Estrogen metabolism and formation of estrogen-DNA adducts in estradiol-treated MCF-10F cells. The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin induction and catechol-O-methyltransferase inhibition

Formation of estrogen metabolites that react with DNA is thought to be a mechanism of cancer initiation by estrogens. The estrogens estrone (E₁) and estradiol (E₂) can form catechol estrogen (CE) metabolites, catechol estrogen quinones [E₁(E₂)-3,4-Q], which react with DNA to form predominantly depurinating adducts. This may lead to mutations that initiate cancer. Catechol-O-methyltransferase (COMT) catalyzes an inactivation (protective) pathway for CE. This study investigated the effect of inhibiting COMT activity on the levels of depurinating 4-OHE₁(E₂)-1-N3Ade and 4-OHE₁(E₂)-1-N7Gua adducts in human breast epithelial cells. MCF-10F cells were treated with TCDD, a cytochrome P450 inducer, then with E₂ and Ro41-0960, a COMT inhibitor. Estrogen metabolites and depurinating DNA adducts in culture medium were analyzed by HPLC with electrochemical detection. Pre-treatment of cells with TCDD increased E₂ metabolism to 4-OHE₁(E₂) and 4-OCH₃E₁(E₂). Inclusion of Ro41-0960 and E₂ in the medium blocked formation of methoxy CE, and depurinating adducts were observed. With Ro41-0960, more adducts were detected in MCF-10F cells exposed to 1 μM E₂, whereas without the inhibitor, no increases in adducts were detected with E₂ ≤ 10 μM. We conclude that low COMT activity and increased formation of depurinating adducts can be critical factors leading to initiation of breast cancer.     

Competitive product inhibition of aromatase by natural estrogens

In order to better understand the function of aromatase, we carried out kinetic analyses to asses the ability of natural estrogens, estrone (E₁), estradiol (E₂), 16-OHE₁, and estriol (E₃), to inhibit aromatization. Human placental microsomes (50 μg protein) were incubated for 5 min at 37°C with [1β-³H]testosterone (1.24 × 10³ dpm ³H/ng, 35–150 nM) or [1β-³H,4-¹⁴C]androstenedione (3.05 × 10³ dpm ³H/ng, ³H/¹⁴C = 19.3, 7–65 nM) as substrate in the presence of NADPH, with and without natural estrogens as putative inhibitors. Aromatase activity was assessed by tritium released to water from the 1β-position of the substrates. Natural estrogens showed competitive product inhibition against androgen aromatization. The K_i of E₁, E₂, 16-OHE₁, and E₃ for testosterone aromatization was 1.5, 2.2, 95, and 162 μM, respectively, where the K_m of aromatase was 61.8 ± 2.0 nM (n = 5) for testosterone. The K_i of E₁, E₂, 16-OHE₁, and E₃ for androstenedione aromatization was 10.6, 5.5, 252, and 1182 μM, respectively, where the K_m of aromatase was 35.4 ± 4.1 nM (n = 4) for androstenedione. These results show that estrogens inhibit the process of andrigen aromatization and indicate that natural estrogens regulate their own synthesis by the product inhibition mechanism in vivo. Since natural estrogens bind to the active site of human placental aromatase P-450 complex as competitive inhibitors, natural estrogens might be further metabolized by aromatase. This suggests that human placental estrogen 2-hydroxylase activity is catalyzed by the active site of aromatase cytochrome P-450 and also agrees with the fact that the level of catecholestrogens in maternal plasma increases during pregnancy. The relative affinities and concentration of androgens and estrogens would control estrogen and catecholestrogen biosynthesis by aromatase.     

In vitro short-term test evaluation of catecholestrogens genotoxicity

Estrogens are indicated as being the most important etiological factors for the development and progression of breast cancer. The implication of estrogen in breast cancer has been associated mostly with the estrogen receptors that mediate cell proliferation. Evidence also exists to support the hypothesis of a direct role of estrogens as tumor initiators. However, the role of estrogen genotoxicity in breast cancer is still questionable.

