca2+ channels. This may be due to the fact that Bcl2 mitigates cell depolarization, thus diminishing the recruitment of L-type Ca2+ channels, the subsequent Ca2+ entry, and mitochondrial Ca2+ overload.