NOX4/Src regulates ANP secretion through activating ERK1/2 and Akt/GATA4 signaling in beating rat hypoxic atria

Nicotinamide adenine dinucleotide phosphate oxidases (NOXs) are the major enzymatic source of reactive oxygen species (ROS). NOX2 and NOX4 are expressed in the heart but its role in hypoxia-induced atrial natriuretic peptide (ANP) secretion is unclear. This study investigated the effect of NOX on ANP secretion induced by hypoxia in isolated beating rat atria. The results showed that hypoxia significantly upregulated NOX4 but not NOX2 expression, which was completely abolished by endothelin-1 (ET-1) type A and B receptor antagonists BQ123 (0.3 µM) and BQ788 (0.3 µM). ET-1-upregulated NOX4 expression was also blocked by antagonists of secreted phospholipase A2 (sPLA2; varespladib, 5.0 µM) and cytosolic PLA2 (cPLA2; CAY10650, 120.0 nM), and ET-1-induced cPLA2 expression was inhibited by varespladib under normoxia. Moreover, hypoxia-increased ANP secretion was evidently attenuated by the NOX4 antagonist GLX351322 (35.0 µM) and inhibitor of ROS N-Acetyl-D-cysteine (NAC, 15.0 mM), and hypoxia-increased production of ROS was blocked by GLX351322. In addition, hypoxia markedly upregulated Src expression, which was blocked by ET receptors, NOX4, and ROS antagonists. ET-1-increased Src expression was also inhibited by NAC under normoxia. Furthermore, hypoxia-activated extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (Akt) were completely abolished by Src inhibitor 1 (1.0 µM), and hypoxia-increased GATA4 was inhibited by the ERK1/2 and Akt antagonists PD98059 (10.0 µM) and LY294002 (10.0 µM), respectively. However, hypoxia-induced ANP secretion was substantially inhibited by Src inhibitor. These results indicate that NOX4/Src modulated by ET-1 regulates ANP secretion by activating ERK1/2 and Akt/GATA4 signaling in isolated beating rat hypoxic atria.     

Pharmacological analysis of the haemodynamic effects of 5-HT1B/D receptor agonists in the normotensive rat

1. The receptors involved in mediating the haemodynamic effects of three 5-HT_1B/D receptor agonists were investigated in pentobarbitone anaesthetized rats (n=6–17 per group).
2. Cumulative intravenous (i.v.) infusions of rizatriptan and sumatriptan (from 0.63 to 2500 μg kg⁻¹; each dose over 5 min) induced dose-dependent and marked hypotension (−42±6 and −34±4 mmHg at the highest dose, respectively; both P<0.05 vs vehicle: +5±3 mmHg) and bradycardia (−85±16 and −44±12 beats min⁻¹ at the highest dose, respectively; both P<0.05 vs vehicle: +16±6 beats min⁻¹). Zolmitriptan evoked only moderate hypotension at the highest dose (−19±9 mmHg; P<0.05 vs vehicle).
3. A high dose of the 5-HT_1B/D receptor antagonist, GR 127935 (0.63 mg kg⁻¹, i.v.), failed to antagonize the hypotension and bradycardia evoked by sumatriptan (−35±6 mmHg and −52±19 beats min⁻¹, respectively; both not significant vs sumatriptan in untreated rats), but moderately reduced the hypotension and bradycardia evoked by rizatriptan (−20±5 mmHg and −30±17 beats min⁻¹, respectively; both P<0.05 vs vehicle and vs rizatriptan in untreated rats).
4. The selective 5-HT_1A receptor antagonist, WAY 100635 (0.16 and 0.63 mg kg⁻¹, i.v.), dose-dependently attenuated the haemodynamic responses evoked by rizatriptan and sumatriptan, which were almost abolished by the higher dose of WAY 100635 (−4±3 mmHg and −15±8 beats min⁻¹; both not significant vs vehicle and P<0.05 vs rizatriptan in untreated rats). A slight but statistically significant reduction in mean arterial pressure (MAP) persisted at the highest dose of sumatriptan (−13±4 mmHg following the higher dose of WAY 100635; P<0.05 vs vehicle).
5. In pithed rats with MAP normalized by angiotensin II, rizatriptan failed to induce hypotension or bradycardia (+5±4 mmHg and −6±16 beats min⁻¹, respectively; both NS vs vehicle and P<0.05 vs rizatriptan in untreated rats). Similarly, sumatriptan failed to induce bradycardia in pithed rats (+5±6 beats min⁻¹; not significant vs vehicle and P<0.05 vs sumatriptan in untreated rats), whereas a slight but statistically significant reduction in MAP, compared to controls, occurred at the highest dose (−9±9 mmHg; P<0.05 vs both vehicle and sumatriptan in untreated rats).
6. In bilaterally vagotomized and atropine-treated (1 mg kg⁻¹, i.v.) rats, the reductions in MAP and heart rate evoked by rizatriptan (−31±4 mmHg and −64 ±9 beats min⁻¹, respectively; both P<0.05 vs vehicle and not significant vs rizatriptan in controls) and sumatriptan (−47±8 mmHg and −56±10 beats min⁻¹, respectively; both P<0.05 vs vehicle and not significant vs sumatriptan in controls) were not statistically significantly different from those observed in controls.
7. In conclusion, the 5-HT_1B/D receptor agonists, rizatriptan and sumatriptan, elicit hypotension and bradycardia in the normotensive anaesthetized rat predominantly via activation of central 5-HT_1A receptors, and a consequent reduction in sympathetic outflow.