In this study the genotoxic activity of catecholestrogens and 16-hydroxy estrone has been investigated by performing Salmonella strain TA98 and TA100 Ames tests, sister chromatide exchange assays (SCE) and micronucleus assays on human peripheral lymphocytes (CBMN and ARA/CBMN). We found a lack of positive results with micronucleus assays, except for 2-hydroxy estradiol (2-OHE₂), which shows a peculiar “bell shaped” trend of micronucleus number versus concentrations. SCE assay suggests weak genotoxic activity of all tested catechol metabolites, except 4-hydroxy estrone (4-OHE₁), which also showed negative results by ARA/CBMN.

In this open debate, our results support the hypothesis of a weak genotoxicity, not correlated with the carcinogenetic potential of estrogens.     

Increased aromatase activity in pubic skin fibroblasts from patients with isolated gynecomastia [Poster 47]

Aromatase activity (AR) was studied in pubic skin fibroblasts from 8 patients with isolated gynecomastia (PSFG) and 5 normal subjects (PSFC) by incubation in the presence of [³H]androstenedione (A) and isolation of aromatized metabolites. After 4 h of incubation, no E₂ formation could be measured in PSFC in contrast to low but significant amounts in PSFG [0.03 ± 0.02 (SEM) fmol/μg DNA, p < 0.01]. In addition, 16-OHE₁ represented 62.5% of total aromatized metabolites in PSFG versus 39% in PSFG. After 24 h, there was no significant difference in E₂ formation between the two groups whereas total aromatization remained significantly higher (p < 0.05) in PFSG; E₃+ epiE₃ represented 50% of the metabolites in both groups.     

The synthesis and biochemical evaluation of new A1 selective adenosine receptor agonists containing 6-hydrazinopurine moieties

The synthesis and SAR of a series of novel derivatives of N-aminoadenosine is described, along with their in vitro effects in biochemical assays. The rat brain A₁ adenosine receptor binding of these compounds is very dependent upon the purine 2-substituent. The novel agonist, 2-chloro-N-[4-(phenylthio)-1-piperidinyl]adenosine, exhibits a L_i value for A₁ receptor binding of <1 nM.     

Acetyl-RYYRIK-NH(2) is a highly efficacious OP(4) receptor agonist in the cardiovascular system of anesthetized rats

Nociceptin, the endogenous ligand of the OP₄ or ORL₁ (opioid receptor-like₁) receptor, decreases blood pressure and heart rate in anesthetized rats. Since the OP₄ receptor antagonist [Phe¹Ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ possesses an agonistic effect in this model, we examined whether other purported OP₄ receptor antagonists, acetyl-RYYRIK-NH₂ and naloxone benzoylhydrazone, antagonize the depressant effects of nociceptin. Acetyl-RYYRIK-NH₂, like nociceptin and [Phe¹Ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ and unlike naloxone benzoylhydrazone, decreased diastolic blood pressure and heart rate (rank order of potencies: nociceptin ≈ acetyl-RYYRIK-NH₂ [Phe¹Ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂). The depressant effects were insensitive to the OP_1–3 receptor antagonist naloxone but diminished by naloxone benzoylhydrazone. In conclusion, the hypotensive and bradycardic effects of nociceptin in the anesthetized rat are mediated via OP₄ receptors, at which acetyl-RYYRIK-NH₂ is a highly potent and efficacious agonist.     

RAT BRAIN FOLATE IDENTIFICATION

Abstract— Endogenous rat brain folates were identified using column chromatography for folate separation, authentic folate standards, and microbiological assay. The total folate content of rat brain was 360 ng per gram brain (wet wt) and consisted of tetrahydropteroylpentaglutamate (H₄ PteGlu₅, 29%), H₄ PteGlu₆ (14%), H₄PteGlu₇ (7%), 5-CH₃-H₄PteGlu₄ (7%), 5-CH₃-H₄PteGlu₅ (13%), and small amounts of formyl-, methyl-, and H₄PteGlu_1-6 (~25%) and non-methylated folates having a glutamate chain over seven glutamates long (4%). Overall folate oxidation state and one carbon forms were H₄PteGlu_n (58%), 5-CH₃-H₄PteGlu_n (18%), 5- and 10-CHO-H₄PteGlu_n (16%), and H₂ PteGlu_n (8%). No PteGlu_n was detected.     

