Search

Numerous studies have shown that low-flux nitric oxide (NO) in tumors produced mainly by inducible nitric oxide synthase (iNOS/NOS2) can signal for angiogenesis, inhibition of apoptosis, and promotion of cell growth, migration, and invasion. Studies in the authors' laboratory have revealed that iNOS-derived NO in various cancer cell types elicits resistance to cytotoxic photodynamic therapy (PDT) and moreover endows PDT-surviving cells with more aggressive proliferation and migration/invasion. In this chapter, we describe how cancer cell iNOS/NO in vitro can be monitored in different PDT model systems (e.g., a targeted cell-bystander cell model) and how pharmacologic interference with basal and PDT-upregulated iNOS/NO can significantly improve PDT outcomes., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Anti-tumor photodynamic therapy (PDT) is a unique oxidative stress-based modality that has proven highly effective on a variety of solid malignancies. PDT is minimally invasive and generates cytotoxic oxidants such as singlet molecular oxygen ( 1 O 2 ). With high tumor site-specificity and limited off-target negative effects, PDT is increasingly seen as an attractive alternative or follow-up to radiotherapy or chemotherapy. Nitric oxide (NO) is a short-lived bioactive free radical molecule that is exploited by many malignant tumors to promote cell survival, proliferation, and metastatic expansion. Typically generated endogenously by inducible nitric oxide synthase (iNOS/NOS2), low level NO can also antagonize many therapeutic interventions, including PDT. In addition to elevating resistance, iNOS-derived NO can stimulate growth and migratory aggressiveness of tumor cells that survive a PDT challenge. Moreover, NO from PDT-targeted cells in any given population is known to promote such aggressiveness in non-targeted counterparts (bystanders). Each of these negative responses to PDT and their possible underlying mechanisms will be discussed in this chapter. Promising pharmacologic approaches for mitigating these NO-mediated responses will also be discussed.

Many malignant tumors use endogenous nitric oxide (NO) to promote survival, growth, and metastatic migration. This NO, which is typically generated by inducible nitric oxide synthase (iNOS), can also antagonize various anti-cancer therapies and its source is most often assumed to be constitutive or pre-existing iNOS. In this paper, we provide evidence (i) that many different cancer cells exhibit resistance to oxidative killing by photodynamic therapy (PDT), and (ii) that cells surviving the challenge grow, migrate and invade more aggressively, as do non-targeted bystander cells. Accompanying these effects are activation or upregulation of pro-survival/progression effector proteins such as NF-κB, Akt, and Survivin. Observed in the author's laboratory, these responses were not attributed to basal iNOS/NO in most cases, but rather to NO from enzyme that was strongly upregulated by photodynamic stress. Each of these effects and how they can be mitigated by inhibitors of iNOS activity or transcription, or by NO scavengers will be discussed. When approved for clinical use, such pharmacologic agents could improve PDT efficacy as well as reduce potentially negative side-effects of this therapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Glioblastoma multiforme is a highly aggressive primary brain malignancy that resists most conventional chemoand radiotherapeutic interventions. Nitric oxide (NO), a short lived free radical molecule produced by inducible NO synthase (iNOS) in glioblastomas and other tumors, is known to play a key role in tumor persistence, progression, and chemo/radiotherapy resistance. Site-specific and minimally invasive photodynamic therapy (PDT), based on oxidative damage resulting from non-ionizing photoactivation of a sensitizing agent, is highly effective against glioblastoma, but resistance also exists in this case. Studies in the authors' laboratory have shown that much of the latter is mediated by iNOS/NO. For example, when glioblastoma U87 or U251 cells sensitized in mitochondria with 5-aminolevulinic acid -induced protoporphyrin IX were exposed to a moderate dose of visible light, the observed apoptosis was strongly enhanced by an iNOS activity inhibitor or NO scavenger, indicating that iNOS/NO had increased cell resistance to photokilling. Moreover, cells that survived the photochallenge proliferated, migrated, and invaded more aggressively than controls, and these responses were also driven predominantly by iNOS/NO. Photostress-upregulated iNOS rather than basal enzyme was found to be responsible for all the negative effects described. Recognition of NO-mediated hyper-resistance/hyper-aggression in PDT-stressed glioblastoma has stimulated interest in how these responses can be prevented or at least minimized by pharmacologic adjuvants such as inhibitors of iNOS activity or transcription. Recent developments along these lines and their clinical potential for improving anti-glioblastoma PDT are discussed., Competing Interests: Conflicts of interest All authors declared that there are no conflicts of interest.

