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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 56 (1991), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Single cell Ca2+ mobilization was studied by non-parametric, quantitative flow cytometry using a sort-selected subclone of PC-12 cells. The response of the parent PC-12 population to bradykinin (BK) was very heterogeneous and of a relatively low magnitude. Cells that exhibited maximal Ca2+ mobilization were singly sorted by flow cytometry, cultured, and reanalyzed. In one subclone, referred to as BK1, BK or the B2-BK receptor agonists Lys-BK and Met-Lys-BK (10 pM-1 μM) induced robust Ca2+ transients in 80% of the cells. All three peptides produced the same maximal responses. The B1-BK receptor agonist Des-Arg9-BK (1 nM-1 μM) failed to elicit Ca2+ mobilization in these cells. The responses to BK (10 and 100 nM) were inhibited by preincubation with the B2-receptor antagonists D-Arg0-Hyp3-thienyl5,8-D-Phe7-BK and D-Arg0-Hyp3-D-Phe7 (0.1 nM-10 μM) in a concentration-dependent manner. Des-Arg9-Leu8-BK, a B1-receptor antagonist, failed to block the BK responses at 0.1–10 μM. The agonist/antagonist profile of the BK responses indicated that the B2-BK receptor mediated the Ca2+ response in the BK1 subclone. Thus, flow cytometric analysis of a receptor-mediated Ca2+ response can be employed to select a homogeneously responsive subclone from a heterogeneous, clonal population that can improve the resolution of receptor-mediated second messenger generation at the single cell level.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 58 (1992), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The effects of angiotensin II (AII) and related peptides on the mobilization of internal Ca2+ were studied in a subclone of NG108–15 cells. The subclone, C1, was prepared by fluorescence-activated cell cloning using a rapid response kinetics and a large response magnitude following stimulation by AII as the selection criteria. Angiotensin I, AII, and angiotensin III (AIII) stimulated Ca2+ mobilization in the C1 cells in a concentration-dependent manner (1 nM-100 μM), yielding EC50 values of 437 ± 80 nM (n = 4; slope = 1.6 ± 0.3), 57 ± 8 nM (n = 12; slope = 1.5 ± 0.3), and 36 ± 5 nM (n = 7; slope = 1.4 ± 0.3), respectively. AIII was significantly more potent than AII (p 〈 0.05). In contrast, Des-Phe8-AII, AII-hexapeptide (AII 3–8), and p-NH2-Phe6-AII (1–10 μM) were inactive as agonists. Although the effects of AII and AIII in C1 and parent NG108–15 cells were totally inhibited by the AT1 receptor-selective nonpeptide antagonist, DUP-753 (0.3–1 μM), the AT2-selective antagonists, EXP-655 and CGP42112A (1–10 μM), failed to block the effects of AII. DUP-753 (0.3–100 nM) produced dextral shifts of the AII-induced concentration-response curves and yielded an estimated affinity constant (pA2) of 8.5 ± 0.2 (n = 16) using single-point analysis involving different concentrations of DUP-753. These data compared well with those obtained for the inhibition of AII-induced aortic contractions by DUP-753 (pA2= 8.5) reported previously by others. These data strongly support the identification of an AT1-angiotensin receptor subtype in the NG108–15. C1 subclone that is coupled to a Ca2+ mobilization signal transduction mechanism.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 50 (1988), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Stereotaxic injection of kainic acid (15 μg) into rat olfactory bulbs was accompanied by a 53% (n = 4; p 〈 0.02) depletion of endogenous thyrotropin-releasing hormone (TRH) as compared to sham-operated controls 2 weeks postlesion. TRH levels remained unaltered in three other caudal regions. Bulbar kainate lesions produced a 58% (n = 5; p 〈 0.001) decrease in TRH receptor binding capacity without affecting the receptor affinity. Kainate lesions also reduced bulbar muscarinic and benzodiazepine receptors by 60% and 48%, respectively. Again, no changes in TRH receptors were apparent in six other brain areas after bulbar kainate treatment. Injection of the dopaminergic neurotoxin, 6-hydroxydopamine (8 μg), into rat bulbs decreased TRH receptors by 35% (n = 4; p 〈 0.05) 1 week postlesion. One month after surgical bulbectomy, TRH and TRH receptor levels in a number of brain areas were unaltered compared to those of control animals. These studies suggest that TRH in the olfactory bulb originates intrinsically and may be produced predominantly for local use. Secondly, TRH receptors in the bulb appear to be postsyn-aptically localized on intrinsic neurons, although a small proportion are also associated with presynaptic elements of dopaminergic noradrenergic neurons. Bulbar TRH receptors exhibited nanomolar affinity and a pharmacological selectivity akin to that of the pituitary gland and other brain regions.