Importance of the route of administration for genetic differences in benzo[a]pyrene-induced in utero toxicity and teratogenicity

C57BL/6N (Ahb/Ahb) mice have a high-affinity Ah receptor in tissues, whereas AKR/J and DBA/2N (Ahd/Ahd) mice have a poor-affinity Ah receptor. The cytochrome P1-450 induction response (enhanced benzo[a]pyrene metabolism) occurs much more readily in Ahb/Ahb and Ahb/Ahd than in Ahd/Ahd mice, at any given dose of the inducer benzo[a]pyrene. Embryos from the AKR/J X (C57BL/6N)(AKR/J)F1 and the reciprocal backcross were studied during benzo[a]pyrene feeding of the pregnant females. Oral benzo[a]pyrene (120 mg/kg/day) given to pregnant Ahd/Ahd mice between gestational day 2 and 10 produces more intrauterine toxicity and malformations in Ahd/Ahd than Ahb/Ahd embryos. This striking allelic difference is not seen in pregnant Ahb/Ahd mice receiving oral benzo[a]pyrene. Pharmacokinetics studies with [3H]benzo[a]pyrene in the diet and high-performance liquid chromatographic analysis of benzo[a]pyrene metabolism in vitro by the maternal intestine, liver, and ovary and the embryos of control and oral benzo[a]pyrene-treated pregnant females are consistent with "first-pass elimination" kinetics and differences in benzo[a]pyrene metabolism by the embryos and/or placentas versus maternal tissues. In the pregnant Ahd/Ahd mouse receiving oral benzo[a]pyrene, little induction of benzo[a]pyrene metabolism occurs in her intestine and liver; this leads to much larger amounts of benzo[a]pyrene reaching her embryos, and genetic differences in toxicity and teratogenesis are manifest. In the pregnant Ahb/Ahd mouse receiving oral benzo[a]pyrene, benzo[a]pyrene metabolism is greatly enhanced in her intestine and liver; this leads to less benzo[a]pyrene reaching her embryos, much less intrauterine toxicity and malformations, and no genetic differences are manifest. More toxic metabolites (especially benzo[a]pyrene 1,6- and 3,6-quinones) are shown to occur in Ahd/Ahd embryos than in Ahb/Ahd embryos. In additional studies, no prenatal or neonatal "imprinting" effect in C57BL/6N mice by 2,3,7,8-tetrachlorodibenzo-p-dioxin or Aroclor 1254 on benzo[a]pyrene metabolism later in life was detectable. These genetic differences in intrauterine toxicity and teratogenicity induced by oral benzo[a]pyrene are just opposite those induced by intraperitoneal benzo[a]pyrene [Shum et al., '79; Hoshino et al., '81). The data in the present report emphasize the importance of the route of administration when the teratogen induces its own metabolism.     

Studies of mechanisms of niridazole-elicited embryotoxicity: evidence against a major role for covalent binding

Studies reported here were designed to examine the hypothesis that covalent binding of reactive intermediates to macromolecules of the conceptus represents a major mechanism for the embryotoxicity of niridazole (NDZ). The roles of embryonic thiol content and oxygenation on: 1) malformation incidence; 2) reductive metabolism; and 3) covalent binding to embryonic macromolecules of metabolites resulting from reductive biotransformation of NDZ were studied. Results were compared with those from studies with the nondysmorphogenic analog of NDZ, 4'-methylniridazole (MNDZ). Day 10 rat embryos were pretreated for 5 hours in vitro with either L-buthionine-S, R-sulfoximine (BSO) or N-acetylcysteine (NAC) to modulate their glutathione (GSH) content. BSO reduced GSH levels, but NAC was ineffective. Following pretreatment, embryos were cultured for an additional 15 hours in the presence of [14C]NDZ or [14C]MNDZ with an initial oxygen concentration of 5%. At the end of the culture period (day 11, AM), those embryos with active heartbeat and vitelline circulation were examined for asymmetric malformations. Drug metabolites were subjected to multiple extractions from the culture medium and subjected to quantitative high-performance liquid chromatography (HPLC) analysis. Homogenates of the embryos were extracted with trichloroacetic acid (TCA) to estimate the covalent binding of radiolabeled parent compound/metabolites. Autoradiographic analyses were performed on other embryos. BSO pretreatment, which reduces embryonic GSH tissue levels, dramatically increased both the conversion of NDZ to 1-thiocarbamoyl-2-imidazolidinone (TCI) (generated via reductive metabolism of NDZ) and covalently bound label but failed to increase embryotoxicity. NAC, by contrast, did not significantly affect embryonic GSH levels, TCI generation, or covalent binding. Because both rates of metabolism of NDZ to TCI and covalent binding could vary independently of malformation incidence, we concluded that they do not represent critical mechanistic factors for the embryotoxicity of NDZ and related nitroheterocycles.     

