Disease-associated nonsense and frame-shift variants resulting in the truncation of the GluN2A or GluN2B C-terminal domain decrease NMDAR surface expression and reduce potentiating effects of neurosteroids.

N-methyl-D-aspartate receptors (NMDARs) play a critical role in normal brain function, and variants in genes encoding NMDAR subunits have been described in individuals with various neuropsychiatric disorders. We have used whole-cell patch-clamp electrophysiology, fluorescence microscopy and in-silico modeling to explore the functional consequences of disease-associated nonsense and frame-shift variants resulting in the truncation of GluN2A or GluN2B C-terminal domain (CTD). This study characterizes variant NMDARs and shows their reduced surface expression and synaptic localization, altered agonist affinity, increased desensitization, and reduced probability of channel opening. We also show that naturally occurring and synthetic steroids pregnenolone sulfate and epipregnanolone butanoic acid, respectively, enhance NMDAR function in a way that is dependent on the length of the truncated CTD and, further, is steroid-specific, GluN2A/B subunit-specific, and GluN1 splice variant-specific. Adding to the previously described effects of disease-associated NMDAR variants on the receptor biogenesis and function, our results improve the understanding of the molecular consequences of NMDAR CTD truncations and provide an opportunity for the development of new therapeutic neurosteroid-based ligands.

Effects of Pregnanolone Glutamate and Its Metabolites on GABAA and NMDA Receptors and Zebrafish Behavior.

Multiple molecular targets have been identified to mediate membrane-delimited and nongenomic effects of natural and synthetic steroids, but the influence of steroid metabolism on neuroactive steroid signaling is not well understood. To begin to address this question, we set out to identify major metabolites of a neuroprotective synthetic steroid 20-oxo-5ß-pregnan-3α-yl l-glutamyl 1-ester (pregnanolone glutamate, PAG) and characterize their effects on GABAA and NMDA receptors (GABARs, NMDARs) and their influence on zebrafish behavior. Gas chromatography-mass spectrometry was used to assess concentrations of PAG and its metabolites in the hippocampal tissue of juvenile rats following intraperitoneal PAG injection. PAG is metabolized in the peripheral organs and nervous tissue to 20-oxo-17α-hydroxy-5ß-pregnan-3α-yl l-glutamyl 1-ester (17-hydroxypregnanolone glutamate, 17-OH-PAG), 3α-hydroxy-5ß-pregnan-20-one (pregnanolone, PA), and 3α,17α-dihydroxy-5ß-pregnan-20-one (17-hydroxypregnanolone, 17-OH-PA). Patch-clamp electrophysiology experiments in cultured hippocampal neurons demonstrate that PA and 17-OH-PA are potent positive modulators of GABARs, while PAG and 17-OH-PA have a moderate inhibitory effect at NMDARs. PAG, 17-OH-PA, and PA diminished the locomotor activity of zebrafish larvae in a dose-dependent manner. Our results show that PAG and its metabolites are potent modulators of neurotransmitter receptors with behavioral consequences and indicate that neurosteroid-based ligands may have therapeutic potential.

Pregnane-based steroids are novel positive NMDA receptor modulators that may compensate for the effect of loss-of-function disease-associated GRIN mutations.

BACKGROUND AND PURPOSE: N-methyl-D-aspartate receptors (NMDARs) play a critical role in synaptic plasticity, and mutations in human genes encoding NMDAR subunits have been described in individuals with various neuropsychiatric disorders. Compounds with a positive allosteric effect are thought to compensate for reduced receptor function. EXPERIMENTAL APPROACH: We have used whole-cell patch-clamp electrophysiology on recombinant rat NMDARs and human variants found in individuals with neuropsychiatric disorders, in combination with in silico modelling, to explore the site of action of novel epipregnanolone-based NMDAR modulators. KEY RESULTS: Analysis of the action of 4-(20-oxo-5ß-pregnan-3ß-yl) butanoic acid (EPA-But) at the NMDAR indicates that the effect of this steroid with a "bent" structure is different from that of cholesterol and oxysterols and shares a disuse-dependent mechanism of NMDAR potentiation with the "planar" steroid 20-oxo-pregn-5-en-3ß-yl sulfate (PE-S). The potentiating effects of EPA-But and PE-S are additive. Alanine scan mutagenesis identified residues that reduce the potentiating effect of EPA-But. No correlation was found between the effects of EPA-But and PE-S at mutated receptors that were less sensitive to either steroid. The relative degree of potentiation induced by the two steroids also differed in human NMDARs carrying rare variants of hGluN1 or hGluN2B subunits found in individuals with neuropsychiatric disorders, including intellectual disability, epilepsy, developmental delay, and autism spectrum disorder. CONCLUSION AND IMPLICATIONS: Our results show novel sites of action for pregnanolones at the NMDAR and provide an opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with glutamatergic system hypofunction.