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Annals of the New York Academy of Sciences 553 (1989), S. 0 
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Neurochemical research 15 (1990), S. 1045-1049 
    ISSN: 1573-6903
    Keywords: TRH ; substance P ; tachykinins ; rat brain ; amygdala
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Binding sites for thyrotropin-releasing hormone (TRH) were labelled with [3H](2-Me-His3)TRH ([3H]MeTRH) on membranes from rat brain regions at 0°C for 5 h. Amygdaloid membranes bound [3H]MeTRH with high-affinity (K d=3.1±0.5 nM (n=4)). Five TRH analogs competed for this binding with the same rank order and with affinities that matched the pharmacological specificity of pituitary TRH receptors. Substance P (SP) and its C-terminal fragments reduced amygdaloid TRH receptor binding in a concentration dependent manner (IC50 for SP=65 μM). The rank order of potency of SP analogs at inhibiting TRH receptor binding was: SP〉nonapeptide (3–11)〉hexapeptide (6–11)〉heptapeptide (5–11)〉pentapeptide (7–11). However, other tachykinins were inactive in this system. SP was a potent inhibitor of [3H]MeTRH binding in hippocampus〉 spinal cord〉retina〉n. accumbens〉hypothalamus〉amygdaloid〉olfactory bulb ≥pituitary〉pons/medulla in parallel assays. In amygdaloid membranes SP (50 μM) reduced the apparent maximum receptor density by 39% (p〈0.01) without altering the binding affinity, and 100 μM SP induced a biphasic dissociation of [3H]MeTRH with kinetics faster than those induced by both TRH (10 μM) and serotonin (100 μM). In contrast, other neuropeptides such as neurotensin, proctolin, angiotensin II, bombesin and luteinizing hormone releasing hormone did not significantly inhibit [3H]MeTRH binding to amydaloid membranes. Thus, the SP site with low affinity in the rat brain is not like any of the previously described tachykinin/neurokinin binding sites but resembles the site found on neuroblastoma cells (108CC15) and on adrenal chromaffin cells that modulate cation permeability and nicotinic receptors respectively. The physiological role of these atypical SP sites in the rat brain remains to be determined.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1573-6903
    Keywords: Dopamine receptors ; autoradiography ; hypertension ; schizophrenia ; Parkinson's disease
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The effects of chronic dietary sodium chloride (NaCl) consumption on renal function and brain dopamine receptors were studied in adult, male normotensive rats. Compared to rats maintained on the normal NaCl (0.33%) diet, animals maintained on the low NaCl (0%) diet for 4 weeks exhibited significant increases in plasma aldosterone, chloride and changes in urinary electrolyte excretion. In contrast, rats maintained on the high NaCl (8%) diet for 4 weeks demonstrated significant increases in urine volume and urinary sodium, chloride and dopamine excretions and water intake. Rats fed the high NaCl diet displayed a 42–59% decrease (p〈0.001–0.05) in D1 binding in the nucleus accumbens (NA), olfactory tubercle (OT) and the striatum (STM), without any effects on D2 binding in these brain regions. Rats maintained on the low NaCl diet also demonstrated decreased D1 binding in the ventral (24%, p〈0.02) and lateral (29%, p〈0.01) STM, but not in the OT, NA, entopeduncular nucleus and substantia nigra. Rats fed low or high NaCl diets exhibited a 35–180% increase (p〈0.01–0.05) in D2 binding in several mid-brain areas (e.g. hypothalamus, thalamus and hippocampus) and hindbrain regions (e.g. superior colliculus and nucleus tractus solitarius) without affecting the D1 binding. These data indicate that chronic modification of dietary salt intake profoundly affects the renal handling of sodium/water excretion and leads to selective up- and/or down-regulation of DA receptor subtypes in different areas of the brain. These findings may have relevance to centrally-mediated hypertension, Parkinson's disease, schizophrenia and other brain disorders involving dopamine and dopamine receptors.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1573-6903