Glioblastomas are highly aggressive brain tumors that can persist after exposure to conventional chemotherapy or radiotherapy. Nitric oxide (NO) produced by inducible NO synthase (iNOS/NOS2) in these tumors is known to foster malignant cell proliferation, migration, and invasion as well as resistance to chemo- and radiotherapy. Minimally invasive photodynamic therapy (PDT) sensitized by 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) is a highly effective anti-glioblastoma modality, but it is also subject to NO-mediated resistance. Studies by the authors have revealed that glioblastoma U87 and U251 cells use endogenous iNOS/NO to not only resist photokilling after an ALA/light challenge, but also to promote proliferation and migration/invasion of surviving cells. Stress-upregulated iNOS/NO was found to play a major role in these negative responses to PDT-like treatment. Our studies have revealed a tight network of upstream signaling events leading to iNOS induction in photostressed cells and transition to a more aggressive phenotype. These events include activation or upregulation of pro-survival/ pro-expansion effector proteins such as NF-κB, phosphoinositide-3-kinase (PI3K), protein kinase-B (Akt), p300, Survivin, and Brd4. In addition to this upstream signaling and its regulation, pharmacologic approaches for directly suppressing iNOS at its activity vs. transcriptional level are discussed. One highly effective agent in the latter category is bromodomain and extra-terminal (BET) inhibitor, JQ1, which was found to minimize iNOS upregulation in photostressed U87 cells. By acting similarly at the clinical level, a BET inhibitor such as JQ1 should markedly improve the efficacy of anti-glioblastoma PDT., Competing Interests: Conflicts of interest The authors have no conflicts of interest to declare.

Nitric oxide (NO) generated endogenously by inducible nitric oxide synthase (iNOS) promotes growth and migration/invasion of glioblastoma cells and also fosters resistance to chemotherapy and ionizing radiotherapy. Our recent studies revealed that glioblastoma cell iNOS/NO also opposes the cytotoxic effects of non-ionizing photodynamic therapy (PDT), and moreover stimulates growth/migration aggressiveness of surviving cells. These negative responses, which depended on PI3K/Akt/NF-κB activation, were strongly suppressed by blocking iNOS transcription with JQ1, a BET bromodomain inhibitor. In the present study, we sought to identify additional molecular events that precede iNOS transcriptional upregulation. Akt activation, iNOS induction, and viability loss in PDT-challenged glioblastoma U87 cells were all strongly inhibited by added l-histidine, consistent with primary involvement of photogenerated singlet oxygen ( 1 O 2 ). Transacetylase p300 not only underwent greater Akt-dependent activation after PDT, but greater interaction with NF-κB subunit p65, which in turn exhibited greater K310 acetylation. In addition, PDT promoted intramolecular disulfide formation and inactivation of tumor suppressor PTEN, thereby favoring Akt and p300 activation leading to iNOS upregulation. Importantly, deacetylase Sirt1 was down-regulated by PDT stress, consistent with the observed increase in p65-acK310 level, which fostered iNOS transcription. This study provides new mechanistic insights into how glioblastoma tumors can exploit iNOS/NO to not only resist PDT, but to attain a more aggressive survival phenotype., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Many studies have shown that low flux nitric oxide (NO) produced by inducible NO synthase (iNOS/NOS2) in various tumors, including glioblastomas, can promote angiogenesis, cell proliferation, and migration/invasion. Minimally invasive, site-specific photodynamic therapy (PDT) is a highly promising anti-glioblastoma modality. Recent research in the authors' laboratory has revealed that iNOS-derived NO in glioblastoma cells elicits resistance to 5-aminolevulinic acid (ALA)-based PDT, and moreover endows PDT-surviving cells with greater proliferation and migration/invasion aggressiveness. In this contribution, we discuss iNOS/NO antagonism to glioblastoma PDT and how this can be overcome by judicious use of pharmacologic inhibitors of iNOS activity or transcription.