Endogenous neurosteroids pregnanolone and pregnanolone sulfate potentiate presynaptic glutamate release through distinct mechanisms.

BACKGROUND AND PURPOSE: Neurosteroids influence neuronal function and have multiple promising clinical applications. Direct modulation of postsynaptic neurotransmitter receptors by neurosteroids is well characterized, but presynaptic effects remain poorly understood. Here, we report presynaptic glutamate release potentiation by neurosteroids pregnanolone and pregnanolone sulfate and compare their mechanisms of action to phorbol 12,13-dibutyrate (PDBu), a mimic of the second messenger DAG. EXPERIMENTAL APPROACH: We use whole-cell patch-clamp electrophysiology and pharmacology in rat hippocampal microisland cultures and total internal reflection fluorescence (TIRF) microscopy in HEK293 cells expressing GFP-tagged vesicle priming protein Munc13-1, to explore the mechanisms of neurosteroid presynaptic modulation. KEY RESULTS: Pregnanolone sulfate and pregnanolone potentiate glutamate release downstream of presynaptic Ca2+ influx, resembling the action of a phorbol ester PDBu. PDBu partially occludes the effect of pregnanolone, but not of pregnanolone sulfate. Calphostin C, an inhibitor that disrupts DAG binding to its targets, reduces the effect PDBu and pregnanolone, but not of pregnanolone sulfate, suggesting that pregnanolone might interact with a well-known DAG/phorbol ester target Munc13-1. However, TIRF microscopy experiments found no evidence of pregnanolone-induced membrane translocation of GFP-tagged Munc13-1, suggesting that pregnanolone may regulate Munc13-1 indirectly or interact with other DAG targets. CONCLUSION AND IMPLICATIONS: We describe a novel presynaptic effect of neurosteroids pregnanolone and pregnanolone sulfate to potentiate glutamate release downstream of presynaptic Ca2+ influx. The mechanism of action of pregnanolone, but not of pregnanolone sulfate, partly overlaps with that of PDBu. Presynaptic effects of neurosteroids may contribute to their therapeutic potential in the treatment of disorders of the glutamate system.

Palmitoylation Controls NMDA Receptor Function and Steroid Sensitivity.

NMDARs are ligand-gated ion channels that cause an influx of Na+ and Ca2+ into postsynaptic neurons. The resulting intracellular Ca2+ transient triggers synaptic plasticity. When prolonged, it may induce excitotoxicity, but it may also activate negative feedback to control the activity of NMDARs. Here, we report that a transient rise in intracellular Ca2+ (Ca2+ challenge) increases the sensitivity of NMDARs but not AMPARs/kainate receptors to the endogenous inhibitory neurosteroid 20-oxo-5ß-pregnan-3α-yl 3-sulfate and to its synthetic analogs, such as 20-oxo-5ß-pregnan-3α-yl 3-hemipimelate (PAhPim). In cultured hippocampal neurons, 30 µm PAhPim had virtually no effect on NMDAR responses; however, following the Ca2+ challenge, it inhibited the responses by 62%; similarly, the Ca2+ challenge induced a 3.7-fold decrease in the steroid IC50 on recombinant GluN1/GluN2B receptors. The increase in the NMDAR sensitivity to PAhPim was dependent on three cysteines (C849, C854, and C871) located in the carboxy-terminal domain of the GluN2B subunit, previously identified to be palmitoylated (Hayashi et al., 2009). Our experiments suggested that the Ca2+ challenge induced receptor depalmitoylation, and single-channel analysis revealed that this was accompanied by a 55% reduction in the probability of channel opening. Results of in silico modeling indicate that receptor palmitoylation promotes anchoring of the GluN2B subunit carboxy-terminal domain to the plasma membrane and facilitates channel opening. Depalmitoylation-induced changes in the NMDAR pharmacology explain the neuroprotective effect of PAhPim on NMDA-induced excitotoxicity. We propose that palmitoylation-dependent changes in the NMDAR sensitivity to steroids serve as an acute endogenous mechanism that controls NMDAR activity.SIGNIFICANCE STATEMENT There is considerable interest in negative allosteric modulators of NMDARs that could compensate for receptor overactivation by glutamate or de novo gain-of-function mutations in neurodevelopmental disorders. By a combination of electrophysiological, pharmacological, and computational techniques we describe a novel feedback mechanism regulating NMDAR activity. We find that a transient rise in intracellular Ca2+ increases NMDAR sensitivity to inhibitory neurosteroids in a process dependent on GluN2B subunit depalmitoylation. These results improve our understanding of the molecular mechanisms of steroid action at the NMDAR and indeed of the basic properties of this important glutamate-gated ion channel and may aid in the development of therapeutics for treating neurologic and psychiatric diseases related to overactivation of NMDARs without affecting normal physiological functions.