    Keywords: NMDA ; [3H]MK-801 ; dog brain ; spinal cord ; rodent brain
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The biochemical and pharmacological properties of [3H]MK-801 binding to the N-methyl-d-aspartate (NMDA) receptor-channel in homogenates of mouse, guinea pig and dog brain, dog cerebral cortex and rat spinal cord were determined using radioligand binding techniques. Specific [3H]MK-801 binding increased linearily with increasing tissue concentration and in general represented 80–93% of the total binding at 6–8 nM radioligand concentration. [3H]MK-801 interacted with brain and spinal homogenates with high affinity. The dissociation constants (K d ) for all tissues studied were similar ranging between 7.9 and 11.9 nM, whereas the maximum number of binding sites (Bmax) showed a wide, tissue-dependent range (0.1–6.75 pmol/mg protein). The rank order of tissue enrichment was found to be as follows: mouse brain〉〉dog cerebral cortex〉〉dog brain〉〉 guinea pig brain〉〉rat spinal cord. Specific [3H]MK-801 binding in rodent and dog brain, dog cerebral cortex and rat spinal cord exhibited a similar pharmacological profile 9correlation coefficients=0.93–0.99). The rank order of potency of unlabelled compounds competing for [3H]MK-801 binding was: (+)MK-801〉(−)MK-801〉phencyclidine〉(−)cyclazocine〉〉(+)cyclazocine ≥ ketamine〉(+)N-allyl-N-normetazocine〉(−)N-allyl-N-normetazocine〉(−)pentazocine〉(+)pentazocine. NMDA, Kainate, quisqualate and several other compounds failed to inhibit [3H]MK-801 binding at 100 μM. In modulation studies conducted on extensively washed dog cortex membranes, Mg2+ ions stimulated [3H]MK-801 binding at 10 μM-1 mM (EC50=91.5 μM) and then inhibited the binding from 1 mM to 10 mM (IC50=3.1 mM). Glycine stimulated [3H]MK-801 binding at 30 nM-1 mM (EC50=256 nM). In contrast, Zn2+ ions inhibited the binding of [3H]MK-801 binding site exhibited similar pharmacological and biochemical properties. These data appear to suggest that the pharmacological profile of the NMDA-receptor-channel is species and tissue independent.
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  • 8
    ISSN: 1573-6903
    Keywords: Dopamine receptors ; quinpirole hypothermia ; D2 receptors ; D1/D2 receptor interaction ; temperature regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The effect of centrally and peripherally administered dopamine D1 and D2 specific compounds on core body temperature in mice was investigated. Quinpirole (LY-17155), a D2 agonist, induced a dose-dependent fall in body temperature (2.4–11.6%; p〈0.003) when injected intraperitoneally (ip, 0.3–3.0 mg/kg) and intracerebroventricularly (icv, 0.1 mg/kg). This quinpirole-induced (1.0 mg/kg, ip) hypothermia was reversed by the central and peripheral administration of the D2 antagonists S-(−)-sulpiride (3.0–30.0 mg/kg, ip; 0.1–3.0 mg/kg, icv) and spiperone (0.03 and 0.1 mg/kg, ip; 0.03–3.0 mg/kg, icv). Domperidone, a D2 antagonist which does not cross the blood brain barrier, had no effect on quinpirole-induced hypothermia (1.0–10.0 mg/kg, ip). Domperidone partially reversed quinpirole-induced hypothermia at 0.1–30.0 mg/kg, icv. The D1 agonist, SKF-38393 at a high dose of 10.0 mg/kg, ip mildly attenuated quinpirole-induced hypothermia (a 1.8% increase in temperature). SKF-38393 at 10.0 mg/kg, icv potentiated quinpirole-induced hypothermia. SCH-23390 (0.1–3.0 mg/kg, ip), a D1 antagonist, had no effect on quinpirole-induced hypothermia and potentiated the hypothermia when administered icv. An ineffective icv dose of spiperone (0.01 mg/kg) in reversing quinpirole-induced hypothermia was rendered effective by prior administration of SCH-23390 (0.1–3.0 mg/kg, icv) but not by SKF-38393 (1.0–10.0 mg/kg, icv). These data suggest a central D2 receptor mechanism mediating hypothermia in mice which is capable of being modulated by the D1 receptor.