Binding activities by propiverine and its N-oxide metabolites of L-type calcium channel antagonist receptors in the rat bladder and brain

The present study was undertaken to characterize the binding activities of propiverine and its N-oxide metabolites (1-methyl-4-piperidyl diphenylpropoxyacetate N-oxide: P-4(N → O), 1-methyl-4-piperidyl benzilate N-oxide: DPr-P-4(N → O)) toward L-type calcium channel antagonist receptors in the rat bladder and brain. Propiverine and P-4(N → O) inhibited specific (+)-[³H]PN 200–110 binding in the rat bladder in a concentration-dependent manner. Compared with that for propiverine, the K_i value for P-4(N → O) in the bladder was significantly greater. Scatchard analysis has revealed that propiverine increased significantly K_d values for bladder (+)-[³H]PN 200–110 binding. DPr-P-4(N → O) had little inhibitory effects on the bladder (+)-[³H]PN 200–110 binding. Oxybutynin and N-desethyl-oxybutynin (DEOB) also inhibited specific (+)-[³H]PN 200–110 binding in the rat bladder. Propiverine, oxybutynin and their metabolites inhibited specific [N-methyl-³H]scopolamine methyl chloride ([³H]NMS) binding in the rat bladder. The ratios of K_i values for (+)-[³H]PN 200–110 to [³H]NMS were markedly smaller for propiverine and P-4(N → O) than oxybutynin and DEOB. Propiverine and P-4(N → O) inhibited specific binding of (+)-[³H]PN 200–110, [³H]diltiazem and [³H]verapamil in the rat cerebral cortex in a concentration-dependent manner. The K_i values of propiverine and P-4(N → O) for [³H]diltiazem were significantly smaller than those for (+)-[³H]PN 200–110 and [³H]verapamil. Further, their K_i values for [³H]verapamil were significantly smaller than those for (+)-[³H]PN 200–110. The K_i values of propiverine for each radioligand in the cerebral cortex were significantly (P < 0.05) smaller than those of P-4(N → O). In conclusion, the present study has shown that propiverine and P-4(N → O) exert a significant binding activity of L-type calcium channel antagonist receptors in the bladder and these effects may be pharmacologically relevant in the treatment of overactive bladder after oral administration of propiverine.     

Chele- and anticheleselectivity IV. Reaction-accelerating neighboring-group effects in the cross-coupling of vinylbromides with alkyl-transition metal reagents

The cross-coupling of vinylbromides with Me₄FeLi₂ or Me₄MnLi₂ is distinctly accelerated if the vinylbromide is provided with an hydroxy group in the -, β- or γ-position to the bromine atom (compounds 2–4). This was elucidated by intermolecular competition experiments and is very probably due to chelate formation as the first reaction step. Corresponding neighboring-group effects as seen in the reaction with 2–4 were observed by treating 2 with Bu₄MnLi₂, the -methoxy vinylbromide (5) with Me₄MnLi₂, the secondary amino vinylbromide (6) with Me₄FeLi₂, the tertiary amino vinylbromide (7) with Me₄FeLi₂ or Me₄MnLi₂, and the β-cyano vinylbromide (8) with Me₄FeLi₂. These effects allow very chemoselective cross-coupling. The observed cross-coupling with vinyl- bromides are believed to occur by oxidative addition (intramolecular due to preceding chelate formation) with subsequent reductive elimination.     

Effects of the Dopamine D3 Antagonist PD 58491 and Its Interaction with the Dopamine D3 Agonist PD 128907 on Brain Dopamine Synthesis in Rat