Endogenous nitric oxide (NO) generated by inducible NO synthase (iNOS) promotes glioblastoma cell proliferation and invasion and also plays a key role in glioblastoma resistance to chemotherapy and radiotherapy. Non-ionizing photodynamic therapy (PDT) has anti-tumor advantages over conventional glioblastoma therapies. Our previous studies revealed that glioblastoma U87 cells up-regulate iNOS after a photodynamic challenge and that the resulting NO not only increases resistance to apoptosis but renders surviving cells more proliferative and invasive. These findings were largely based on the effects of inhibiting iNOS activity and scavenging NO. Demonstrating now that iNOS expression in photostressed U87 cells is mediated by NF-κB, we hypothesized that (i) recognition of acetylated lysine (acK) on NF-κB p65/RelA by bromodomain and extra-terminal (BET) protein Brd4 is crucial; and (ii) by suppressing iNOS expression, a BET inhibitor (JQ1) would attenuate the negative effects of photostress. The following evidence was obtained. (i) Like iNOS, Brd4 protein and p65-acK levels increased severalfold in photostressed cells. (ii) JQ1 at minimally toxic concentrations had no effect on Brd4 or p65-acK up-regulation after PDT but strongly suppressed iNOS, survivin, and Bcl-xL up-regulation, along with the growth and invasion spurt of PDT-surviving cells. (iii) JQ1 inhibition of NO production in photostressed cells closely paralleled that of growth/invasion inhibition. (iv) Finally, at 1% the concentration of iNOS inhibitor 1400W, JQ1 reduced post-PDT cell aggressiveness to a far greater extent. This is the first evidence for BET inhibitor targeting of iNOS expression in cancer cells and how such targeting can markedly improve therapeutic efficacy., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Many malignant tumors employ iNOS-derived NO to resist eradication by chemotherapeutic agents or ionizing radiation. In this study, we determined whether human breast carcinoma MDA-MB-231 cells in vitro and in vivo as tumor xenografts would exploit endogenous iNOS/NO to resist the cytotoxic effects of 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT). Broad band visible irradiation of ALA-treated cells resulted in a marked after-light upregulation of iNOS protein which persisted for at least 24 h. Apoptotic killing of ALA/light-challenged cells was significantly enhanced by iNOS inhibitors (1400W, GW274150) and a NO trap (cPTIO), implying that stress-induced iNOS/NO was acting cytoprotectively. We found that cells surviving the photostress proliferated and migrated more rapidly than controls in 1400W- and cPTIO-inhibitable fashion, indicating iNOS/NO involvement. Female SCID mice bearing MDA-MB-231 tumors were used for animal model experiments. ALA-PDT with a 633 nm light source caused a significant reduction in post-irradiation tumor growth relative to light-only controls, which was further reduced by administration of 1400W or GW274150, whereas 1400W had little or no effect on controls. Immunoblot analyses of tumor samples revealed a progressive post-PDT upregulation of iNOS, which reached >5-times the control level after six days. Correspondingly, the nitrite/nitrate level in post-PDT tumor samples was substantially higher than that in controls. In addition, a 1400W-inhibitable upregulation of pro-survival/progression effector proteins such as Bcl-xL, Survivin, and S100A4 was observed after in vitro and in vivo ALA-PDT. This is the first known study to demonstrate iNOS/NO-induced resistance to PDT in an in vivo human tumor model., Competing Interests: statement The authors have no conflicts of interest to declare., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The bystander effects of anti-cancer ionizing radiation have been widely studied, but far less is known about such effects in the case of non-ionizing photodynamic therapy (PDT). In the present study, we tested the hypothesis that photodynamically-stressed prostate cancer PC3 cells can elicit nitric oxide (NO)-mediated pro-growth/migration responses in non-stressed bystander cells. A novel approach was used whereby both cell populations existed on a culture dish, but made no physical contact with one other. Visible light irradiation of target cells sensitized with 5-aminolevulinic acid-induced protoporphyrin IX resulted in a striking upregulation of inducible nitric oxide synthase (iNOS) along with NO, the level of which increased after irradiation. Slower and less pronounced iNOS/NO upregulation was also observed in bystander cells. Activation of transcription factor NF-κB was implicated in iNOS induction in both targeted and bystander cells. Like surviving targeted cells, bystanders exhibited a significant increase in growth and migration rate, both responses being strongly attenuated by an iNOS inhibitor (1400W), a NO scavenger (cPTIO), or iNOS knockdown. Incubating bystander cells with conditioned medium from targeted cells failed to stimulate growth/migration, ruling out involvement of relatively long-lived stimulants. The following post-irradiation changes in pro-survival/pro-growth proteins were observed in bystander cells: upregulation of COX-2 and activation of protein kinases Akt and ERK1/2, NO again playing a key role. This is the first reported evidence for NO-enhanced bystander aggressiveness in the context of PDT. In the clinical setting, such effects could be averted through pharmacologic use of iNOS inhibitors as PDT adjuvants., (Copyright © 2016. Published by Elsevier Inc.)

Ionizing radiation of specifically targeted cells in a given population is known to elicit pro-death or pro-survival responses in non-targeted bystander cells, which often make no physical contact with the targeted ones. We have recently demonstrated a similar phenomenon for non-ionizing photodynamic therapy (PDT), showing that prostate cancer cells subjected to targeted photodynamic stress stimulated growth and migration of non-stressed, non-contacting bystander cells. Diffusible nitric oxide (NO) generated by stress-upregulated inducible nitric oxide synthase (iNOS) was shown to play a dominant role in these responses. Moreover, target-derived NO stimulated iNOS/NO induction in bystanders, suggesting a NO-mediated feed-forward field effect driven by targeted cells surviving the photodynamic challenge. In this research highlight, we will review these findings and discuss their potential negative implications on clinical PDT outcomes and how these might be mitigated through pharmacologic use of select iNOS inhibitors., Competing Interests: Conflicting interests The authors have declared that no conflict of interests exist.

Gliomas are aggressive brain tumors that are resistant to conventional chemotherapy and radiotherapy. Much of this resistance is attributed to endogenous nitric oxide (NO). Recent studies revealed that 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) has advantages over conventional treatments for glioblastoma. In this study, we used an in vitro model to assess whether NO from glioblastoma cells can interfere with ALA-PDT. Human U87 and U251 cells expressed significant basal levels of neuronal NO synthase (nNOS) and its inducible counterpart (iNOS). After an ALA/light challenge, iNOS level increased three- to fourfold over 24 h, whereas nNOS remained unchanged. Elevated iNOS resulted in a large increase in intracellular NO. Extent of ALA/light-induced apoptosis increased substantially when an iNOS inhibitor or NO scavenger was present, implying that iNOS/NO was acting cytoprotectively. Moreover, cells surviving a photochallenge exhibited a striking increase in proliferation, migration and invasion rates, iNOS/NO again playing a dominant role. Also observed was a large iNOS/NO-dependent increase in matrix metalloproteinase-9 activity, decrease in tissue inhibitor of metalloproteinase-1 expression and increase in survivin and S100A4 expression, each effect being consistent with accelerated migration/invasion as a prelude to metastasis. Our findings suggest introduction of iNOS inhibitors as pharmacologic adjuvants for glioblastoma PDT., (© 2016 The American Society of Photobiology.)