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  • 9
    ISSN: 1573-6903
    Keywords: Thyrotropin-releasing hormone ; TRH receptor ; TRH analogs ; CG3509 ; CG3703 ; RX77368 ; MK-771
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract [3H](3-Me-His2) thyrotropin-releasing hormone ([3H]MeTRH) bound to TRH receptors in rodent, rabbit and dog brain and spinal cord (SC), and in rat, sheep, bovine and dog anterior pituitary (PIT) glands, with high affinity (dissociation constants, Kds=5–9 nM; n=3–4) but to different densities of these sites (B max range 6–145 fmol/mg protein) (rabbit SC〉sheep PIT≫G.pig brain〉dog brain〉rat brain〉bovine and dog PIT). Various TRH analogs competitively inhibited [3H]MeTRH binding in these tissues with a similar rank order of potency: MeTRH〉TRH〉 CG3703≥RX77368≥MK-771〉TRH Glycinamide〉Glu1-TRH≫CG3509≥NVal2-TRH〉〉〉TRH free acid〉〉〉and cyclo-His-Pro, indicating a pharmacological similarity of CNS and pituitary TRH receptors. While most TRH analogs displaced [3H]MeTRH binding with a similar potency in the different species, TRH exhibited a 2-fold lower affinity in the rat and G.pig brain than in other tissues of other species. Similarly, CG3703 was 2.4–4.5 times more active in the rabbit brain than in the rodent and dog brain, and also more potent in the rabbit brain as compared to the sheep PIT. However, MK-771 and RX77368 had a similar affinity for the brain TRH receptors in the different species but RX77368 was 2-fold more active in the SC preparations and ≈3–4-fold less active in the sheep PIT when compared to the brain homogenates. RX 77368 exhibited the highest affinity for the dog PIT TRH receptor. In contrast, MK-771 showed a similar affinity for the brain, SC and PIT TRH receptor apart from in the rat PIT where it had the highest affinity. Similarly, TRH glycinamide was more active in the dog brain than rodent and rabbit brain. These data suggest that while the rank order of potency of TRH analogs is similar in the species examined, certain analogs appear to be more potent in certain tissues of some species than in others. In addition, the current results have shown that CG3703 is almost equipotent with RX77368 and MK-771 in most species but is substantially more active than its related analog, CG3509 in the brain, SC and PIT. Taken together, these observations may have some relevance to the future clinical applications of these metabolically stabilized TRH analogs.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1573-6903
    Keywords: Bradykinin ; receptors ; phosphoinositides ; PI turnover ; receptor binding ; HSDM1C1 cells ; fibrosarcoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Bradykinin (BK) and its analogs (1 nM-100 μM) stimulated phosphoinositide (PI) turnover in murine fibrosarcoma (HSDM1C1) cells in a concentration-dependent manner. The relative potencies (EC50) were: BK=48±4 nM; Lys-BK=39±3 nM; Met-Lys-BK=158±33 nM; Des-Arg9-BK=2617±598 nM (means±SEM, n=3–14). All these analogs were full agonists and they produced up to 5.4±0.4-fold stimulation of PI turnover at the highest concentration (10–100 μM) of the peptides. In contrast, the analogs [D-Arg0-HYP3-Thienyl5,8-D-Phe7]-BK (HYP3-antagonist), [D-Arg0-HYP3-Thienyl,5,8-D-Phe7]-BK (Thienyl antagonist) and Des-Arg9-Leu8-BK were inactive, as agonists, at 0.1 nM-1 μM in this system. These data suggested that BK-induced PI turnover in these cells was mediated via B2-type of BK receptors. This was confirmed further by the fact that both the B2-selective Hyp3- and Thienyl-antagonists inhibited BK-induced PI turnover with KBS of 369±51 nM and 368±118 nM respectively while the B1-selective antagonist, Des-Arg9-Leu8-BK, was inactive at 1 μM. [3H]BK receptor binding studies revealed two binding sites, one with high affinity (Kd=0.24±0.06 nM; Bmax=1.4±0.4 pmol/g tissue) and the other with low affinity (Kd=18.5±0.95 nM; Bmax=25.1±0.52 pmol/g tissue), on HSDM1C1 cell homogenates. The rank order of affinity of BK analogs at inhibiting specific [3H]BK binding was similar to that found for PI turnover. Taken together, these data have provided evidence for the presence of two B2-type BK binding sites on the HSDM1C1 cells. Based on the affinity parameters, the low-affinity component of [3H]BK binding in HSDM1C1 cells appears to be coupled to the phospholipase C-induced PI turnover mechanism. The high-affinity component has been previously shown to mediate the production of prostaglandins by activation of phospholipase A2.
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