Abstract: The dopamine (DA) D₃ receptor antagonist PD 58491 {3-[4-[1-[4-[2-[4-(3-diethylaminopropoxy)phenyl]-benzoimidazol-1-yl-butyl]-1 H -benzoimidazol-2-yl]-phenoxy]propyl]diethylamine} bound with high affinity and selectivity to recombinant human DA D₃ versus D_2L and D_4.2 receptors transfected into Chinese hamster ovary cells: K _i values of 19.5 n M versus 2,362 and >3,000 n M , respectively. In contrast, the putative DA D₃ receptor antagonist (+)-AJ76 displayed low affinity and selectivity for D₃ versus D_2L and D_4.2 receptors (91 n M vs. 253 and 193 n M , respectively). In vitro, PD 58491 (1 n M −1µ M ) exhibited D₃ receptor antagonist activity, reversing the quinpirole (10 n M )-induced stimulation of [³H]thymidine uptake in D₃ CHOpro-5 cells, but did not have any significant intrinsic activity by itself in this assay. PD 58491 did not decrease the γ-butyrolactone-induced increase in DA synthesis ( l -3,4-dihydroxyphenylalanine accumulation) in rat striatum, indicating that the compound possessed no in vivo DA D₂/D₃ receptor agonist action at DA autoreceptors. PD 58491 (3–30 mg/kg, i.p.) generally did not alter DA or serotonin synthesis in either the striatum or mesolimbic region of rat brain. The D₃-preferring agonist PD 128907 decreased DA synthesis in striatum and mesolimbic regions, and this effect was attenuated by pretreatment with PD 58491. These findings support the hypothesis that DA D₃ autoreceptors may in part modulate the synthesis and release of DA in striatum and mesolimbic regions.     

A Monoclonal Antibody to Oestradiol Potentiates the Stimulation of the Specific Activity of the Brain Type Creatine Kinase by Oestrogen in vivo and in vitro

We described previously the in vivo immunoneutralization effects of a high affinity anti-oestradiol antibody clone 15 in blocking ovulation and synaptic remodeling in cycling female rats. In the present study we report the enhancing effects of this antibody. Treatment of ovariectomized female rats or female derived skeletal cell cultures in vitro with anti-E₂ 15 plus oestrogen (E₂) potentiated the specific activity of the brain type creatine kinase (CK) response to E₂ in the rat tissues or skeletal cells. The enhancing CK response of anti E₂ 15 plus E₂ was time- and dose-dependent in the uterus, thymus, epiphysis and diaphysis of ovariectomized female rats. In the pituitary, on the other hand, anti-E₂ 15 blocked the stimulatory CK response to E₂. Two other high affinity anti-E₂ anti-bodies, clones 8D₉ and 11B₆, had no effect in augmenting the response of CK to E₂ in rat tissues. Moreover, the enhancing CK response in rat tissues was specific to anti-E₂ 15 plus E₂ since the intact anti-E₂ in the presence of other oestrogen mimetics, such as oestriol or stilbestrol or tamoxifen did not potentiate the CK response in rat tissues. In this model system the Fab' monomer of anti-E₂ 15 abolished the CK response to E₂ in rat tissues and not to anti-E₂ 15 plus E₂ whereas tamoxifen completely blocked the CK response to anti E₂ plus E₂. Anti E₂ 15 may therefore serve as a specific carrier in delivering E₂ to oestrogen sensitive rat tissues or cells containing functional oestrogen receptors and thereby increasing the magnitude of E₂ effects in vivo and in vitro.     

Beta(3)-adrenoceptor agonists for the treatment of frequent urination and urinary incontinence: 2-[4-(2-[[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl)phenoxy]-2-methylpropionic acid.

In a search for novel analogues of β₃-adrenoceptor (AR) agonists relaxing the bladder for treatment of urinary dysfunction, 2-[4-(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino}ethyl)phenoxy]-2-methylpropionic acids (1a–e), into which a fibrate-like structure had been incorporated, were synthesised. Compound 1a was found to be a selective β₃-AR agonist in functional assays using the ferret detrusor (β₃-AR), rat uterus (β₂-AR), and rat atrium (β₁-AR); β₃: EC₅₀=7.8 nM, β₂: IC₅₀=7,300 nM, β₁: EC₂₀=23,000 nM. The introduction of a chlorine atom or methyl substituent at the ortho-position on the phenyl ring of 1a further improved β₃-AR selectivity. In an in vivo study, 1a lowered intrabladder pressure (ED₅₀=31 μg/kg) in rats, without increasing heart rate, in keeping with the in vitro results. Consequently, it is proposed that 1a and its analogues (1b–e), possess β₃-AR agonistic activity in the absence of undesirable β₁- or β₂-AR mediated actions, and may be useful for clinical treatment and pharmacological studies.     

