Decreases in peripheral-type benzodiazepine receptors in postmortem brains of chronic schizophrenics

We measured the peripheral-type benzodiazepine receptors (PBRs), a marker of gliosis, in 26 brain areas (cerebral cortex, thalamus and extrapyramidal system) of the postmortem brains of 13 chronic schizophrenics and 10 controls, using [3H] PK 11195 as a ligand for the receptor assay. The specific [3H] PK 11195 binding was significantly decreased in three brain areas (superior parietal cortex, primary visual area and putamen) of schizophrenics, although there were no changes in the binding in the other brain areas. Scatchard analysis revealed that there were decreases in both the Bmax and Kd of [3H] PK 11195 binding in the brain areas. These results were almost in accordance with a number of neuropathological studies reporting that there was no change or reduction in glial cells in the brain regions of schizophrenics and suggested that the decreased density of PBRs in the brain may be involved in the pathophysiology of schizophrenia, associated with reduced production of neurosteroids coupled to PBRs.     

Ovarian steroids and stress produce changes in peripheral benzodiazepine receptor density

Although past research has described changes in the density of the peripheral benzodiazepine receptor in brain and in peripheral organs in response to stressors and steroid hormone exposure, their combined influence had yet to be determined. This study examined the effect of swim-stress as a function of ovarian hormone administration on the binding of an isoquinoline carboxamide derivative, [3H]PK 11195, in brain and peripheral tissues. In olfactory bulb and adrenal gland, stress increased peripheral benzodiazepine receptor density in ovariectomized rats with and without estradiol and progesterone replacement injection, even when compared with unstressed animals treated with hormones, where estradiol + progesterone decreased peripheral benzodiazepine receptor number in olfactory bulb, but estradiol and estradiol + progesterone increased it in adrenal gland. In frontal cortex, stress decreased peripheral benzodiazepine receptor number, an effect that was reversed by estradiol. In hippocampus estradiol decreased peripheral benzodiazepine receptor density in unstressed animals and estradiol + progesterone decreased peripheral benzodiazepine receptor number in unstressed and stressed animals. In cerebellum, stress, estradiol and estradiol + progesterone alone decreased peripheral benzodiazepine receptor density. In uterus of unstressed controls, estradiol + progesterone increased peripheral benzodiazepine receptor density, and stress produced a further increase in steroid-treated females. Stress did not affect peripheral benzodiazepine receptor density in kidney, except in animals that received estradiol + progesterone injections, where swim-stress produced a significant decrease in peripheral benzodiazepine receptor density. Thus, steroid hormones regulate peripheral benzodiazepine receptor density in endocrine organs and brain, and the hormonal state of the organism modifies the peripheral benzodiazepine receptor response to stress in a tissue- and brain region-specific manner, suggesting that the peripheral benzodiazepine receptor may play a pivotal role in an integrated response to stress.     

Involvement of peripheral-type benzodiazepine receptors in the intracellular transport of heme and porphyrins

To investigate the involvement of peripheral-type benzodiazepine receptors (PBR) in heme metabolism, we examined the interaction of [55Fe]heme with PBR. Transfection of the cloned mouse PBR-isoquinoline carboxamide-binding protein (PBR/IBP) cDNA into monkey kidney Cos-1 cells resulted in a 2.5-fold increase in [55Fe]hemin binding sites, concomitant with the increase in [3H]PK11195 binding sites, as compared with those seen in antisense PBR/IBP cDNA-transfected cells. The binding of hemin to the transfected receptors exhibited a relatively high affinity with a Kd of 12 nM, and was inhibited by several benzodiazepine ligands, including PK11195, Ro 5-4864, diazepam and protoporphyrin IX. When mouse liver mitochondria were incubated with [55Fe]hemin, the binding to PBR had a Kd of 15 +/- 1.8 nM. The Bmax of [55Fe]hemin binding to the mitochondria was 6.88 +/- 0.76 pmol/mg of protein, a value consistent with that of [3H]PK11195 binding, with a lower affinity. Coproporphyrinogen III, a precursor porphyrin produced in the cytosol, is translocated into mitochondria, then is converted to protoporphyrinogen IX; this conversion decreased in the presence of benzodiazepine ligands. To examine whether this decrease was related to a decrease in the binding of coproporphyrinogen to the mitochondria, the effects of benzodiazepines on the binding of coproporphyrinogen were examined. As the binding was dose-dependently inhibited by PK11195, Ro 5-4864, and diazepam, porphyrins are likely to be endogenous ligands for PBR. We propose that PBR play a role in the intracellular transport of porphyrins and heme.     