Photodynamic therapy (PDT) is a unique site-specific treatment for eradicating a variety of solid tumors, including prostate, lung, bladder, and brain tumors. PDT is a three-component modality involving (i) administration of a photosensitizing agent (PS), (ii) PS photoexcitation by visible or near-infrared light, and (iii) molecular oxygen. Upon photoexcitation, PS gives rise to tumor-damaging reactive oxygen species, most prominently singlet oxygen (1O2). Previous studies revealed that endogenous nitric oxide (NO) in various mouse tumor models significantly reduced PDT effectiveness. Recent studies in the authors' laboratory indicated that NO produced by photostressed tumor cells per se can elicit anti-PDT effects. For example, breast cancer COH-BR1 and prostate cancer PC3 cells exhibited a rapid and prolonged upregulation of inducible nitric oxide synthase (iNOS) after sensitization with 5- aminolevulinic acid (ALA)-induced protoporphyrin-IX, followed by broad-band visible irradiation. Use of iNOS inhibitors and NO scavengers demonstrated that iNOS/NO played a key role in cell resistance to apoptotic photokilling. Moreover, cells surviving an ALA/light challenge proliferated, migrated, and invaded more rapidly than controls, again in iNOS/NOdependent fashion. Thus, NO was found to play a crucial role in various manifestations of enhanced aggressiveness exhibited by remaining live cells. Recent work has revealed that induced NO in PDT-targeted PC3 cells can also translocate and increase aggressiveness of non-targeted bystander cells. These negative and potentially tumor-promoting side effects of NO in PDT may be averted through use of iNOS inhibitors as adjuvants. Each of the above aspects of PDT antagonism by NO will be discussed in this review.

Employing an in vitro model for 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT), we recently reported that human prostate cancer PC3 cells rapidly and persistently overexpressed inducible nitric oxide synthase (iNOS) and nitric oxide (NO) after a moderate ALA/light challenge. The upregulated iNOS/NO was shown to play a key role in cell resistance to apoptotic photokilling and also in the dramatic growth spurt observed in surviving cells. In the present study, we found that PC3 cells surviving an ALA/light insult not only proliferated faster than non-stressed controls, but migrated and invaded faster as well, these effects being abrogated by an iNOS inhibitor or NO scavenger. Photostressed prostate DU145 cells exhibited similar behavior. Using in-gel zymography, we showed that PC3 extracellular matrix metalloproteinase-9 (MMP-9) was strongly activated 24 h after ALA/light treatment and that MMP-9 inhibitor TIMP-1 was downregulated, consistent with MMP-9 involvement in enhanced invasiveness. We also observed a photostress-induced upregulation of α6 and β1 integrins, implying their involvement as well. The MMP-9, TIMP-1, and integrin effects were strongly attenuated by iNOS inhibition, confirming NO's role in photostress-enhanced migration/invasion. This study reveals novel, potentially tumor-promoting, side-effects of prostate cancer PDT which may be averted through use of iNOS inhibitors as PDT adjuvants., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Voltage-gated T-type Ca(2+) channel Ca(v)3.2 (α(1H)) subunit, responsible for T-type Ca(2+) current, is expressed in different tissues and participates in Ca(2+) entry, hormonal secretion, pacemaker activity, and arrhythmia. The precise subcellular localization and regulation of Ca(v)3.2 channels in native cells is unknown. Caveolae containing scaffolding protein caveolin-3 (Cav-3) localize many ion channels, signaling proteins and provide temporal and spatial regulation of intracellular Ca(2+) in different cells. We examined the localization and regulation of the Ca(v)3.2 channels in cardiomyocytes. Immunogold labeling and electron microscopy analysis demonstrated co-localization of the Ca(v)3.2 channel and Cav-3 relative to caveolae in ventricular myocytes. Co-immunoprecipitation from neonatal ventricular myocytes or transiently transfected HEK293 cells demonstrated that Ca(v)3.1 and Ca(v)3.2 channels co-immunoprecipitate with Cav-3. GST pulldown analysis confirmed that the N terminus region of Cav-3 closely interacts with Ca(v)3.2 channels. Whole cell patch clamp analysis demonstrated that co-expression of Cav-3 significantly decreased the peak Ca(v)3.2 current density in HEK293 cells, whereas co-expression of Cav-3 did not alter peak Ca(v)3.1 current density. In neonatal mouse ventricular myocytes, overexpression of Cav-3 inhibited the peak T-type calcium current (I(Ca,T)) and adenovirus (AdCa(v)3.2)-mediated increase in peak Ca(v)3.2 current, but did not affect the L-type current. The protein kinase A-dependent stimulation of I(Ca,T) by 8-Br-cAMP (membrane permeable cAMP analog) was abolished by siRNA directed against Cav-3. Our findings on functional modulation of the Ca(v)3.2 channels by Cav-3 is important for understanding the compartmentalized regulation of Ca(2+) signaling during normal and pathological processes.