Nicotine-induced embryonic malformations mediated by apoptosis from increasing intracellular calcium and oxidative stress

BACKGROUND: Tobacco smoking by women during pregnancy increases the risk of congenital birth defects in the infants. Among the smoke products, nicotine is believed to be the major teratogenic factor that perturbs embryonic development. However, the role of nicotine in embryonic malformations has not been addressed, and the mechanisms by which nicotine affects embryonic development remain to be delineated. METHODS: To investigate the effects of nicotine on early embryogenesis, murine embryos at embryonic day (E) 8.5 were dissected out of the uteri, cultured in a roller bottle system, and treated with nicotine (0.6-6 microM) or vehicle. Embryonic morphogenesis and growth were examined in terms of structural morphology and crown/rump length, respectively. Programmed cell death (apoptosis) was assessed using LysoTracker Red staining of whole mount embryos and TUNEL assay of tissue sections. Changes in intracellular calcium concentration ([Ca2+]i) and reactive oxygen species (ROS) production were assessed using fluorescent dyes (Flu-4, AM; H2DCFDA, respectively) under a confocal microscope. To further investigate the role of intracellular calcium and ROS in nicotine-induced embryopathy, embryos were treated with BAPTA-AM (2 microM) to inhibit [Ca2+]i elevation and ascorbic acid (vitamin C; 100 microg/ml) to scavenge ROS in presence of nicotine (6 microM). RESULTS: The embryos treated with nicotine in 3-6 microM were smaller than those treated with vehicle. Most of the embryos had open neural tube in the anterior (brain) regions. The embryos treated with 6 microM nicotine also exhibited severe defects in the posterior trunk, resembling caudal dysplasia. Excessive apoptosis was observed in the deformed structures. Significant increases in [Ca2+]i and ROS were seen in the tissues that had higher levels of apoptosis. Furthermore, inhibition of [Ca2+]i and scavenging of ROS significantly reduced embryonic malformation and apoptotic rates in the embryos. CONCLUSIONS: Nicotine affects embryonic development in a concentration-dependent manner. The nicotine-induced embryonic malformations are, in part, a result of excessive cell death. Nicotine increases [Ca2+]i and ROS level, which play a role in nicotine-induced embryonic apoptosis and malformations. These studies identify the molecular pathway of nicotine action in embryonic apoptosis and malformations, and provide a promising approach for ameliorating the teratogenic effects of nicotine.     

Placental calcium provision in a lizard with prolonged oviductal egg retention

A prominent scenario for the evolution of viviparity and placentation in reptiles predicts a step-wise pattern with an initial phase of prolonged oviductal egg retention accompanied by progressive reduction in eggshell thickness culminating in viviparity; calcium placentotrophy evolves secondarily to viviparity. Saiphos equalis is an Australian scincid lizard with a reproductive mode that is uncommon for squamates because eggs are retained in the oviduct until late developmental stages, and the embryonic stage at oviposition varies geographically. We studied calcium mobilization by embryos in two populations with different oviductal egg retention patterns to test the hypothesis that the pattern of nutritional provision of calcium is independent of the embryonic stage at oviposition. Females from one population are viviparous and oviposit eggs containing fully formed embryos, whereas embryos in oviposited eggs of the second population are morphologically less mature, and these eggs hatch several days later. The reproductive mode of this population is denoted as prolonged oviductal egg retention. Yolk provided the highest proportion of calcium to hatchlings in both populations. Eggs of both populations were enclosed in calcified eggshells, but shells of the population with prolonged egg retention had twice the calcium content of the viviparous population and embryos recovered calcium from these eggshells. Placental transfer accounted for a substantial amount of calcium in hatchlings in both populations. Hatchling calcium concentration was higher in the population with prolonged egg retention because these embryos mobilized calcium from yolk, the eggshell and the placenta. This pattern of embryonic calcium provision in which both a calcified eggshell and placentotrophy contribute to embryonic nutrition is novel. The reproductive pattern of S. equalis illustrates that calcified eggshells are compatible with prolonged oviductal egg retention and that viviparity is not requisite to calcium placentotrophy.     