Neuroprotective properties of lifarizine compared with those of other agents in a mouse model of focal cerebral ischaemia

1. Changes in the peripheral type benzodiazepine binding site density following middle cerebral artery occlusion in the mouse, have been used as a marker of neuronal damage. These sites can be identified using the selective ligand [3H]-PK 11195 located on non neuronal cells, macrophages and astroglia, within the CNS. Glial cell proliferation and macrophage invasion is an unvoidable sequelae to cerebral ischaemic injury, secondary to neuronal loss. Following occlusion of the left middle cerebral artery (left MCA) a reproducible lesion was found in the parietal cortex within 7 days which gave rise to a significant increase in [3H]-PK 11195 binding. 2. Treatment of animals with the sodium channel blocker, lifarizine, significantly reduced the ischaemia-induced increase in [3H]-PK 11195 binding when given either 30 min pre-ischaemia and three times daily for 7 days at 0.5 mg kg-1, i.p. (P < 0.01) or delayed until 15 min post-ischaemia and three times daily for 7 days at 0.5 mg kg-1, i.p. (P < 0.001). Lifarizine was an effective neuroprotective agent in this model of focal ischaemia in the mouse. 3. Lifarizine also showed a dose-related protection against the ischaemia-induced increase in [3H]-PK 11195 binding with significant protection at doses of 0.1 mg kg-1, i.p. (P < 0.05), 0.25 mg kg-1, i.p. (P < 0.01) or 0.5 mg kg-1, i.p. (P < 0.01) 15 min post-ischaemia and b.i.d. for 7 days. No significant change is seen in the Kd for [3H]-PK 11195. The first dose could be delayed for up to 4 h after cerebralartery cauterization and protection was maintained.4. Phenytoin (28 mg kg-1, i.v. 15 min and 24 h post-ischaemia) was also neuroprotective in this model(P<0.01). This agent is thought to interact with voltage-dependent sodium channels to effect its anticonvulsantactions and this mechanism may also underlie its neuroprotective actions in focal cerebralischaemia.5. Agents with other mechanisms of action were also shown to have significant neuroprotection in this model. The non-competitive NMDA antagonist, MK 801, showed significant neuroprotection in the model when given at 0.5 mg kg-1, i.p. 30 min pre-ischaemia with t.i.d. dosing for 7 days (P< 0.001). The dihydropyridine calcium antagonist, nimodipine was not protective when given using the same dosing protocol as MK 801, 0.5 mg kg-1 30 min pre-occlusion and three times daily for 7 days but showed significant protection when given at 0.05 mg kg-1 15 min post-ischaemia and three times daily for 7days. The lipid peroxidation inhibitor, tirilazad (single dose 1 mg kg-1, i.v.) showed significant neuroprotection when given 5 min post-ischaemia but not when the first dose was delayed for 4 h.     

Identification and characterization of a high-affinity peripheral-type benzodiazepine receptor in rabbit urinary bladder

The present study used radioligand binding and in vitro contractility experiments to identify and characterize a peripheral-type benzodiazepine receptor PBR in rabbit urinary bladder. [3H]PK11195 bound to bladder membranes with high-affinity and density (Kd = 5.2 nM., Bmax = 268 fmol./mg. protein), indicating the presence of a PBR. [3H]flunitrazepam bound with high-affinity and density (Kd = 1.2 nM., Bmax = 48 fmol./mg. protein). The rank order potency of various benzodiazepines and isoquinoline carboxamides in displacing the binding of [3H]PK11195 was Ro5-4864 > diazepam = flunitrazepam > Ro15-1788 = clonazepam. Ro5-4864 and PK11195 inhibited nerve-evoked contractions in a concentration-dependent manner (IC50 = 42 microM. and 56 microM., respectively). Carbachol- and KCl-induced contractions were also inhibited by Ro5-4864 and PK11195. KCl-induced contractions were inhibited to a greater extent than carbachol-induced or field-stimulated contractions with all the drugs tested. Both Ro5-4864 and PK11195 significantly increased the ED50 for calcium-induced contractions following a cholinergic stimulus compared with control. These data demonstrate the presence of a PBR in urinary bladder capable of altering contractility in vitro through modulation of calcium activity.     