In Vitro Properties of a High Affinity Selective Antagonist of the VIP1 Receptor

Gourlet, P., P. De Neef, J. Cnudde, M. Waelbroeck and P. Robberecht. In vitro properties of a high affinity selective antagonist of the VIP₁ receptor. Peptides 18(10) 1555–1560, 1997.—A selective high affinity VIP₁ receptor antagonist [Acetyl-His¹, D-Phe², Lys¹⁵, Arg¹⁶, Leu¹⁷] VIP(3-7)/GRF(8-27) or PG 97-269 was synthesized, by analogy with recently obtained selective VIP₁ receptor agonists. The properties of the new peptide were evaluated on Chinese hamster ovary (CHO) cell membranes expressing either the rat VIP₁-, rat VIP₂- or the human VIP₂- recombinant receptors and on LoVo cell membranes expressing exclusively the human VIP₁ receptor. The IC₅₀ values of ¹²⁵I-VIP binding inhibition by PG 97-269 were 10, 2000, 2 and 3000 nM on the rat VIP₁-, rat VIP₂-, human VIP₁- and human VIP₂ receptors, respectively. PG 97-269 had a negligible affinity for the PACAP I receptor type. It did not stimulate adenylate cyclase activity, but inhibited competitively effect of VIP on the VIP₁ receptor mediated stimulation of adenylate cyclase activity. The K_i values were respectively of 15 ± 5 nM and 2 ± 1 nM for the rat and human VIP₁ receptors. Thus the described molecule in the first reported VIP antagonist with an affinity in the nM range and with a high selectivity for the VIP₁ receptor subclass. It may be useful for evaluation of the physiological role of VIP in rat and human tissues.     

Inhibition of Vitamin D3 metabolism enhances VDR signalling in androgen-independent prostate cancer cells

Induction of growth arrest and differentiation by 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) occurs in non-malignant cell types but is often reduced in cancer cells. For example, androgen-independent prostate cancer cells, DU-145 and PC-3, are relatively insensitive to the anti-proliferative action of 1,25-(OH)₂D₃. This appears to be due to increased 1,25-(OH)₂D₃-metabolism, as a result of CYP24 enzyme-induction, which in turn leads to decreased anti-proliferative efficacy. In the in vitro rat kidney mitochondria assay, the 2-(4-hydroxybenzyl)-6-methoxy-3,4-dihydro-2H-naphthalen-1-one (4) was found to be a potent inhibitor of Vitamin D₃ metabolising enzymes (IC₅₀ 3.5 μM), and was shown to be a more potent inhibitor than the broad spectrum P450 inhibitor ketoconazole (IC₅₀ 20 μM). The combination of the inhibitor and 1,25-(OH)₂D₃ caused a greater inhibition of proliferation in DU-145 cells than when treated with both agents alone. Examination of the regulation of VDR target gene mRNA in DU-145 cells revealed that co-treatment of 1,25-(OH)₂D₃ plus inhibitor of Vitamin D₃ metabolising enzymes co-ordinately upregulated CYP24, p21^waf1/cip1 and GADD45.     

Correlative Regulation of Nerve Growth Factor Level and Choline Acetyltransferase Activity by Thyroxine in Particular Regions of Infant Rat Brain

Abstract: Effects of thyroxine (T₄) on nerve growth factor (NGF) level and choline acetyltransferase (ChAT) activity of rat brains were investigated. Repetitive intraperitoneal administration of T₄ caused increases in both NGF level and ChAT activity in the frontal cortex, septum, hippocampus, and striatum and decreases in the cerebellum in 2-day-old rats. Only ChAT activity was elevated in the olfactory bulb, and the NGF level remained unchanged there. No changes were observed in the midbrain and pons/medulla. Furthermore, T₄ was effective on the post-natal rats only up to day 11. These results suggest that T₄ plays a role in the developmental regulation of NGF level and ChAT activity in rat brain in a region- and/or stage-specific manner. That (1) changes in NGF level and ChAT activity occurred in regions nearly identical to those that contained NGF-responding neurons, and (2) the change in NGF level in the hippocampus and frontal cortex was followed by the change of ChAT activity after a single injection of T₄ suggest that the effects of T₄ on cholinergic differentiation are, at least in part, mediated via NGF, which itself is quantitatively regulated by T₄.     