Characterization of hyaluronic acid interaction with calcium oxalate crystals: implication of crystals faces, pH and citrate

Interaction between hyaluronic acid (HA) present at the surface of tubular epithelial cells and calcium oxalate monohydrate (COM) crystals is thought to play an important role in kidney stone formation. AFM-based force spectroscopy, where HA is covalently attached to AFM-probes, was used to quantify the interaction between HA and the surfaces of COM crystals. The work of adhesion of the HA-probe as well as the rupture force of single HA molecules were quantified in order to understand the molecular regulation of HA binding to COM crystals. Our results reveal that HA adsorbs to the crystal surface in physiological conditions. We also observed increased adhesion when the pH is lowered to a value that increases the risk of kidney stone formation. HA adhesion to the COM crystal surface can be suppressed by citrate, a physiological inhibitor of stone retention currently used in the treatment and prevention of kidney stone formation. Interestingly, we also observed preferential binding of HA onto the [100] face versus the [010] face, suggesting a major contribution of the [100] faces in the crystal retention process at the surface of tubular epithelial cells and the promotion of stone formation. Our results clearly establish a direct role for the glycosaminoglycan HA present at the surface of kidney tubular epithelium in the process of COM crystal retention.     

Adenosine uptake sites in dog heart and brain; interaction with calcium antagonists

[3H] Nitrobenzylthioinosine (NBI) binding is characterized in dog heart and brain. Evidence is presented suggesting that [3H]NBI is binding to the adenosine uptake site in both tissues. Physiologic studies in open-chested dogs clearly demonstrate that NBI acts as a coronary vasodilator, consistent with an action at the adenosine uptake site. The binding is reversible, saturable and of high affinity (KD = 0.78 +/- .06 nM for heart and 0.52 +/- .05 nM for brain). Both dipyridamole and hexobendine are high potency inhibitors of [3H]NBI binding in heart and brain while other antihypertensives and vasodilators such as propranolol and nitroglycerin have no effect. The inhibition of [3H]NBI binding observed with dipyridamole was competitive indicating that both agents are acting at the same site. The dihydropyridine calcium antagonists also inhibited binding with a lower potency than the adenosine uptake blockers. Non-dihydropyridine calcium antagonists were much less potent in this regard. The inhibition of [3H]NBI binding observed with the dihydropyridine calcium antagonists was non-competitive suggesting that the calcium channel and adenosine uptake site may be coupled to each other.     

Importance of genetic predisposition and maternal environment for the occurrence of congenital malformations in offspring of diabetic rats

Previous experimental studies have implicated a genetic component in the induction of malformations in the offspring of diabetic rats. We have compared the outcome of diabetic pregnancy in two outbred (sub)strains of Sprague-Dawley rats (with low incidence [H] and high incidence [U] of skeletal malformations in the offspring) and hybrids between them. The fetuses of diabetic H mothers had no skeletal malformations and the lowest frequency of resorptions (8-9%), regardless of embryo type (H/H or H/U). When the diabetic mother was U or from the hybrid strain (H/U) and the offspring were of the mixed H/U type, we found increased resorption (16-21%) and skeletal malformation (3-5%) rates. If instead the embryos contained a major U genome [either U/U or U/(H/U)], further increased resorptions (23-30%) and skeletal malformations (17-19%) resulted. The H/H and U/U embryonic susceptibility to defined teratogens (3-6 mg/ml D-glucose, 4-8 mM B-hydroxy-butyrate) were compared in whole embryo culture and found to be similar, suggesting that the malformations occurring in vivo may have a different etiology than those found in vitro. In the rat model studied, diabetes in the mother appears to cause a disturbance of early stages of embryogenesis in genetically predisposed embryos. This early disturbance results in skeletal malformations and seems to require inducing factor(s) in addition to increased levels of D-glucose and B-hydroxybutyrate. The findings are in concert with the notion of a mixed genetic-environmental etiology of malformations in (diabetic) pregnancy.     

Protein synthesis in brine shrimp embryos. Dormant and developing embryos of Artemia salina contain equivalent amounts of chain initiation factors 2.