     

Pharmacological activation of heme oxygenase (HO)-1/carbon monoxide pathway prevents the development of peripheral neuropathic pain in Wistar rats

Recent studies have emphasized the contribution of neuroinflammation and oxido-nitrosative stress to neuropathic pain. Both, heme oxygenase (HO)-1 and carbon monoxide (CO) play an important role in regulating free radical generation and inflammation. Herein, we investigated the role of HO-1/CO pathway, by using hemin, a selective HO activator, and CO-releasing molecule (CORM)-2, a CO-releasing agent, in rat sciatic nerve chronic constriction injury (CCI)-induced neuropathic pain. CCI rats exhibited full development of behavioral hypersensitivity symptoms, including cold allodynia, mechanical and thermal hyperalgesia and also exhibit of a significant increase in spinal cord pro-inflammatory cytokines (TNF-α and IL-1β) and oxido-nitrosative stress markers, both in spinal cord and ipsilateral sciatic nerve homogenate. Spinal (10 and 30 μg/rat, intrathecal (i.t.)), but not systemic (5 and 10 mg/kg, subcutaneous (s.c.)), administration of hemin for 14 days significantly prevented the development of behavioral hypersensitivity. Further, simultaneous administration of hemin via spinal (10 μg/rat, i.t.) and systemic (5 mg/kg, s.c.) routes led to a more pronounced inhibition of the development of behavioral hypersensitivity. Further, administration of CORM-2 (1 and 5 mg/kg, s.c.), dose-dependently and most effectively, prevented the development of behavioral hypersensitivity. Both hemin and CORM-2 produced ameliorative beneficial effects that paralleled with the extent of reduction of oxido-nitrosative stress and pro-inflammatory cytokines. Also, hemin and CORM-2 significantly improved the levels of HO-1 and activity of anti-oxidant enzymes such as superoxide dismutase and catalase. Thus, it may be concluded that chronic pharmacological activation of HO-1/CO pathway may prevent the development of behavioral symptoms of neuropathic pain, through an activation of anti-inflammatory and anti-oxidant mechanisms.     

Exogenous carbon monoxide suppresses Escherichia coli vitality and improves survival in an Escherichia coli-induced murine sepsis model

Aim:

Endogenous carbon monoxide (CO) has been shown to modulate inflammation and inhibit cytokine production both in vivo and in vitro. The aim of this study was to examine whether exogenous carbon monoxide could suppress the vitality of Escherichia coli (E coli) and improve the survival rate in an E coli-induced murine sepsis model.

Methods:

ICR mice were infected with E coli, and immediately injected intravenously with carbon monoxide releasing molecule-2 (CORM-2, 8 mg/kg) or inactive CORM-2 (8 mg/kg). The survival rate was monitored 6 times daily for up to 36 h. The blood samples, liver and lung tissues were collected at 6 h after the infection. Bacteria in peritoneal lavage fluid, blood and tissues were enumerated following culture. Tissue iNOS mRNA expression was detected using RT-PCR. NF-κB expression was detected with Western blotting.