Temporal changes in glial fibrillary acidic protein messenger RNA and [3H]PK11195 binding in relation to imidazoline-I2-receptor and alpha 2-adrenoceptor binding in the hippocampus following transient global forebrain ischaemia in the rat

Immunohistochemical studies have demonstrated that following global forebrain ischaemia the selective neuronal loss that occurs in the CA1 pyramidal cell layer of the hippocampus is accompanied by a reactive astrocytosis, characterized by increases in glial fibrillary acidic protein, and activation of microglia. In this study the spatial changes in glial fibrillary acidic protein messenger RNA levels in the hippocampus have been mapped four, eight, 12, 16 and 20 days following 10 min of global forebrain ischaemia in the rat and related to changes in [3H]PK11195 binding to peripheral benzodiazepine receptors, a putative marker of activated microglia. Recent studies have suggested that the imidazoline-I2-receptor, one of a class of non-adrenergic receptors related to, but structurally and functionally distinct from alpha 2-adrenoceptors, may have a functional role in controlling the expression of glial fibrillary acidic protein. To explore this possibility further we have also mapped changes in imidazoline-I2-receptor and alpha 2-adrenoceptor binding sites. Following transient ischaemia there was a marked, biphasic increase in glial fibrillary acidic protein messenger RNA levels throughout the vulnerable CA1 region of the hippocampus, peaking four days after ischaemia and then increasing gradually during the remainder of the study period. There was also a sustained increase in [3H]PK11195 binding, however, in contrast to the initial increase in glial fibrillary acidic protein messenger RNA levels that peaked four days after ischaemia the density of [3H]PK11195 binding increased rapidly in all strata of the CA1 region over the first eight days and then increased more slowly throughout days 12 to 20. Despite the marked increase in glial fibrillary acidic protein messenger RNA levels there was no concomitant alteration in imidazoline-I2-receptor binding sites detected using the specific radioligand, [3H]2-(2-benzofuranyl)-2-imidazoline, although alpha 2-adrenoceptor binding was decreased at eight days after ischaemia and did not recover. The time-course and biphasic nature of the changes in the astrocytic marker, glial fibrillary acidic protein messenger RNA, in the hippocampus following ischaemia may reflect different functions of glial fibrillary acidic protein-reactive astrocytes in the post-ischaemic period. Differences in temporal expression of glial fibrillary acidic protein messenger RNA and [3H]PK11195 binding support the proposed localization of peripheral benzodiazepine receptors on activated microglia, as distinct from reactive astrocytes. There was no evidence in the present study that imidazoline-I2-receptors are functionally linked to glial fibrillary acidic protein expression as the reactive astrocytosis in the hippocampus following ischaemia was not associated with changes in imidazoline-I2-receptor binding site density.     

Catabolite inactivation of the yeast maltose transporter requires ubiquitin-ligase npi1/rsp5 and ubiquitin-hydrolase npi2/doa4

Peripheral benzodiazepine receptors have been shown in some endocrine tissues, namely the testis, the adrenal gland, and the pituitary gland. In this work we evaluated whether peripheral benzodiazepine receptors can be found in the purified human pancreatic islets and whether they may have a role in insulin release. Binding of the isoquinoline compound [3H]1-(2-chlorophenyl-N-methyl-1-methyl-propyl)-3- isoquinolinecarboxamide (13H]PK-11195) a specific ligand of peripheral benzodiazepine receptors, to cellular membranes was saturable and Scatchard's analysis of the saturation curve demonstrated the presence of a single population of binding sites, with an affinity constant value of 9.20 +/- 0.80 nM and a maximum number of binding sites value of 8913 +/- 750 fmol/mg of proteins. PK-11195 and 7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4- benzodiazepine-2-on (Ro 5-4864) significantly potentiated insulin secretion from freshly isolated human islets at 3.3 mM glucose. These results show the presence of peripheral benzodiazepine receptors in purified human pancreatic islets and suggest their role in the mechanisms of insulin release.     