Cholesterol modulates presynaptic and postsynaptic properties of excitatory synaptic transmission.

Cholesterol is a structural component of cellular membranes particularly enriched in synapses but its role in synaptic transmission remains poorly understood. We used rat hippocampal cultures and their acute cholesterol depletion by methyl-ß-cyclodextrin as a tool to describe the physiological role of cholesterol in glutamatergic synaptic transmission. Cholesterol proved to be a key molecule for the function of synapses as its depletion resulted in a significant reduction of both NMDA receptor (NMDAR) and AMPA/kainate receptor-mediated evoked excitatory postsynaptic currents (eEPSCs), by 94% and 72%, respectively. We identified two presynaptic and two postsynaptic steps of synaptic transmission which are modulated by cholesterol and explain together the above-mentioned reduction of eEPSCs. In the postsynapse, we show that physiological levels of cholesterol are important for maintaining the normal probability of opening of NMDARs and for keeping NMDARs localized in synapses. In the presynapse, our results favour the hypothesis of a role of cholesterol in the propagation of axonal action potentials. Finally, cholesterol is a negative modulator of spontaneous presynaptic glutamate release. Our study identifies cholesterol as an important endogenous regulator of synaptic transmission and provides insight into molecular mechanisms underlying the neurological manifestation of diseases associated with impaired cholesterol synthesis or decomposition.

Site of Action of Brain Neurosteroid Pregnenolone Sulfate at the N-Methyl-D-Aspartate Receptor.

N-methyl-D-aspartate receptor (NMDAR) hypofunction has been implicated in several neurodevelopmental disorders. NMDAR function can be augmented by positive allosteric modulators, including endogenous compounds, such as cholesterol and neurosteroid pregnenolone sulfate (PES). Here we report that PES accesses the receptor via the membrane, and its binding site is different from that of cholesterol. Alanine mutagenesis has identified residues that disrupt the steroid potentiating effect at the rat GluN1 (G638; I642) and GluN2B (W559; M562; Y823; M824) subunit. Molecular dynamics simulation indicates that, in the absence of PES, the GluN2B M1 helix residue W559 interacts with the M4 helix residue M824. In the presence of PES, the M1 and M4 helices of agonist-activated receptor rearrange, forming a tighter interaction with the GluN1 M3 helix residues G638 and I642. This stabilizes the open-state position of the GluN1 M3 helices. Together, our data identify a likely binding site for the NMDAR-positive allosteric modulator PES and describe a novel molecular mechanism by which NMDAR activity can be augmented.SIGNIFICANCE STATEMENT There is considerable interest in drugs that enhance NMDAR function and could compensate for receptor hypofunction associated with certain neuropsychiatric disorders. Positive allosteric modulators of NMDARs include an endogenous neurosteroid pregnenolone sulfate (PES), but the binding site of PES on the NMDAR and the molecular mechanism of potentiation are unknown. We use patch-clamp electrophysiology in combination with mutagenesis and in silico modeling to describe the interaction of PES with the NMDAR. Our data indicate that PES binds to the transmembrane domain of the receptor at a discrete group of residues at the GluN2B membrane helices M1 and M4 and the GluN1 helix M3, and that PES potentiates NMDAR function by stabilizing the open-state position of the GluN1 M3 helices.