Results:

Addition of CORM-2 (200 and 400 μmol/L) into culture medium concentration-dependently suppressed the growth of E coli and decreased the colony numbers, but inactive CORM-2 had no effect. Treatment of the infected mice with CORM-2 significantly increased the survival rate to 55%, while all the infected mice treated with inactive CORM-2 died within 36 h. E coli infection caused severe pathological changes in liver and lungs, and significantly increased serum transaminases, lipopolysaccharide, TNF-α and IL-1β levels, as well as myeloperoxidase activity, TNF-α and IL-1β levels in the major organs. Meanwhile, E coli infection significantly increased the number of colonies and the expression of iNOS mRNA and NF-κB in the major organs. All these abnormalities were significantly attenuated by CORM-2 treatment, while inactive CORM-2 was ineffective.

Conclusion:

In addition directly suppressing E coli, CORM-2 protects the liver and lungs against E coli-induced sepsis in mice, thus improving their survival.     

Bisphenol A activates Maxi-K (KCa1.1) channels in coronary smooth muscle

Background and purpose:

Bisphenol A (BPA) is used to manufacture plastics, including containers for food into which it may leach. High levels of exposure to this oestrogenic endocrine disruptor are associated with diabetes and heart disease. Oestrogen and oestrogen receptor modulators increase the activity of large conductance Ca²⁺/voltage-sensitive K⁺ (Maxi-K; K_Ca1.1) channels, but the effects of BPA on Maxi-K channels are unknown. We tested the hypothesis that BPA activates Maxi-K channels through a mechanism that depends upon the regulatory β1 subunit.

Experimental approach:

Patch-clamp recordings of Maxi-K channels were made in human and canine coronary smooth muscle cells as well as in AD-293 cells expressing pore-forming α or α plus β1 subunits.

Key results:

BPA (10 µM) activated an outward current in smooth muscle cells that was inhibited by penitrem A (1 µM), a Maxi-K blocker. BPA increased Maxi-K activity in inside-out patches from coronary smooth muscle, but had no effect on single channel conductance. In AD-293 cells with Maxi-K channels composed of α subunits alone, 10 µM BPA did not affect channel activity. When channels in AD-293 cells contained β1 subunits, 10 µM BPA increased channel activity. Effects of BPA were rapid (<1 min) and reversible. A higher concentration of BPA (100 µM) increased Maxi-K current independent of the β1 subunit.

Conclusions and implications:

Our data indicate that BPA increased the activity of Maxi-K channels and may represent a basis for some potential toxicological effects.     

Cloning,expression and pharmacology of a truncated splice variant of the human 5-HT7 receptor (h5-HT7(b))

1. The rat 5-hydroxytryptamine (5-HT)₇ receptor displays two splice variations, a long form, and a truncated splice isoform, arising from the introduction of a stop codon near the carboxy-terminus. The human 5-HT₇ receptor gene contains at least two introns and encodes a 445 amino acid 5-HT receptor.
2. A truncated splice variation in the human 5-HT₇ receptor was isolated from a human placental cDNA library. In accordance with current NC-IUPHAR nomenclature guidelines, it is suggested that this receptor be denoted as the h5-HT_7(b) receptor and the long form of the receptor as h5-HT_7(a).
3. The h5-HT_7(b) receptor was stably expressed in HEK 293 cells and ligand affinities were determined by displacement of [³H]-5-carboxyamidotryptamine (5-CT; K_d=0.28±0.06 nM, B_max=7.3±1.7 pmol mg⁻¹ protein). The rank order of affinities (pK_i) for a series of ligands was: 5-carboxamidotryptamine (5-CT, 9.65)>5-hydroxytryptamine (5-HT, 9.41)>methiothepin (8.87)>mesulergine (7.87)>8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT, 6.85)>ketanserin (6.44).
4. The h5-HT_7(b) receptor coupled positively to adenylyl cyclase in HEK 293 cells. This response was elicited by a number of agonists with the following order of potency (pEC₅₀): 5-CT (8.7±0.11)>5-MeOT (5-methoxytryptamine; 8.1±0.20)>5-HT (7.5±0.13)>tryptamine (5.6±0.36)>8-OH-DPAT (5.3±0.28)>5-methoxytryptamine (5.0±0.06). This rank order was comparable to that observed in the radioligand binding studies.
5. In a similar fashion to that described for the 5-HT_7(a) receptor, PCR studies suggested that the 5-HT_7(b) receptor mRNA is found in great abundance throughout the brain, in the small intestine and aorta.
6. It is concluded that the h5-HT₇ receptor, like the rat receptor, exists as splice variants exhibiting similar pharmacology, signal transduction and distribution. It is thus likely that there exists a complex physiological role for alternate splicing products of the 5-HT₇ receptor gene.