Modification of cardiac actions of RO 05-4864 by PK 11195 and flumazenil in the perfused rat heart

The benzodiazepine analogue Ro 05-4864 [chlorodiazepam] (2.10[-5] to 4.10[-4] M) induced a concentration-dependent increase of coronary flow rate (Emax 82.4% [+/- 2.2 SEM]) and an increase of contraction force (Emax 68.3% [+/- 4.7 SEM]) of the retrograde perfused, isolated Langendorff rat heart. The influence of PK 11195, antagonist of the peripheral type benzodiazepine receptor, and flumazenil (Anexate), antagonist of the central type benzodiazepine receptor, on these responses to Ro 5-4864 was studied. In concentrations of 10(-7) to 5.10(-5) M, PK 11195 significantly reduced both the increase of coronary flow rate and of contraction force, without affecting these functions by itself; the positive inotropic response produced by Ro 05-4864 was even abolished in the presence of 5.10(-5) M PK 11195. The Emax values of Ro 05-4864 on both coronary flow and inotropy were reduced significantly by PK 11195. In the presence of flumazenil, 10(-7) to 10(-5) M, both the vasodilatory and the positive inotropic response induced by Ro 05-4864 were significantly counteracted as well. The Emax values of Ro 05-4864 were reduced significantly. In conclusion, the results support earlier suggestions that it is tempting to involve peripheral type benzodiazepine receptors in cardiac actions of benzodiazepines. The finding that the centrally acting benzodiazepine antagonist flumazenil reduced the cardiac actions of Ro 05-4864 is as yet difficult to explain. On the other hand concentrations of both agonist and antagonist employed are so-much high that interference of other receptors than benzodiazepine receptors must be considered as well.     

The attenuation of kainate-induced neurotoxicity by chlormethiazole and its enhancement by dizocilpine, muscimol, and adenosine receptor agonists

Systemically administered kainate (10 mg.kg-1) caused neuronal loss in both the hippocampus and the entorhinal regions of the rat brain. This resulted in a loss of 68.3 +/- 13.8 and 53.3 +/- 12.8% of pyramidal neurones in the hippocampal CA1 and CA3a regions, respectively. Chlormethiazole attenuated the loss of neurones in the hippocampal cell layers CA1 (cell loss 10 +/- 3.2%) and CA3a (cell loss 10 +/- 7.7%). The neuroprotective activity of chlormethiazole was apparent in the presence or absence of a low dose of clonazepam (200 micrograms.kg-1 i.p.). The kainate-induced damage could also be measured by the increase in binding of the peripheral benzodiazepine ligand ([3H]PK11195) in the hippocampus. In kainate-treated rats there was a 350-500% increase in binding indicative of reactive gliosis. Chlormethiazole prevented this elevation in a dose- and time-dependent manner, with an ED50 of 10.64 mg.kg-1 and an effective therapeutic window from 1 to 4 h posttreatment. Dizocilpine also attenuated damage significantly. The GABAA agonist muscimol was also able to attenuate the increase in [3H]PK11195 binding in a dose-dependent manner, with an ED50 of approximately 0.1 mg.kg-1. If muscimol, dizocilpine, or the adenosine A1 receptor agonist R-N6-phenylisopropyl-adenosine were administered together with chlormethiazole at their respective ED25 doses, a potentiation was apparent in the degree of neuroprotection. It is concluded that the combination of neuroprotective agents with different mechanisms of action can lead to a synergistic protection against excitotoxicity.     

Cardiovascular characterization of pyrrolo[2,1-d][1,5]benzothiazepine derivatives binding selectively to the peripheral-type benzodiazepine receptor (PBR): from dual PBR affinity and calcium antagonist activity to novel and selective calcium entry blockers