7-Methoxyderivative of tacrine is a 'foot-in-the-door' open-channel blocker of GluN1/GluN2 and GluN1/GluN3 NMDA receptors with neuroprotective activity in vivo.

N-methyl-d-aspartate receptors (NMDARs) are ionotropic glutamate receptors that mediate excitatory neurotransmission in the mammalian central nervous system (CNS), and their dysregulation results in the aetiology of many CNS syndromes. Several NMDAR modulators have been used successfully in clinical trials (including memantine) and NMDARs remain a promising pharmacological target for the treatment of CNS syndromes. 1,2,3,4-Tetrahydro-9-aminoacridine (tacrine; THA) was the first approved drug for Alzheimer's disease (AD) treatment. 7-methoxyderivative of THA (7-MEOTA) is less toxic and showed promising results in patients with tardive dyskinesia. We employed electrophysiological recordings in HEK293 cells and rat neurones to examine the mechanism of action of THA and 7-MEOTA at the NMDAR. We showed that both THA and 7-MEOTA are "foot-in-the-door" open-channel blockers of GluN1/GluN2 receptors and that 7-MEOTA is a more potent but slower blocker than THA. We found that the IC50 values for THA and 7-MEOTA exhibited the GluN1/GluN2A < GluN1/GluN2B < GluN1/GluN2C = GluN1/GluN2D relationship and that 7-MEOTA effectively inhibits human GluN1/GluN2A-M817V receptors that carry a pathogenic mutation. We also showed that 7-MEOTA is a "foot-in-the-door" open-channel blocker of GluN1/GluN3 receptors, although these receptors were not inhibited by memantine. In addition, the inhibitory potency of 7-MEOTA at synaptic and extrasynaptic hippocampal NMDARs was similar, and 7-MEOTA exhibited better neuroprotective activity when compared with THA and memantine in rats with NMDA-induced lesions of the hippocampus. Finally, intraperitoneal administration of 7-MEOTA attenuated MK-801-induced hyperlocomotion and pre-pulse inhibition deficit in rats. We conclude that 7-MEOTA may be considered for the treatment of diseases associated with the dysfunction of NMDARs.

Surface Expression, Function, and Pharmacology of Disease-Associated Mutations in the Membrane Domain of the Human GluN2B Subunit.

N-methyl-D-aspartate receptors (NMDARs), glutamate-gated ion channels, mediate signaling at the majority of excitatory synapses in the nervous system. Recent sequencing data for neurological and psychiatric patients have indicated numerous mutations in genes encoding for NMDAR subunits. Here, we present surface expression, functional, and pharmacological analysis of 11 de novo missense mutations of the human hGluN2B subunit (P553L; V558I; W607C; N615I; V618G; S628F; E657G; G820E; G820A; M824R; L825V) located in the pre-M1, M1, M2, M3, and M4 membrane regions. These variants were identified in patients with intellectual disability, developmental delay, epileptic symptomatology, and autism spectrum disorder. Immunofluorescence microscopy indicated that the ratio of surface-to-total NMDAR expression was reduced for hGluN1/hGluN2B(S628F) receptors and increased for for hGluN1/hGluN2B(G820E) receptors. Electrophysiological recordings revealed that agonist potency was altered in hGluN1/hGluN2B(W607C; N615I; and E657G) receptors and desensitization was increased in hGluN1/hGluN2B(V558I) receptors. The probability of channel opening of hGluN1/hGluN2B (V558I; W607C; V618G; and L825V) receptors was diminished ~10-fold when compared to non-mutated receptors. Finally, the sensitivity of mutant receptors to positive allosteric modulators of the steroid origin showed that glutamate responses induced in hGluN1/hGluN2B(V558I; W607C; V618G; and G820A) receptors were potentiated by 59-96% and 406-685% when recorded in the presence of 20-oxo-pregn-5-en-3ß-yl sulfate (PE-S) and androst-5-en-3ß-yl hemisuccinate (AND-hSuc), respectively. Surprisingly hGluN1/hGluN2B(L825V) receptors were strongly potentiated, by 197 and 1647%, respectively, by PE-S and AND-hSuc. Synaptic-like responses induced by brief glutamate application were also potentiated and the deactivation decelerated. Further, we have used homology modeling based on the available crystal structures of GluN1/GluN2B NMDA receptor followed by molecular dynamics simulations to try to relate the functional consequences of mutations to structural changes. Overall, these data suggest that de novo missense mutations of the hGluN2B subunit located in membrane domains lead to multiple defects that manifest by the NMDAR loss of function that can be rectified by steroids. Our results provide an opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with hypofunction of the glutamatergic system.