     

Renal effects of intrathecally injected tachykinins in the conscious saline-loaded rat: receptor and mechanism of action

1. The effects of intrathecally (i.t.) injected substance P (SP), neurokinin A (NKA), [β-Ala⁸]NKA (4–10) and [MePhe⁷]neurokinin B (NKB) at T₁₃ thoracic spinal cord level were investigated on renal excretion of water, sodium and potassium in the conscious saline-loaded rat. Antagonists selective for NK₁ (RP 67580), NK₂ (SR 48968) and NK₃ (R 820; 3-indolylcarbonyl-Hyp-Phg-N(Me)-Bzl) receptors were used to characterize the spinal effect of SP on renal function.
2. Saline gavage (4.5% of the body weight) enhanced renal excretion of water, sodium and potassium over the subsequent hour of measurement. Whereas these renal responses were not affected by 0.65 nmol SP, the dose of 6.5 nmol SP blocked the natriuretic response (aCSF value 3.9±0.8; SP value 0.7±0.3 μmol min⁻¹, P<0.01) as well as the renal excretion of water (aCSF value 48.9±5.8; SP value 14.5±4.0 μl min⁻¹, P<0.01) and potassium (aCSF value 4.8±0.6; SP value 1.5±0.6 μmol min⁻¹, P<0.01) at 30 min post-injection. SP had no significant effect on urinary osmolality. The SP-induced renal inhibitory effects during the first 30 min were abolished in rats subjected to bilateral renal denervation 1 week earlier or in rats injected i.t. 5 min earlier with 6.5 nmol RP 67580. In contrast, the co-injection of SR 48968 and R 820 (6.5 nmol each) did not affect the inhibitory responses to SP. On their own, these antagonists had no direct effect on renal excretion function. Since SP induced only transient changes in mean arterial blood pressure (−18.8±3.8 mmHg at 1 min and +6.3±2.4 mmHg at 5 min post-injection), it is unlikely that the renal effects of SP are due to systemic haemodynamic changes.
3. NKA (6.5 nmol but not 0.65 nmol) produced a transient drop in renal excretion of water (aCSF value 31.2±5.1; NKA value 11.3±4.2 μl min⁻¹, P<0.05), sodium (aCSF value 1.7±0.8; NKA value 0.4±0.2 μmol min⁻¹, P<0.05) and potassium (aCSF value 4.1±0.7; NKA value 1.5±0.4 μmol min⁻¹, P<0.05) at 15 min post-injection. However, the same doses (6.5 nmol) of selective agonists for tachykinin NK₂ ([β-Ala⁸]NKA(4-10)) and NK₃ ([MePhe⁷]NKB) receptors were devoid of renal effects.
4. This study provided functional evidence that tachykinins may be involved in the renal control of water and electrolyte excretion at the level of the rat spinal cord through the activation of NK₁ receptors and the sympathetic renal nerve.

     