Development of High Affinity Selective VIP1 Receptor Agonists

Gourlet, P., A. Vandermeers, P. Vertongen, J. Rathe, P. De Neef, J. Cnudde, M. Waelbroeck and P. Robberecht. Development of high affinity selective VIP₁ receptor agonists. Peptides 18(10) 1539–1545, 1997.—The biological effects of VIP are mediated by at least two VIP receptors: the VIP₁ and the VIP₂ receptors that were cloned in rat, human and mice. As the mRNA coding for each receptor are located in different tissues, it is likely that each receptor modulates different functions. It is therefore of interest to obtain selective agonists for each receptor subtype. In the present work, we achieved the synthesis of two VIP₁ receptor selective agonists derived from secretin and GRF. [R¹⁶]chicken secretin had IC₅₀ values of binding of 1, 10,000, 20, and 3000 nM for the rat VIP₁-, VIP₂-, secretin- and PACAP receptors, respectively. This peptide, however, had a weaker affinity for the human VIP₁ receptor (IC₅₀ of 60 nM). The chimeric, substituted peptide [K¹⁵,R¹⁶,L²⁷]VIP(1-7)/GRF(8-27) had IC₅₀ values of binding of 1, 10,000, 10,000 and 30,000 nM for the rat VIP₁-, VIP₂-, secretin- and PACAP receptors, respectively. Furthermore, its also showed an IC₅₀ of 0.8 nM for the human VIP₁ receptor and a low affinity for the human VIP₂ receptor. It is unlikely that this GRF analogue interacted with a high affinity to the pituitary GRF receptors as it did not stimulate rat pituitary adenylate cyclase activity. The two described analogues stimulated maximally the adenylate cyclase activity on membranes expressing each receptor subtype.     

Cocoa procyanidins protect PC12 cells from hydrogen-peroxide-induced apoptosis by inhibiting activation of p38 MAPK and JNK

Oxidative stress induced by reactive oxygen species has been strongly associated with the pathogenesis of neurodegenerative disorders, including Alzheimer's disease. In this study, we investigated the possible protective effects of a cocoa procyanidin fraction (CPF) and procyanidin B2 (epicatechin-(4β-8)-epicatechin) – a major polyphenol in cocoa – against apoptosis of PC12 rat pheochromocytoma (PC12) cells induced by hydrogen peroxide (H₂O₂). CPF (1 and 5 μg/ml) and procyanidin B2 (1 and 5 μM) reduced PC12 cell death caused by H₂O₂, as determined by MTT and trypan blue exclusion assays. CPF and procyanidin B2 attenuated the H₂O₂-induced fragmentation of nucleus and DNA in PC12 cells. Western blot data demonstrated that H₂O₂ induced cleavage of poly(ADP-ribose)polymerase (PARP), downregulated Bcl-X_L and Bcl-2 in PC12 cells. Pretreatment with CPF or procyanidin B2 before H₂O₂ treatment diminished PARP cleavage and increased Bcl-X_L and Bcl-2 expression compared with those only treated with H₂O₂. Activation of caspase-3 by H₂O₂ was inhibited by pretreatment with CPF or procyanidin B2. Furthermore, H₂O₂-induced rapid and significant phosphorylation of c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and both of these effects were attenuated by CPF or procyanidin B2 treatment. These results suggest that the protective effects of CPF and procyanidin B2 against H₂O₂-induced apoptosis involve inhibiting the downregulation of Bcl-X_L and Bcl-2 expression through blocking the activation of JNK and p38 MAPK.     