The synthesis and cardiovascular characterization of a series of novel pyrrolo[2,1-d][1,5]-benzothiazepine derivatives (54-68) are described. Selective peripheral-type benzodiazepine receptor (PBR) ligands, such as PK 11195 and Ro 5-4864, have recently been found to possess low but significant inhibitory activity of L-type calcium channels, and this property is implicated in the cardiovascular effects observed with these compounds. In functional studies both PK 11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxa mide) and Ro 5-4864 (4'-chlorodiazepam) did not display selectivity between cardiac and vascular tissue. Therefore, several 7-(acyloxy)-6-arylpyrrolo[2,1-d][1,5]benzothiazepines, potent and selective peripheral-type benzodiazepine receptor ligands recently developed by us (3, 7-20), were subjected to calcium channel receptor binding assay. Some of these compounds showed an unexpected potency in displacing the binding of [3H]nitrendipine from L-type calcium channels, much higher than that reported for PK 11195 and Ro 5-4864 and equal to or higher than that of reference calcium antagonists such as verapamil and (+)-cis-diltiazem. Specifically, in rat cortex homogenate, our prototypic PBR ligand 7-acetoxy-6-(p-methoxyphenyl)pyrrolo[2,1-d][1,5]benzothiazepine (3) showed an IC50 equal to 0.13 nM for inhibition of [3H]nitrendipine binding. Furthermore, in functional studies this compound displayed a clear-cut selectivity for cardiac over vascular tissue. Comparison of calcium antagonist activity on guinea pig aorta strips with the negative inotropic activity, determined by using isolated guinea pig left atria, revealed that 3 displayed higher selectivity than the reference (+)-cis-diltiazem. Thus, the pyrrolobenzothiazepine 3 might represent a new tool for characterizing the relationship between the PBR and cardiac function. Furthermore, we have also investigated the structural dependence of binding to PBR and L-type calcium channels, and this study allowed us to identify a new class of potent calcium channel blockers selective for cardiac over vascular tissue, with no affinity for PBR. A number of structure-activity relationship trends have been identified, and a possible explanation is advanced in order to account for the observed differences in selectivity. Three structural features, namely, (i) the saturation of the C(6)-C(7) double bond, with a consequent higher molecular flexibility, (ii) the presence of a substituent in the benzofused ring, and (iii) a basic side chain at C-10 of the pyrrolobenzothiazepine ring system, were found to be responsible for potent L-type calcium channel antagonism and clear-cut selectivity for cardiac over vascular tissue. Among the synthesized compounds the pyrrolobenzothiazepine 62 was found to be the most promising selective calcium channel blocker. Additionally, the molecular structure determination of the key intermediate 48 by X-ray diffraction, molecular modeling, and NMR analysis is reported.     

Induction of peripheral-type benzodiazepine receptors in mouse brain following thioacetamide-induced acute liver failure

To investigate the possible role of peripheral-type benzodiazepine receptors (PBR) in hepatic encephalopathy, we examined expression of PBR in mouse brain following thioacetamide (TAA)-induced acute liver failure. Treatment of mice with TAA resulted in an increase in the number of binding sites of the PBR ligand [3H] Ro5-4864 to brain homogenates, with no significant change in affinity of the ligand. The order of potency of different ligands to compete against [3H] Ro5-4864 binding in the brain of TAA-treated mice was Ro5-4864 > PK11195 > diazepam > protoporphyrin IX, findings similar to those in the control. Northern blot analysis revealed an increase in PBR/isoquinoline binding protein (PBR/IBP) mRNA in mouse brain following TAA treatment, in a time- and dose-dependent manner. These results indicate that the increased number of PBR in the brains of TAA-treated mice relates to the induction of PBR/IBP expression and suggest that the induction of PBR in brain may contribute to pathogenesis of hepatic encephalopathy.     

Up-regulation of estrogen receptor mRNA and estrogen receptor activity by estradiol in liver of rainbow trout and other teleostean fish

[125I]- and [123I]NNC 13-8241 were prepared from the trimethyltin precursor and radioactive iodide using the chloramine-T method. The total radiochemical yields of [125I]- and [123I]NNC 13-8241 were 60-70% and 40-50% respectively, with radiochemical purity higher than 98%. In binding studies with [125I]NNC 13-8241 in rats in vitro and in vivo a high uptake of radioactivity was demonstrated in brain regions known to have a high density of benzodiazepine (BZ) receptors such as the occipital and frontal cortex. SPECT examination with [123I]NNC 13-8241 in a Cynomolgus monkey demonstrated a high uptake of radioactivity in the occipital and frontal cortex. After displacement with flumazenil radioactivity in these brain regions was reduced to the level of a central region including the pons. Four hours after injection about 80% of the radioactivity in monkey plasma represented unchanged radioligand. This low degree of metabolism indicates that NNC 13-8241 is metabolically more stable than the radioligands hitherto developed for imaging of BZ-receptors in the primate brain.     

In vitro and in vivo primate evaluation of carbon-11-etomidate and carbon-11-metomidate as potential tracers for PET imaging of the adrenal cortex and its tumors