Dormant and developing embryos of Artemia salina contain equivalent amounts of eIF-2, the eukaryotic initiation factor which forms a ternary complex with GTP and Met-tRNAf. The factor was purified from 0.5 M NH4Cl ribosomal washes by (NH4)2SO4 fractionation, followed by chromatography on heparin-Sepharose, DEAE-cellulose, hydroxyapatite and phosphocellulose. Purified preparations from dormant and developing embryos have similar specific activities and nucleotide requirements. The mobility of both proteins in dodecylsulfate gel electrophoresis is indistinguishable, and each contains three major polypeptide chains of molecular weight 52 000, 45 000 and 42 000. Both proteins are also immunologically identical, and each stimulates amino acid incorporation in a cell-free system of protein synthesis. The binding of [35S]Met-tRNAf to 40-S ribosomal subunits is catalyzed by eIF-2 isolated from dormant or developing embryos and is dependent upon GPT and AUG. Binding of [35S]Met-tRNAf to 40-S ribosomal subunits, and ternary complex formation with eIF-2, GTP, and [35S]Met-tRNAf is stimulated 2--3-fold by a factor present in the 0.5 M NH4Cl ribosomal wash and which elutes from DEAE-cellulose at 50 mM KCl. This protein does not exhibit GTP-dependent binding of [35S]Met-tRNAf. Binding of GDP and GTP was investigated with purified eIF-2 from developing embryos. The factor forms a binary complex with GDP or GTP, and eIF-2-bound [3H]GDP exchanges very slowly with free nucleotides. Our results suggest that eIF-2 does not limit resumption of embryo development following encystment, nor does it limit mRNA translation in extracts from dormant embryos.     

3H]serotonin binding to blastula, gastrula, prism, and pluteus sea urchin embryo cells

1. The presence of serotonin binding sites in blastula, gastrula, prism, and pluteus embryos of the sea urchin, Arbacia punctulata, was investigated by the binding of radiolabelled serotonin to dissociated embryo cells. 2. [3H]serotonin binding sites were identified in prism, early pluteus, and advanced pluteus larvae, but not in blastula or gastrula embryos. 3. The ontogeny of [3H]serotonin binding activity closely parallels that of serotonin content as previously reported in Paracentrotus lividus embryos (Toneby, 1977a). 4. Results of this study support a regulatory role of serotonin in developmental processes in postgastrula sea urchin embryos.     

Reversible Dihydropyridine Isothiocyanate Binding to Brain Calcium Channels

Voltage-dependent calcium channels from ileal smooth muscle can be affinity-labeled with a [3H]dihydropyridine isothiocyanate radioligand. We examined the binding of this agent to brain membranes, to compare the properties of calcium channel drug binding sites in brain with those previously described in ileum. In brain, the [3H]dihydropyridine isothiocyanate labels sites that correspond in number and pharmacologic characteristics to binding sites for the classic calcium entry blocker, [3H]nitrendipine. However, in contrast to the covalent nature of dihydropyridine isothiocyanate binding in ileum, brain calcium channels are labeled reversibly. This difference in binding properties may reflect structural variations in voltage-dependent calcium channels in different tissues.     

3H]nitrendipine binding to adrenal capsular membranes

The physiologic regulation of aldosterone secretion is dependent on extracellular calcium and appears to be mediated by increases in cytosolic free calcium concentration in the zona glomerulosa cell. A specific role for voltage-dependent calcium channels was suggested by previous studies with the calcium channel antagonist verapamil. We therefore studied the [3H]nitrendipine calcium channel binding site in adrenal capsules. These studies revealed a single class of saturable, high affinity sites with KD = .26 +/- .04 nM and Bmax = 105 +/- 5.7 fmol/mg protein. Specific binding of [3H]nitrendipine was inhibited by calcium channel antagonists with potencies nitrendipine = nifedipine much greater than verapamil, while diltiazem had no inhibitory effect. In the rat, binding sites for [3H]nitrendipine were located in the adrenal capsule and medulla and were undetectable in the zona fasciculata. Physiologic studies with collagenase-dispersed adrenal glomerulosa cells demonstrated that nifedipine selectively inhibited angiotensin-II and potassium-stimulated steroidogenesis. These observations suggest both a pharmacologic and physiologic role for the nitrendipine binding site in aldosterone production.     