Gaboxadol, a selective extra synaptic GABA(A) receptor agonist, has been in clinical development for the treatment of insomnia. Development of tolerance to therapeutic effects (e.g. hypnotic and anticonvulsant and sedative) and withdrawal symptoms (e.g. REM sleep rebound and reduced seizure threshold) upon treatment discontinuation is reported for GABA(A) receptor allosteric modulators acting via the benzodiazepine binding site, e.g. zolpidem and indiplon. We conducted a head to head comparison in rats of the hypnotic (sleep EEG after 21 daily doses and 24 and 48 h after the last dose) and seizure threshold modifying (bicuculline assay 24 h after 28 daily doses) effects of gaboxadol and benzodiazepine ligands. Furthermore, we investigated in further details a previously reported apparent rapid development of tolerance to gaboxadol's effects in a rat rotarod motor coordination assay and related this effect to CNS exposure levels and in vitro potency at extra synaptic GABA(A) receptors. Sleep EEG studies demonstrated lack of tolerance and withdrawal effects after 28 daily doses with gaboxadol, whereas zolpidem produced both tolerance and withdrawal effects under a similar dosing regimen. Daily dosing with gaboxadol, zolpidem or indiplon for 28 days and acute discontinuation of treatment left the threshold to bicuculline-induced seizures unchanged. The rapidly attenuated effect of repeated gaboxadol dosing was confirmed in the rotarod model. However, re-challenge of gaboxadol insensitive animals with gaboxadol produced a maximum response, ruling out that receptor desensitisation accounts for these effects. By comparing CNS exposure at rotarod responses and concentration response relation at cloned GABA(A) receptors expressed in Xenopus oocytes it appears that the decline in response in the rotarod model coincides with the steep part of the concentration response curve for gaboxadol at extra synaptic GABA(A) receptors. In conclusion, rat sleep EEG repeated dose studies of gaboxadol confirm a hypnotic-like profile and no withdrawal effects, whereas tolerance and withdrawal effects were shown with zolpidem. Withdrawal from gaboxadol, zolpidem and indiplon did not affect the seizure threshold to bicuculline. Gaboxadol's apparent rapid development of tolerance in the rotarod assay appears to be kinetically determined.

Extended treatment with human immunodeficiency virus (HIV) protease inhibitors (HPIs) is standard in HIV/AIDS therapy. While these drugs have helped decrease the overall incidence of AIDS defining illnesses, the relative prevalence of HIV/AIDS dementia has increased. HPIs may cause induction of blood-brain barrier (BBB) drug transporters (P-glycoprotein; P-gp) and thereby limit entry of HPIs into brain tissue, increasing the probability that the brain could become an HIV sanctuary site. Using bovine brain microvessel endothelial cells (BMEC) as an in-vitro model of the BBB, the potential for the HIV protease inhibitor ritonavir to cause induction of P-gp activity and expression was examined. BMEC were isolated from fresh cow brain by enzymatic digest and density centrifugation. Primary culture BMEC were co-incubated with ritonavir or vehicle control for 120 h. Quantitative drug accumulation of rhodamine 123 (Rh123) and fluorescence microscopy were used as measures of P-gp activity. P-gp expression was assessed using quantitative Western blotting. Ritonavir decreased Rh123 cell accumulation and increased P-gp immunoreactive protein in a concentration-dependent manner. Fluorescent microscopy mirrored Rh123 quantitative studies. In BMEC pretreated with 30 microM ritonavir, Rh123 accumulation was decreased 40% and immunoreactive P-gp protein increased 2-fold. Collectively, a strong correlation between decreased Rh123 BMEC accumulation and increased P-gp immunoreactive protein was observed (Spearman r2 = 0.77, P < 0.0001). Thus extended exposure of BMEC to ritonavir caused a concentration-dependent increase in P-gp activity and expression. Similar findings may occur at the clinical level with prolonged HIV protease inhibitor use, giving insight into the central nervous system as an HIV sanctuary site and eventual development of HIV dementia.

To assess benzodiazepine tolerance in aged animals, lorazepam or vehicle was administered chronically to male Crl: CD-1(ICR)BR mice. Pharmacodynamic and neurochemical endpoints were examined on days 1 and 14 of drug administration. There was no age-related significant difference in plasma lorazepam levels. Young and middle-aged animals demonstrated behavioral tolerance to lorazepam, while the aged animals showed a similar trend which failed to reach significance. In addition, aged animals also showed a trend toward tolerance to the anticonvulsant effects of lorazepam. There were no changes in alpha1 mRNA levels in cortex or hippocampus following administration of lorazepam when compared to vehicle-treated animals in any age group. Aged animals, however, had an initial increase in alpha1 mRNA expression in cortex and hippocampus on day 1 of vehicle treatment followed by decreased expression on day 14. These age-related changes were abolished by lorazepam administration. In summary, age-related sensitivity to the effects of lorazepam was not demonstrated in the present study. However, comparison of these data to other studies indicates that the effect of chronic benzodiazepine treatment may be specific to the benzodiazepine administered, the technique used to quantify mRNA expression changes, the subunits of the GABA(A) receptor investigated and the brain region analyzed. The phenomenon of benzodiazepine sensitivity in the elderly is an area of research which remains controversial and may well be compound specific. Determining benzodiazepines that do not produce pharmacodynamic sensitivity, such as lorazepam, may allow more careful prescribing and dosing of these drugs, and perhaps even the development of specific agents which could avoid this sensitivity.