Preferential Inhibition of Tonically over Phasically Activated NMDA Receptors by Pregnane Derivatives.

Postsynaptic N-methyl-d-aspartate receptors (NMDARs) phasically activated by presynaptically released glutamate are critical for synaptic transmission and plasticity. However, under pathological conditions, excessive activation of NMDARs by tonically increased ambient glutamate contributes to excitotoxicity associated with various acute and chronic neurological disorders. Here, using heterologously expressed GluN1/GluN2A and GluN1/GluN2B receptors and rat autaptic hippocampal microisland cultures, we show that pregnanolone sulfate inhibits NMDAR currents induced by a prolonged glutamate application with a higher potency than the NMDAR component of EPSCs. For synthetic pregnanolone derivatives substituted with a carboxylic acid moiety at the end of an aliphatic chain of varying length and attached to the steroid skeleton at C3, the difference in potency between tonic and phasic inhibition increased with the length of the residue. The steroid with the longest substituent, pregnanolone hemipimelate, had no effect on phasically activated receptors while inhibiting tonically activated receptors. In behavioral tests, pregnanolone hemipimelate showed neuroprotective activity without psychomimetic symptoms. These results provide insight into the influence of steroids on neuronal function and stress their potential use in the development of novel therapeutics with neuroprotective action. SIGNIFICANCE STATEMENT: Synaptic activation of N-methyl-d-aspartate receptors (NMDARs) plays a key role in synaptic plasticity, but excessive tonic NMDAR activation mediates excitotoxicity associated with many neurological disorders. Therefore, there is much interest in pharmacological agents capable of selectively blocking tonically activated NMDARs while leaving synaptically activated NMDARs intact. Here, we show that an endogenous neurosteroid pregnanolone sulfate is more potent at inhibiting tonically than synaptically activated NMDARs. Further, we report that a novel synthetic analog of pregnanolone sulfate, pregnanolone hemipimelate, inhibits tonic NMDAR currents without inhibiting the NMDAR component of the EPSC and shows neuroprotective activity in vivo without inducing psychomimetic side effects. These results suggest steroids may have a clinical advantage over other known classes of NMDAR inhibitors.

Block of NMDA receptor channels by endogenous neurosteroids: implications for the agonist induced conformational states of the channel vestibule.

N-methyl-D-aspartate receptors (NMDARs) mediate synaptic plasticity, and their dysfunction is implicated in multiple brain disorders. NMDARs can be allosterically modulated by numerous compounds, including endogenous neurosteroid pregnanolone sulfate. Here, we identify the molecular basis of the use-dependent and voltage-independent inhibitory effect of neurosteroids on NMDAR responses. The site of action is located at the extracellular vestibule of the receptor's ion channel pore and is accessible after receptor activation. Mutations in the extracellular vestibule in the SYTANLAAF motif disrupt the inhibitory effect of negatively charged steroids. In contrast, positively charged steroids inhibit mutated NMDAR responses in a voltage-dependent manner. These results, in combination with molecular modeling, characterize structure details of the open configuration of the NMDAR channel. Our results provide a unique opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with dysfunction of the glutamate system.

Cholesterol modulates open probability and desensitization of NMDA receptors.

NMDA receptors (NMDARs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the CNS. Although these receptors are in direct contact with plasma membrane, lipid-NMDAR interactions are little understood. In the present study, we aimed at characterizing the effect of cholesterol on the ionotropic glutamate receptors. Whole-cell current responses induced by fast application of NMDA in cultured rat cerebellar granule cells (CGCs) were almost abolished (reduced to 3%) and the relative degree of receptor desensitization was increased (by seven-fold) after acute cholesterol depletion by methyl-ß-cyclodextrin. Both of these effects were fully reversible by cholesterol repletion. By contrast, the responses mediated by AMPA/kainate receptors were not affected by cholesterol depletion. Similar results were obtained in CGCs after chronic inhibition of cholesterol biosynthesis by simvastatin and acute enzymatic cholesterol degradation to 4-cholesten-3-one by cholesterol oxidase. Fluorescence anisotropy measurements showed that membrane fluidity increased after methyl-ß-cyclodextrin pretreatment. However, no change in fluidity was observed after cholesterol enzymatic degradation, suggesting that the effect of cholesterol on NMDARs is not mediated by changes in membrane fluidity. Our data show that diminution of NMDAR responses by cholesterol depletion is the result of a reduction of the open probability, whereas the increase in receptor desensitization is the result of an increase in the rate constant of entry into the desensitized state. Surface NMDAR population, agonist affinity, single-channel conductance and open time were not altered in cholesterol-depleted CGCs. The results of our experiments show that cholesterol is a strong endogenous modulator of NMDARs.