[3H]Nitrendipine Binding to Calcium Channels in Bovine and Rat Pituitary

[3H]Nitrendipine was used to label sites in homogenates of bovine anterior and neurointermediate lobes of the pituitary gland. The amount of specific binding in the anterior lobe was 1.82 +/- 0.30 pmol/g wet weight of tissue and the KD was 1.44 +/- 0.02 X 10(-10) M. Preliminary experiments indicated a similar amount of binding in bovine neurointermediate lobe. In competition studies nimodipine and nisoldipine (two potent voltage-sensitive calcium channel blockers) displayed IC50 values of 1.6 and 6.8 X 10(-10) M, respectively. Verapamil and the verapamil-like calcium channel blockers D-600 and tiapamil competed in a complex manner for the [3H]nitrendipine specific binding to bovine anterior pituitary homogenates. Autoradiographical studies demonstrated specific [3H]nitrendipine binding sites distributed approximately equally in the anterior and posterior lobes, but not in the intermediate lobe of the rat pituitary. In general the properties of [3H]nitrendipine binding in the pituitary tissue resemble strongly the properties of [3H]nitrendipine binding in the brain which is believed to be to voltage-sensitive calcium channels. These results provide support for the hypothesis that calcium channels are involved in pituitary hormone secretion and that drugs that interact with calcium channels may modulate the secretory process directly at the level of the pituitary.     

Localization and binding of transforming growth factor-beta isoforms in mouse preimplantation embryos and in delayed and activated blastocysts.

The stage and cell-specific accumulation of mammalian isoforms of transforming growth factor-beta (TGF-beta 1, TGF-beta 2, and TGF-beta 3) and TGF-beta binding were examined in the preimplantation embryo and in progesterone (P4)-treated delayed or P4 plus estradiol-17 beta (E2)-treated activated blastocysts in the mouse. Immunocytochemical studies revealed that while all three immunoreactive TGF-beta isoforms were present in one-cell embryos, very little or no immunostaining was observed in two-cell embryos. However, distinct immunostaining of these isoforms was again observed in four-cell embryos and persisted through the blastocyst stage. Among the isoforms studied, TGF-beta 2 immunostaining showed a unique pattern in late morulae. In many of these morulae, the staining was primarily observed in outside cells. However, in blastocysts, immunostaining for all three isoforms was present both in the inner cell mass (ICM) and trophectoderm (Tr). Immunostaining in sectioned blastocysts and immunosurgically isolated ICMs confirmed immunostaining in Tr and ICM cells. To ascertain whether preimplantation embryos can produce TGF-beta isoforms, immunostaining was performed in embryos grown in vitro from two-cell stage in simple balanced salt solution. Immunoreactive TGF-beta s 1-3 were present in embryos at all stages of development examined (four-cell embryos through blastocysts). The virtual absence of immunoactive TGF-beta s in two-cell embryos but their accumulation in embryos at later stages of development in vitro provides evidence that these growth factors were produced by embryos. In order to assess at what stages of development preimplantation embryos could be responsive to TGF-beta s, specific binding of [125I]TGF-beta 1 and [125I]TGF-beta 2 was performed in embryos and examined by autoradiography. Low levels of binding were first detected in eight-cell embryos. The binding increased in morulae followed by a further increase in blastocysts. Analysis of binding of [125I]TGF-beta 2 in immunosurgically isolated ICMs indicated that binding was primarily evident in Tr cells. Affinity labeling of TGF-beta 1 or TGF-beta 2 in Day 4 blastocysts revealed three classes of binding proteins with approximate molecular sizes of 65 kDa (type I), 90 kDa (type II), and greater than 250 kDa (type III), in addition to a doublet of 130 and 140 kDa proteins. This observation is similar to those reported for other cell types. The data suggest that embryos are likely to be responsive to TGF-beta s after the third cleavage.(ABSTRACT TRUNCATED AT 400 WORDS)     

Homogeneity of [3H]ouabain-binding sites in rat soleus muscle.

Homogeneity or heterogeneity of rat soleus-muscle Na,K-ATPase (Na+ + K+-dependent ATPase) with respect to affinity for [3H]ouabain was evaluated. Since the standard method for measuring specific [3H]ouabain binding to rat skeletal-muscle samples includes subtraction of a value for non-specific [3H]ouabain uptake and retention, and a wash-out in the cold to remove [3H]ouabain from the extracellular phase, it was possible that these procedures could hide a class of [3H]ouabain-binding sites either with low affinity or with a rapid dissociation of [3H]ouabain. However, measurements of [3H]ouabain uptake and retention over the range 0.1-5 mM, as well as the omission of wash-out, gave no evidence for heterogeneity of [3H]ouabain-binding sites in rat soleus muscle. Furthermore, the observation of agreement between the uptake and retention of non-specific [3H]ouabain and of [14C]sucrose gave no evidence for the existence of a major pool of [3H]ouabain-binding sites with low affinity for [3H]ouabain. Assuming homogeneity, the total concentration of [3H]ouabain binding sites in soleus-muscle samples from 12-week-old rats is 278-359 pmol/g wet wt.     