METHODS: With the purpose of developing a PET imaging agent for tumors of the adrenal cortex, we developed syntheses for 11C-etomidate and its methyl analog, 11C-metomidate. (R)-[O-ethyl-1-11C]Etomidate and (R)-[O-methyl-11C]metomidate were prepared by reaction of the appropriate respective 11C-labeled alkyl iodide and the tetrabutylammonium salt of the carboxylic acid derivative. The specificity of binding to the adrenal cortex was tested through the use of frozen section autoradiography of different tissues of the rat, pig and human. Inhibition of tracer binding was evaluated with etomidate, ketoconazole and metyrapone, well-known inhibitors of enzymes for steroid synthesis. Tracer binding to different human tumor samples was compared to immunohistochemical staining with antibodies for the steroid synthesis enzymes P450 11beta (11beta-hydroxylase), P450 scc (cholesterol side-chain cleavage enzyme), P450 C21 (21 -hydroxylase) and P450 17alpha (17alpha-hydroxylase). Three PET investigations, one with 11C-etomidate and two with 11C-metomidate, were performed in rhesus monkey sections, including the adrenals, liver and kidneys. Time-activity curves were generated from measured tracer uptake in these organs. RESULTS: In frozen section autoradiography of various tissues, high binding was seen in the adrenal cortex from all species, as well as in the tumors of adrenal cortical origin. The level of liver binding was about 50% of that in the adrenals, whereas that of all other organs was <10% of the adrenal binding. The adrenal binding was blocked by etomidate and ketoconazole at low doses but not by metyrapone. The binding in the adrenal tumor samples correlated with immunostaining for P450 11beta . PET studies in the monkey demonstrated high uptake in the adrenals with excellent visualization. The uptake increased with time without indication of washout. Slightly lower uptake was seen in the liver as compared to the adrenals, and in the late images, no organs other than adrenals and liver were seen. CONCLUSION: These investigations indicate that 11C-etomidate and 11C-metomidate have the potential to be useful specific agents for the visualization of the normal adrenal cortex and to provide positive identification of adrenal cortical tumors.     

In vivo and in vitro biotransformation of the lithium salt of gamma-linolenic acid by three human carcinomas

Lipid metabolism has been considered recently as a novel target for cancer therapy. In this field, lithium gamma-linolenate (LiGLA) is a promising experimental compound for use in the treatment of human tumours. In vivo and in vitro studies allowed us to assess the metabolism of radiolabelled LiGLA by tumour tissue and different organs of the host. In vitro studies demonstrated that human pancreatic (AsPC-1), prostatic (PC-3) and mammary carcinoma (ZR-75-1) cells were capable of elongating GLA from LiGLA to dihomo-gamma-linolenic acid (DGLA) and further desaturating it to arachidonic acid (AA). AsPC-1 cells showed the lowest delta5-desaturase activity on DGLA. In the in vivo studies, nude mice bearing the human carcinomas were given Li[1-(14)C]GLA (2.5 mg kg(-1)) by intravenous injection for 30 min. Mice were either sacrificed after infusion or left for up to 96 h recovery before sacrifice. In general, the organs showed a maximum uptake of radioactivity 30 min after the infusion started (t = 0). Thereafter, in major organs the percentage of injected radioactivity per g of tissue declined below 1% 96 h after infusion. In kidney, brain, testes/ovaries and all three tumour tissues, labelling remained constant throughout the experiment. The ratio of radioactivity in liver to tumour tissues ranged between 16- and 24-fold at t = 0 and between 3.1- and 3.7-fold at 96 h. All tissues showed a progressive increase in the proportion of radioactivity associated with AA with a concomitant decrease in radiolabelled GLA as the time after infusion increased. DGLA declined rapidly in liver and plasma, but at a much slower rate in brain and malignant tissue. Seventy-two hours after the infusion, GLA was only detected in plasma and tumour tissue. The sum of GLA + DGLA varied among tumour tissues, but it remained 2-4 times higher than in liver and plasma. In brain, DGLA is the major contributor to the sum of these fatty acids. Data showed that cytotoxic GLA and DGLA, the latter provided either by the host or by endogenous synthesis, remained in human tumours for at least 4 days.     

Novel ligands specific for mitochondrial benzodiazepine receptors: 6-arylpyrrolo[2,1-d][1,5]benzothiazepine derivatives. Synthesis, structure-activity relationships, and molecular modeling studies

A novel class of ligands specific for MBR receptors has been identified: 6-arylpyrrolo[2,1-d][1,5]benzothiazepine derivatives. The majority of newly synthesized esters 37-64 as well as some intermediate ketones showed micro- or nanomolar affinity for [3H]PK 11195 binding inhibition. A SAR study on 42 compounds and a molecular modeling approach led to a preliminary structural selectivity profile: the 6,7-double bond, the carbamoyloxy, alcanoyloxy, and mesyloxy side chains at the 7-position, and the prospective chloro substitution at the 4-position seemed to be the most important structural features improving affinity. Therefore, 7-[(dimethylcarbamoyl)oxy]- and 7-acetoxy-4-chloro-6-phenylpyrrolo[2,1-d][1,5]benzothiazepine (43 and 57) were synthesized. With 7-[(dimethylcarbamoyl)oxy]-6-(p-methoxyphenyl)pyrrolo[2,1- d][1,5]benzothiazepine (65), these were the most promising compounds with IC50s of respectively 9, 8, and 9 nM, under conditions where PK 11195 had an IC50 of 2 nM.     