Zolpidem is chemically unrelated to classical benzodiazepines but has demonstrated relatively high affinity binding to the alpha(1) GABA(A) receptor. To assess pharmacodynamic and neurochemical effects of zolpidem, open-field behavior, pentylenetetrazole-induced seizure threshold and benzodiazepine receptor binding in vitro were evaluated in the same animal following a single dose of zolpidem. Zolpidem (2, 5 and 10 mg/kg), lorazepam (2 mg/kg) or vehicle was administered intraperitoneally in male CD-1 mice. Behavioral activity, assessed by three open-field parameters, was decreased following the two highest doses of zolpidem (5 and 10 mg/kg), and reached significance at the 10 mg/kg dose. Locomotor activity was also decreased significantly by lorazepam as expected. Pentylenetetrazole-induced seizure threshold was increased with the administration of 2 and 10 mg/kg zolpidem as well as with lorazepam. Apparent affinity (K(D)) of [3H]flunitrazepam, a non-selective ligand, for the benzodiazepine receptor in cortical membrane preparations was not significantly changed, while receptor number (Bmax) was decreased at all doses of zolpidem, reaching significance at the 10 mg/kg dose. These results confirm that the behavioral effects of zolpidem are similar to those of classical benzodiazepines. In addition, zolpidem had no significant effect on the affinity of the benzodiazepine receptor for [3H]flunitrazepam, but did decrease the density of receptor binding sites.

Previously we reported delayed cell death, defined by clear-cut cell loss 60 days after a nitrite-induced hypoxic episode. The loss of cells was not apparent two weeks after the treatment, although some changes in cellular appearance were observed at that time. A similar delayed loss of neurons in the hippocampus after hypoxia induced by blood vessel occlusion has also been found. In addition, we reported that the amount of methemoglobinemia induced by the sodium nitrite can be reduced by the stress produced by handling and the injection of saline 2 or 24 h before the nitrite administration. The degree of methemoglobin formed is directly related to cell death in certain areas of the brain, including regions within the hippocampus. Considering the many effects that can be produced by chronic and acute stress of several kinds and the length of time during which these effects manifest themselves, we undertook to determine the histologic effects of the stresses of transport on the neuroanatomic effects of sodium nitrite administration 60 days post administration. Comparisons were made of the effects of two methods of transport from the laboratory in which the animals (male CD-1 mice) were injected with the sodium nitrite or saline (Tufts Medical School) to the laboratory in which the histologic evaluations were made (Binghamton University). The animals began their travel several hours after the injections. One transport method was by commuter airline and the other was by automobile. All animals had the same transport from the supplier to the Boston location (truck). Thus, the stress of experimental interest occurred after the nitrite administration. Upon arrival at Binghamton University, the animals were housed at the University in their own colony room for 60 days before sacrifice. After sacrifice, sections from their brains were subjected to a number of histologic staining procedures, including PTAH, the Bielschowsky silver method, GFAP, and the standard Nissl procedure. Although special attention was paid to hippocampal areas, changes in cells in the habenulae and the linings of ventricular areas were also prominent. Surprisingly, the nitrite treatment before transport to Binghamton offered partial protection against the very substantial and lasting effects of the injections, transport, and handling found in the control animals. Differential effects caused by the two methods of transport were also noted.

To assess pharmacodynamic and neurochemical aspects of tolerance, lorazepam (2 mg/kg/day), or vehicle was administered chronically to male Crl: CD-1(ICR)BR mice via implantable osmotic pump. Open-field behavior, benzodiazepine receptor binding in vitro, receptor autoradiography, and muscimol-stimulated chloride uptake were examined at both 1 and 14 days. Open-field activity was depressed in lorazepam-treated animals on Day 1. On Day 14, open-field parameters were indistinguishable from those of vehicle-treated animals, indicating behavioral tolerance. Benzodiazepine binding, as determined by the specific binding of [125I]diazepam, was also decreased in cortex on Day 14. Hippocampal binding was unchanged following chronic lorazepam exposure. Apparent affinity in cortical membrane preparations was unchanged, indicating that altered ligand uptake was due to decreased receptor number. Muscimol-stimulated chloride uptake into cortical synaptoneurosomes from lorazepam-treated animals was not significantly different on Day 1 or Day 14 compared to vehicle-treated animals. These results confirm that down-regulation of benzodiazepine receptor binding is closely associated with behavioral tolerance to benzodiazepines. These observed changes in binding are not necessarily associated with robust changes in receptor function.