HISTAMINE-SENSITIVE ADENYLATE CYCLASE IN HYPOTHALAMUS OF RAT BRAIN: H1 AND H2 RECEPTORS

Abstract— In in vitro experiments on rat hypothalamic homogenates the effects of biogenic amines such as histamine (HA), noradrenaline (NA), dopamine (DA), serotonin (5-HT) and drugs such as isoprenaline (ISP), 2-(2-pyridyl)ethylamine (H₂ stimulant—H_ls), 4-methyl-histamine (H₂ stimulant H_2s), mepyramine (H₁ antagonistp H_la), cimetidine (H₂ antagonist—H_2a) were tested on adenylate cyclase activity. HA possessed a powerful stimulating effect on hypothalamic adenylate cyclase activity, higher than that shown by the other substances.
The stimulating effect of HA was greatest in hypothalamic tissue from male rats, while tissue from females showed only a modest stimulation. H_2s, induced a greater stimulation of adenylate cyclase than H_ls. On the other hand, the H_2a inhibited HA stimulation to a greater extent than the H_la, H_la and H_2a, when used together, completely inhibited the HA stimulation. HA may have a neurotrans-mitter role in the hypothalamus, and in this area there appears to be a mixed population of H₁ and H₂ receptors, with a majority of H₂ receptors.     

Characterization of somatostatin receptor subtypes controlling rat gastric acid and pancreatic amylase release

An examination of the binding characteristics of a large number of somatostatin analogues with respect to the five known somatostatin receptor subtypes has recently resulted in the discovery of several peptides with some selectivity for types 2, 3, and 4 and little affinity for type 1 or 5 receptor. A panel of these peptides has thus far implicated type 2 receptors in the inhibition of release of pituitary growth hormone and type 4 receptors in inhibiting pancreatic insulin release. In the present article, we have examined the inhibitory effects of the same group of peptides on in vivo rat gastric acid and pancreatic amylase release and binding to rat pancreatic acinar cells. The type 2-selective ligand NC-8–12 was a potent inhibitor of gastric acid release (EC₅₀s in the 1.5 nM region) whereas the type 4-selective ligand, DC-23–99, elicited little response. However, some involvement of type 3 receptors could not be ruled out because the type 3-selective analoueg, DC-25–20, exhibited inhibitory effects at higher dose levels (EC₅₀ > 10 nM). Conversely, the type 4 analogue was a potent inhibitor of amylase release (EC₅₀ 1.1 nM) whereas the type 3 analogue had no significant effects at doses tested. DC-23–99 also bound with high affinity to rat acinar cells (EC₅₀ 3.8 nM), whereas DC-25-20 exhibited more than 10-fold less affinity. Thus, these two major biological functions of somatostatin appear to be controlled by different receptors and, furthermore, effects on both endocrine and exocrine pancreas appear to be type 4 receptor mediated.     

Regulation of Serotonin2A Receptor Expression by an Antisense Oligodeoxynucleotide

Abstract: The regulation of 5-HT_2A receptor expression by an antisense oligodeoxynucleotide, complementary to the coding region of rat 5-HT_2A receptor mRNA, was examined in a cortically derived cell line and in rat brain. Treatment of A₁A₁ variant cells, which express the 5-HT_2A receptor coupled to the stimulation of phosphatidylinositol (PI) hydrolysis, with antisense oligodeoxynucleotide decreased the maximal stimulation of PI hydrolysis by the partial agonist quipazine and the number of 5-HT_2A receptor sites as measured by the binding of 2-[¹²⁵I]-iodolysergic acid diethylamide. Treatment of cells with random, sense, or mismatch oligodeoxynucleotide did not alter the stimulation of PI hydrolysis by quipazine or 5-HT_2A receptor number. Intracerebroventricular infusion of antisense, but not mismatch, oligodeoxynucleotide for 8 days resulted in a significant increase in cortical 5-HT_2A receptor density and an increase in headshake behavior induced by the 5-HT₂ receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane. The density of cortical 5-HT_2A receptors was not altered by administration of antisense oligodeoxynucleotide for 1, 2, or 4 days. We hypothesize that in brain this antisense oligodeoxynucleotide relieved some form of translational suppression, resulting in an increase in 5-HT_2A receptor expression.