Key amino acid residues within the third membrane domains of NR1 and NR2 subunits contribute to the regulation of the surface delivery of N-methyl-D-aspartate receptors.

N-methyl-d-aspartate (NMDA) receptors are glutamate ionotropic receptors that play critical roles in synaptic transmission, plasticity, and excitotoxicity. The functional NMDA receptors, heterotetramers composed mainly of two NR1 and two NR2 subunits, likely pass endoplasmic reticulum quality control before they are released from the endoplasmic reticulum and trafficked to the cell surface. However, the mechanism underlying this process is not clear. Using truncated and mutated NMDA receptor subunits expressed in heterologous cells, we found that the M3 domains of both NR1 and NR2 subunits contain key amino acid residues that contribute to the regulation of the number of surface functional NMDA receptors. These key residues are critical neither for the interaction between the NR1 and NR2 subunits nor for the formation of the functional receptors, but rather they regulate the early trafficking of the receptors. We also found that the identified key amino acid residues within both NR1 and NR2 M3 domains contribute to the regulation of the surface expression of unassembled NR1 and NR2 subunits. Thus, our data identify the unique role of the membrane domains in the regulation of the number of surface NMDA receptors.

Neurosteroid modulation of N-methyl-D-aspartate receptors: molecular mechanism and behavioral effects.

Glutamate is the main neurotransmitter released at synapses in the central nervous system of vertebrates. Its excitatory role is mediated through activation of specific glutamatergic ionotropic receptors, among which the N-methyl-D-aspartate (NMDA) receptor subtype has attracted considerable attention in recent years. Substantial progress has been made in elucidating the roles these receptors play under physiological and pathological conditions and in our understanding of the functional, structural, and pharmacological properties of NMDA receptors. Many pharmacological compounds have been identified that affect the activity of NMDA receptors, including neurosteroids. This review summarizes our knowledge about molecular mechanisms underlying the neurosteroid action at NMDA receptors as well as about the action of neurosteroids in animal models of human diseases.

Luminescence study of singlet oxygen production by meso-tetraphenylporphine.

The research in the field of the photodynamic therapy of cancer (PDT) is focused on a development of photosensitizers exhibiting high quantum yield of singlet oxygen production. Direct time-resolved spectroscopic observation of singlet oxygen phosphorescence can provide time constants of its population and depopulation as well as photosensitizer phosphorescence lifetime and relative quantum yields. In our contribution, a study of time and spectral resolved phosphorescence of singlet oxygen photosensitized by meso-tetraphenylporphine in acetone together with the photosensitizer phosphorescence is presented. Time constants of singlet oxygen population and depopulation were determined at wide range of photosensitizer concentrations. The time constant of singlet oxygen generation (0.28 +/- 0.01) micros is slightly shorter then the lifetime of photosensitizer's triplet state (0.32 +/- 0.01) micros. It is caused by lower ability of TPP aggregates to transfer excitation energy to oxygen. The lifetime of singlet oxygen (approximately 50 micros) decreases with increasing photosensitizer concentration. Therefore, the photosensitizer acts also as a quencher of oxygen singlet state, similarly to the effects observed in [A. A. Krasnovsky, P. Cheng, R. E. Blankenship, T. A. Moore, and D. Gust (1993). Photochem. Photobiol. 57, 324-330; H. Küpper, R. Dedic, A. Svoboda, J. Hála, and P. M. H. Kroneck (2002). Biochim. Biophys. Acta Gen. Subj. 1572, 107-113]. Moreover, the increasing concentration of the photosensitizer causes a slight hypsochromic shift of the singlet oxygen luminescence maximum.