Previous work in our laboratory has demonstrated region-specific effects for chronic alprazolam on binding and function at the GABA(A) receptor. The present study evaluated regional changes in mRNA expression of several subunits of the GABA(A) receptor following chronic alprazolam administration that might underlie these effects. Mice received alprazolam (2 mg/kg/day) or vehicle via subcutaneously implanted osmotic pumps for 1, 7, 14 or 28 days. In situ hybridization histochemistry was performed on tissue sections using [35S]dATP oligonucleotide probes corresponding to the alpha1 and gamma2 subunits of the GABA(A) receptor. Specific hybridization was clearly demonstrated and alpha1 subunit mRNA expression in frontoparietal cortex (layers II-IV) on day 1 of infusion was reduced in animals receiving alprazolam compared to vehicle. On subsequent days, there were no alterations in the levels of alpha1 subunit mRNA in the frontoparietal cortex, hippocampus or dentate gyrus. Expression of gamma2 subunit mRNA was increased on day 1 in the frontoparietal cortex (layer VI), hippocampus and dentate gyrus. mRNA expression was also increased in the dentate gyrus on day 28 of infusion. Comparison of the present study with the results of chronic treatment with other benzodiazepines clearly demonstrates that the pattern of mRNA subunit alterations obtained is both treatment- and region-specific. This makes a definitive conclusion regarding benzodiazepines and their interactions with GABA(A) receptors difficult at best.

To characterize the potential interaction between the excitatory and inhibitory neurotransmitter systems, the effects of dizocilpine, CPP, and lorazepam on open-field behavior and pentylenetetrazol-induced seizures were evaluated in mice. Dizocilpine (0.01-0.1 mg/kg), CPP (1-10 mg/kg), or vehicle was administered intraperitoneally 15 min prior to lorazepam (0.2-2 mg/kg) or vehicle. Behavioral monitoring began 25 min after the lorazepam injection. Upon completion of testing, unrestrained mice were infused intravenously with pentylenetetrazole until the onset of a full tonic-clonic seizure. The highest dose of dizocilpine by itself significantly increased the average distance traveled, the number of rears, and the number of stereotypies during the test period. Lorazepam alone dose dependently decreased activity on all behavioral parameters. Lorazepam also completely antagonized the hyperactivity produced by dizocilpine when the two compounds were coadministered. This antagonism is most likely due to an interaction in the regulation of dopaminergic tone which underlies motor activity. Lorazepam exerted a dose-dependent anticonvulsant effect. Dizocilpine alone had no effect on seizure induction and did not potentiate the anticonvulsive effect of lorazepam when coadministered with lorazepam. CPP reduced the number of rears and the number of stereotypies during the test period. CPP did not alter the pentylenetetrazol-induced seizure threshold and did not influence the anticonvulsant effect of lorazepam.

We previously demonstrated that ketoconazole is a potent inhibitor of triazolam biotransformation in vitro and in vivo. Despite significant elevations in triazolam plasma levels with coadministration of ketoconazole, the pharmacodynamic enhancement was lower than predicted based on plasma levels of triazolam. The present study examines the effects of ketoconazole on benzodiazepine receptor binding in vitro as well as on open-field behavior in male CD-1 mice. Triazolam alone inhibited [3H]flunitrazepam binding with an IC50 value of 0.85 nM and a Ki value of 0.50 nM. Ketoconazole alone also competitively antagonized [3H]flunitrazepam binding in a concentration-dependent manner with an IC50 value of 1.56 microM and a Ki value of 1.17 microM. In the presence of 1, 3 or 9 microM ketoconazole, the IC50 value of triazolam was increased to 1.11, 1. 58 and 5.73 nM, respectively, whereas maximal binding was reduced by 36%, 69% and 89%. Coadministration of 50 mg/kg ketoconazole and triazolam (0.1-0.3 mg/kg) to intact animals significantly elevated plasma and brain triazolam levels. Ketoconazole could be measured in mouse brain at levels averaging 31% of those in plasma. Ketoconazole alone had minimal or no effect on open field activity, but it significantly potentiated the decreased activity seen with triazolam administration. The ability of ketoconazole to inhibit triazolam displacement of [3H]flunitrazepam binding may explain the muted pharmacodynamic effect of this benzodiazepine in the presence of ketoconazole. Based on these results, it is likely that ketoconazole acts as a neutral ligand at the benzodiazepine receptor.

Pregnenolone sulfate (PS) has been reported to selectively augment glutamate-induced depolarizations mediated by the NMDA subtype of the glutamate receptor. The present study examines the ability of this neuroactive steroid to potentiate NMDA-mediated increases in intracellular calcium in cultured chick cortical neurons using the fluorescent dye Fura2. PS, in the absence of NMDA and glycine, significantly elevated intracellular calcium at 250 and 500 microM. This increase in free calcium was significantly attenuated at 250 microM PS by the prior addition of 50 microM CNQX, 10 microM dizocilpine or 1 microM nimodipine. NMDA and glycine, when added to the cells in saturating concentrations of 500 and 50 microM, respectively, consistently increased intracellular free calcium over baseline levels. In the presence of NMDA and glycine, both 50 and 100 microM PS produced a further significant rise in intracellular free calcium. The prior addition of CNQX, dizocilpine or both compounds together significantly inhibited this elevation in free calcium. The application of the endogenous polyamine spermine (250 microM) significantly potentiated the response of chick cortical neuronal cells to NMDA and glycine. PS, in the presence of NMDA, glycine and spermine, produced a further increase in intracellular free calcium at concentrations of 50 and 100 microM. The prior application of CNQX, dizocilpine or both compounds together significantly attenuated this rise in free calcium. These data confirm that PS is a positive allosteric modulator of the NMDA receptor and provide evidence that this neurosteroid does not interact with the polyamine modulatory site.