Dihydropyridine [methyl-3H]PN 200–110 Binding and Myogenesis in Intact Muscle Cells In Vitro

The radioligand dihydropyridine [methyl-3H]PN 200-110 binds to contracting myotubes in culture derived from chick embryo pectoralis muscle. [methyl-3H]PN 200-110 binds specifically to high-affinity sites, with nonspecific binding only between 15 and 30% of the total binding. A Scatchard plot of the specific binding revealed a single high-affinity binding site with a KD (dissociation constant) of 0.5 nM +/- 0.2 nM and Bmax (number of binding sites) of 100 fmol/10(6) nuclei. We employed this sensitive assay to probe the appearance of high-affinity [methyl-3H]PN 200-110 binding sites during myogenesis. The time course of appearance of high-affinity binding sites lags behind that of fusion. Low-calcium media prevented the differentiation of myoblasts and blocked the appearance of high-affinity sites. Chelation of intracellular calcium before or after fusion of myoblasts with the calcium indicator Quin 2 prevented the appearance of dihydropyridine binding sites. These findings are consistent with the view that the expression of dihydropyridine receptors is modulated by the intracellular calcium.     

A new class of calcium entry blockers defined by 1,3-diphosphonates. Interactions of SR-7037 (belfosdil) with receptors for calcium channel ligands

Tetrabutyl-2(2-phenoxyethyl)-1,3-propylidene diphosphonate (SR-7037) completely displaced dihydropyridine [( 3H]PN200-110), phenylalkylamine [( 3H]D888), and benzothiazepine [( 3H]diltiazem) ligands from brain L-type calcium channels. Half-maximal inhibition of [3H]PN200-110 binding occurred at 19 nM with a Hill coefficient of 0.96. SR-7037 primarily decreased the affinity for [3H]PN200-110 with a small, but significantly, effect on the maximal binding capacity. Kinetic studies showed that this was due to an increased radioligand dissociation rate from 0.04 min-1 to 0.43 min-1 in the presence of the diphosphonate. Displacement of [3H]D888 by SR-7037 was biphasic with respective IC50 of 44 and 8400 nM. Likewise, unlabeled (-)-D888 identified two sites with IC50 values of 0.9 and 27 nM. Both SR-7037 (1000 nM) and D888 (200 nM) accelerated radioligand dissociation about 2-fold. [3H]Diltiazem binding was inhibited by SR-7037 with an IC50 value of 29 nM. The inhibition of dihydropyridine binding by SR-7037 is enhanced by most divalent cations at millimolar concentrations with the following potency: Mn2+ greater than Mg2+ greater than Ca2+ greater than Co2+. Barium has the opposite effect. The half-maximal effect of calcium occurred at 6 microM free ion. Specific binding of [3H]D888 was antagonized in the presence of 1 mM CaCl2. It is concluded that SR-7037 has allosteric interactions with the dihydropyridine receptor of the L-type calcium channel. The differential effect of Ca2+ on the potency of D888 and diltiazem relative to that of SR-7037 indicates that the three drugs may bind to nonequivalent sites. These results support specific calcium channel inhibition, possibly at a novel site, as the primary mechanism of the diphosphonate's pharmacological actions.     