Peripheral type benzodiazepine receptor in T lymphocyte rich preparation

Some types of mood disorders and drugs are suggested to affect peripheral type benzodiazepine receptors (PBR), but their mechanisms are unclear. The isolation of pure lymphocytes is requisite for the investigation of the function of PBR on lymphocytes, since platelets and monocytes also have many PBR. The objective of this study was to establish a method of binding assay for PBR using pure T lymphocytes. Mononuclear cells and T lymphocytes were prepared by using a density gradient material and magnetic beads, respectively. The cells were analyzed using flow cytometry and a counting chamber. Binding studies were performed using T lymphocytes from 10 normal volunteers. The T lymphocytes were incubated with [3H]PK11195, harvested on glass fiber filters, and counted with a plate scintillation counter. The binding data were analyzed by the Scatchard method. With the magnetic bead technique, pure T cells were selected that contained only 1.5% monocytes and platelet/cell ratio of 1.4. The Scatchard plot of the data indicated that only one type of specific binding site was involved in the binding. The dissociation constant (Kd) was 3.8+/-1.3nM (mean+/-SD), and the Bmax was 379+/-124 fmol//10(6) cells (mean+/-SD). The density gradient- magnetic beads technique can be used as an appropriate method of preparation of T cells for PBR binding assay.     

Evaluation and management of men whose radical prostatectomies failed: results of an international survey

1. The responses of the electrically-driven right ventricle strip of the guinea-pig heart to diazepam were recorded in the absence and in the presence of different selective cyclic nucleotide phosphodiesterase (PDE) inhibitors. 2. Diazepam, at concentrations ranging from 1 microM to 100 microM, was devoid of effect on the contractile force in this preparation. 3. Conversely, diazepam (5 microM-100 microM) produced a consistent positive inotropic response in the presence of a concentration (1 microM), that was without effect in the absence of diazepam, of either of the selective PDE 3 inhibitors milrinone or SK&F 94120, but not in the presence of the selective PDE 4 inhibitor rolipram. 4. This effect of diazepam was not gamma-aminobutyric acid (GABA)-dependent, since it was neither mimicked nor potentiated by GABA, and was not affected by either a high concentration (5 microM) of the antagonists of the benzodiazepine/GABA/channel chloride receptor complex, picrotoxin, flumazenil and beta-carboline-3-carboxylic acid methyl ester (betaCCMe), or by the inverse agonists, beta-carboline-3-carboxylic acid N-methylamide (betaCCMa) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM, 0.1 microM). Furthermore, a specific antagonist of the peripheral benzodiazepine receptors, PK 11195 (5 microM), did not influence the effect of diazepam. 5. Biochemical studies with isolated PDEs, confirmed that diazepam selectively inhibits type 4 PDE from guinea-pig right ventricle rather than the other PDEs present in that tissue. The compound inhibited this enzyme in a non-competitive manner. Diazepam was also able to inhibit PDE 5, the cyclic GMP specific PDE absent from cardiac muscle, with a potency close to that shown for PDE 4. 6. Diazepam displaced the selective type 4 PDE inhibitor, rolipram from its high affinity binding site in rat brain cortex membranes, and also potentiated the rise in cyclic AMP levels induced by isoprenaline in guinea-pig eosinophils, where only type 4 PDE is present. 7. The PDE inhibitory properties of diazepam were shared, although with lower potency, by other structurally-related benzodiazepines, that also displaced [3H]-rolipram from its high affinity binding site. The order of potency found for these compounds in these assays was not related to their potencies as modulators of the GABA receptor through its benzodiazepine binding site. 8. The pharmacological and biochemical data presented in this study indicate that diazepam behaves as a selective type 4 PDE inhibitor in cardiac tissue and this effect seems neither to be mediated by the benzodiazepine/GABA/channel chloride receptor complex nor by peripheral type benzodiazepine receptors.     