Interleukin-1 is a polypeptide cytokine involved in the acute-phase response. We examined the effects of IL-1 on the major inhibitory and excitatory neurotransmitter systems in brain mediated by GABA and glutamate, respectively. IL-1 enhanced the effects of GABA as determined by GABA-dependent chloride uptake at 1 and 10 ng/ml in cortical synaptoneurosomes. This effect was attenuated by pretreatment with the IL-1 receptor antagonist. Neither IL-6 nor TNF was effective in this system. IL-1 decreased the effect of NMDA and glycine on intracellular calcium concentrations in cultured chick cortical neurons in both the presence and absence of the endogenous polyamine spermine. This effect was attenuated by pretreatment with the AMPA/kainate antagonist CNQX. These data indicate that IL-1 modulates both inhibitory and excitatory neurotransmitter function in brain.

Polyamine potentiation and inhibition of N-methyl-D-aspartate (NMDA) receptor-mediated Ca2+ changes was studied in cultured chick cortical neurons. Spermidine and spermine potentiated the effect of saturating concentrations of NMDA and glycine. No effect of spermidine or spermine was observed in the absence of NMDA or in the presence of either kainate or quisqualate. Similarly, antagonism of the NMDA receptor complex with dizocilpine (an open channel blocker), or with competitive antagonists to the NMDA or glycine binding sites greatly attenuated or completely abolished the combined effects of polyamines plus NMDA and glycine. N-Acetylspermine and N-acetylspermidine, in the presence or absence of NMDA and glycine, were without effect. These data strongly suggest that spermidine and spermine are potent and selective agonists at the polyamine binding site. Putrescine and diethylenetriamine were ineffective as antagonists of NMDA-mediated intracellular free Ca2+ increases in the presence or absence of added spermine or spermidine. Arcaine and 1,10-diaminodecane, however, antagonized NMDA-mediated intracellular free Ca2+ increases in the presence and absence of spermine and spermidine, and therefore appear to act either as inverse agonists at the polyamine binding site or as open channel blockers of the NMDA receptor.

Release of endogenous polyamines may contribute to neuronal loss in ischemia and related conditions. Primary cortical neurons were exposed to spermine and spermidine and subsequently assayed for [3H]ouabain binding to quantify neuronal loss. Neuronal survival was significantly decreased in the presence of spermine at 24 h (500 microM), 48 h (250 microM and 500 microM) and 72 h (10-500 microM) relative to controls. Co-application of 250 microM spermine and 10 microM dizocilpine for 48 h completely inhibited the effect of spermine alone. Spermidine exposure (10-500 microM) did not alter neuronal survival at any of the time points. These data indicate that the polyamine spermine is toxic to neurons in vitro and that toxicity is prevented by the NMDA-associated channel antagonist dizocilpine.

This study was undertaken to evaluate the effects of pretreatment with calcium channel antagonists (nimodipine or verapamil) on the formation of methemoglobin produced by sodium nitrite. Unexpectedly, the pretreatment of animals with control injections of physiological saline 2 hours before the nitrite administration reduced the amount of methemoglobin found in the blood 25 minutes later. When either of the calcium channel antagonists was given 2 hours before the administration of sodium nitrite, the saline effect was eliminated. When the injections of physiological saline or either of the calcium channel blockers were divided 24 hours before the nitrite administration, all reduced the amount of methemoglobin formed relative to rats that received no pretreatment. A tentative hypothesis is that the reduction of the nitrite-induced methemoglobin can be induced by the stress of handling and intraperitoneal injection and that this stress effect can last at least 24 hours. It is likely that whatever stress-related mechanism is involved in reducing methemoglobin levels, this effect can be reduced by the presence of "L channel" voltage-sensitive calcium antagonists or their active metabolites.

Adult Binghamton Heterogeneous male mice received either nimodipine (5 mg/kg in saline) or vehicle 10 minutes prior to ethanol (1.02 g/kg) administration. Blood samples were obtained at 2, 10, 30, 60 and 120 minutes following the alcohol injection for determination of ethanol content. Although mean blood alcohol concentrations (BACs) were similar for both groups, nimodipine increased the variance in blood alcohol levels. Therefore, while nimodipine may alter alcohol pharmacokinetics through its interaction(s) with the genetic characteristics of the individual animal, the ability of this calcium slow channel blocking agent to enhance the psychotropic effects of alcohol cannot be due to altered absorption or elimination of the intoxicant.