Ontogeny of Calcium Antagonist Binding Sites in Chick Brain and Heart

The ontogeny of chick brain and heart ventricle calcium antagonist binding sites was determined, using [3H]nitrendipine ([3H]NDP), as the ligand. The binding of [3H]NDP to adult heart and brain was kinetically very similar, with the former displaying a KD of 0.28 +/- 0.02 nM and a Bmax of 138 +/- 17 fmol/mg protein, and the latter a KD of 0.30 +/- 0.02 nM and a Bmax of 160 +/- 12 fmol/mg protein. The binding site in both brain and heart was highly specific for dihydropyridine calcium antagonists, such as nifedipine, nimodipine, and nisoldipine, since these drugs were several orders of magnitude more potent as inhibitors of [3H]NDP binding than verapamil, methoxyverapamil, or diltiazem. The developmental appearance of [3H]NDP binding sites in brain was rather gradual, with adult levels being attained just prior to birth. This was in contrast to the profile in heart ventricle which showed essentially adult levels at seven days gestation. The acquisition of [3H]NDP binding sites in chick brain roughly paralleled the onset of neuronal maturation and functional activity. In both chick brain and heart, verapamil and methoxyverapamil were weak inhibitors of [3H]NDP binding. However, the inhibition of binding in both tissues was markedly biphasic, with only 50% of the binding sites being susceptible to inhibition by each agent, suggesting that multiple calcium antagonist binding sites may exist in both tissues.     

Properties of Quisqualate-Sensitive L-[3H]Glutamate Binding Sites in Rat Brain as Determined by Quantitative Autoradiography

Quisqualate, a glutamate analogue, displaced L-[3H]glutamate binding in a biphasic manner, corresponding to "high-affinity" and "low-affinity" binding sites. High-affinity quisqualate sites were termed "quisqualate-sensitive L-[3H]glutamate" binding sites. Quisqualate-sensitive L-[3H]glutamate binding was regionally distributed, with the highest levels present in the cerebellar molecular layer. This binding was stimulated by millimolar concentrations of chloride and calcium. The stimulatory effects of calcium required the presence of chloride ions, whereas chloride's stimulatory effects did not require calcium. All of the L-[3H]glutamate binding stimulated by chloride/calcium was quisqualate sensitive and only weakly displaced by N-methyl-D-aspartate, L-aspartate, or kainate. At high concentrations (1 mM), the anion blockers 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid both reduced, by 41 and 43%, respectively, the stimulatory effects of chloride. At concentrations of 100 microM, kynurenate, L-aspartate, (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and L-2-amino-4-phosphonobutyric acid (L-APB) failed to displace quisqualate-sensitive L-[3H]glutamate binding in the cerebellar molecular layer. In the presence of KSCN, however, 100 microM AMPA displaced 44% of binding. Quisqualate-sensitive L-[3H]glutamate binding was not sensitive to freezing, and, in contrast to other chloride- and calcium-dependent L-[3H]glutamate binding sites that have been reported, quisqualate-sensitive binding observed by autoradiography was enhanced at 4 degrees C compared with 37 degrees C. Quisqualate-sensitive L-[3H]glutamate binding likely represents binding to the subclass of postsynaptic neuronal glutamate receptors known as quisqualate receptors, rather than binding to previously described APB receptors, chloride-driven sequestration into vesicles, or binding to astrocytic membrane binding sites.     

Selective changes of neurotransmitter receptors in middle-aged gerbil brain

Age-related alterations in major neurotransmitter receptors and voltage dependent calcium channels were analyzed by receptor autoradiography in the gerbil brain. [³H]Quinuclidinyl benzilate (QNB). [³H]cyclohexyladenosine (CHA), [³H]muscimol, [³H]MK-801, [³H]SCH 23390, [³H]naloxone, and [³H]PN200-110 were used to label muscarinic acetylcholine receptors, adenosine A₁ receptors, γ-aminobutyric acid_A (GABA_A) receptors, (NMDA) receptors, dopamine D₁ receptors, opioid receptors, and voltage dependent calcium channels, respectively. In middle-aged gerbils (16 months old), the hippocampus exhibited a significant elevation in [³H]QNB, [³H]MK-801, [³H]SCH 23390, [³H]naloxone, and [³H]PN200-110 binding, whereas [³H]CHA and [³H]muscimol binding showed a significant reduction in this area, compared with that of young animals (1 month). On the other hand, the cerebellum showed a significant alteration in [³H]QNB, [³H]CHA, and [³H]naloxone binding and the striatum also exhibited a significant alteration in [³H]SCH 23390 and [³H]CHA binding in middle-aged gerbils. The neocortex showed a significant elevation only in [³H]CHA binding in middle-aged animals. The nucleus accumbens and thalamus also showed a significant alteration only in [³H]muscimol binding. However, the hypothalamus and substantia nigra exhibited no significant alteration in these bindings in middle-aged gerbils. These results demonstrate the age-related alterations of various neurotransmitter receptors and voltage dependent calcium channels in most brain regions. Furthermore, they suggest that the hippocampus is most susceptible to aging processes and is altered at an early stage of senescence.