The specific binding of the platelet-activating factor (PAF) receptor antagonist WEB 2086 and the benzodiazepine flunitrazepam to rat hepatocytes

Thieno-triazolodiazepines WEB 2086 and BN 50739 have been described as the potent PAF receptor antagonists. Binding of radiolabeled [3H]WEB 2086 has been widely employed to characterize PAF receptors in different cells. In a search for a PAF receptor in isolated rat hepatocytes, we discovered that the binding of [3H]WEB to rat hepatocytes was highly specific but had a relatively low affinity with a Kd of 113 nM and Bmax of 0.65 pmol/10(6) cells in freshly isolated cell suspension and Kd of 1.65 muM and Bmax of 2.0 pmol/plate in cultured hepatocytes. No consistent specific binding of [3H]PAF itself was found in the same cell preparations. The binding of [3H]flunitrazepam in the presence of the peripheral type of benzodiazepine receptor antagonist Ro 5-4864 was saturated and exhibited a K(i) of 3.8 nM and Bmax of 3.5 pmol/plate. The central type of benzodiazepine receptor antagonist clonazepam was competed for the [3H]flunitrazepam binding, however with a much lower affinity. Various antagonists inhibited the binding of [3H]WEB 2086 with a rank order BN 50739>Ro 5-4864 > or = clonazepam. Interestingly, bicuculline, specific antagonist of GABA(A) recognition sites, also significantly reduced the binding of [3H]WEB 2086. The binding of [3H]flunitrazepam was inhibited with a rank potency BN 50739>WEB 2086. Taken together, these findings suggest that the specific binding of PAF receptor antagonists WEB 2086 and BN 50739 in rat hepatocytes does not involve PAF receptors and occurs via peripheral benzodiazepine and, possibly GABA(A) receptor sites.     

Effect of peripheral benzodiazepine receptor ligands on lipopolysaccharide-induced tumor necrosis factor activity in thioglycolate-treated mice

To investigate the effect of peripheral and central benzodiazepine receptor ligands on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) activity in mouse macrophages, three types of ligands, 4'-chlorodiazepam (pure peripheral), midazolam (mixed), and clonazepam (pure central), were compared. Midazolam and 4'-chlorodiazepam significantly suppressed LPS (1-microgram/ml)-induced TNF activity in thioglycolate-elicited mouse macrophages. In every concentration examined (0.001 to 100 microM), 4'-chlorodiazepam was the most effective agent, clonazepam was the least effective agent, and midazolam had an effect intermediate between those of the other two ligands. The peripheral benzodiazepine receptor ligands had a dose-dependent suppressive effect, and the 50% inhibitory concentrations were 0.01 microM for 4'-chlorodiazepam and 5 microM for midazolam. Concomitant use of PK 11195 (10 microM), an antagonist of the peripheral benzodiazepine receptor, reversed this suppressive effect with 4'-chlorodiazepam (10 microM) or midazolam (10 microM). PK 11195 showed this antagonistic effect in a dose-dependent manner. Intravenous 4'-chlorodiazepam (5 mg/kg of body weight) significantly suppressed LPS (100-micrograms)-induced TNF activity of sera (2 h postchallenge with LPS) from thioglycolate-treated mice. The present findings suggest that the peripheral benzodiazepine receptor plays an important role in modulating LPS-induced TNF activity in mouse macrophages.     

Unchanged [3H]MK-801 binding and increased [3H]flunitrazepam binding in turtle forebrain during anoxia

In order to determine if functional changes in N-methyl-D-aspartate receptors and GABAA receptors play a role in the remarkable anoxia tolerance of freshwater turtle brain, we used autoradiographic techniques to assay [3H]MK-801 and [3H]flunitrazepam binding in turtle forebrain after turtles had been subjected to anoxia for 2 or 6 h. The effects of glutamate, glycine, competitive N-methyl-D-aspartate antagonists, glycine antagonists, polyamines, magnesium, and zinc on [3H]MK-801 binding were the same in anoxic and control turtle forebrains. These results indicate that NMDA receptor regulation plays no role in the adaptive responses to anoxia in turtle brain. In contrast, [3H]flunitrazepam binding was significantly increased in the anoxic dorsal cortex and striatum. The most parsimonious explanation for elevated benzodiazepine receptor binding is that the rise in extracellular GABA levels known to accompany anoxia enhances benzodiazepine receptor affinity. It is possible, however, that GABAA receptor upregulation during anoxia increases the effectiveness of the inhibitory action of released GABA and contributes to the anoxia tolerance of turtles.