Contributions of HO-1-Dependent MAPK to Regulating Intestinal Barrier Disruption

The mitogen-activated protein kinase (MAPK) pathway controls intestinal epithelial barrier permeability by regulating tight junctions (TJs) and epithelial cells damage. Heme oxygenase-1 (HO-1) and carbon monoxide (CO) protect the intestinal epithelial barrier function, but the molecular mechanism is not yet clarified. MAPK activation and barrier permeability were studied using monolayers of Caco-2 cells treated with tissue necrosis factor α (TNF-α) transfected with FUGW-HO-1 or pLKO.1-sh-HO-1 plasmid. Intestinal mucosal barrier permeability and MAPK activation were also investigated using carbon tetrachloride (CCl₄) administration with CoPP (a HO-1 inducer), ZnPP (a HO-1 inhibitor), CO releasing molecule 2 (CORM-2), or inactived-CORM-2-treated wild-type mice and mice with HO-1 deficiency in intestinal epithelial cells. TNF-α increased epithelial TJ disruption and cleaved caspase-3 expression, induced ERK, p38, and JNK phosphorylation. In addition, HO-1 blocked TNF-α-induced increase in epithelial TJs disruption, cleaved caspase-3 expression, as well as ERK, p38, and JNK phosphorylation in an HO-1-dependent manner. CoPP and CORM-2 directly ameliorated intestinal mucosal injury, attenuated TJ disruption and cleaved caspase-3 expression, and inhibited epithelial ERK, p38, and JNK phosphorylation after chronic CCl₄ injection. Conversely, ZnPP completely reversed these effects. Furthermore, mice with intestinal epithelial HO-1 deficient exhibited a robust increase in mucosal TJs disruption, cleaved caspase-3 expression, and MAPKs activation as compared to the control group mice. These data demonstrated that HO-1-dependent MAPK signaling inhibition preserves the intestinal mucosal barrier integrity by abrogating TJ dysregulation and epithelial cell damage. The differential targeting of gut HO-1-MAPK axis leads to improved intestinal disease therapy.     

Inhibition of colonic motility and defecation by RS-127445 suggests an involvement of the 5-HT2B receptor in rodent large bowel physiology

Background:

5-HT_2B receptors are localized within the myenteric nervous system, but their functions on motor/sensory neurons are unclear. To explore the role of these receptors, we further characterized the 5-HT_2B receptor antagonist RS-127445 and studied its effects on peristalsis and defecation.

Experimental approach:

Although reported as a selective 5-HT_2B receptor antagonist, any interactions of RS-127445 with 5-HT₄ receptors are unknown; this was examined using the recombinant receptor and Biomolecular Interaction Detection technology. Mouse isolated colon was mounted in tissue baths for isometric recording of neuronal contractions evoked by electrical field stimulation (EFS), or under an intraluminal pressure gradient to induce peristalsis; the effects of RS-127445 on EFS-induced and on peristaltic contractions were measured. Faecal output of rats in grid-bottom cages was measured over 3 h following i.p. RS-127445 and separately, validation of the effective doses was achieved by determining the free, unbound fraction of RS-127445 in blood and brain.

Key results:

RS-127445 (up to 1 µmol·L⁻¹) did not interact with the 5-HT₄ receptor. RS-127445 (0.001–1 µmol·L⁻¹) did not affect EFS-induced contractions of the colon, although at 10 µmol·L⁻¹ the contractions were reduced (to 36 ± 8% of control, n= 4). RS-127445 (0.1–10 µmol·L⁻¹) concentration-dependently reduced peristaltic frequency (n= 4). RS-127445 (1–30 mg·kg⁻¹), dose-dependently reduced faecal output, reaching significance at 10 and 30 mg·kg⁻¹ (n= 6–11). In blood and brain, >98% of RS-127445 was protein-bound.

Conclusions and implications:

High-protein binding of RS-127445 indicates that relatively high doses are required for efficacy. The results suggest that 5-HT_2B receptors tonically regulate colonic motility.     

Purification of 5-hydroxytryptamine3 receptors from porcine brain

1. We demonstrate, for the first time, the purification of the 5-hydroxytryptamine₃ (5-HT₃) receptor from a native tissue source, pig cerebral cortex.
2. From a range of detergents, the non-ionic detergent Triton X-100 was demonstrated to exhibit the least inhibition of [³H]-(S)-zacopride binding to membrane bound 5-HT₃ receptors from pig cerebral cortex at concentrations above its critical micellular concentration (CMC). This detergent was therefore selected to solubilize 5-HT₃ binding sites from homogenates of pig cerebral cortex. Maximum yield (43.8±3.7%, mean±s.e.mean, n=13) was obtained with Triton X-100 at 0.4% (22.1×CMC). Radioligand binding studies with [³H]-(S)-zacopride indicated that the solubilized 5-HT₃ receptor displayed near identical pharmacology to the membrane bound receptor (the correlation coefficient (r) between the pK_i values of structurally unrelated compounds competing for [³H]-(S)-zacopride binding in the membrane bound and solubilized 5-HT₃ receptor preparations was 0.99, B_max=20.7±4.2 fmol mg⁻¹ protein, K_d=1.57±0.53 nM, mean±s.e.mean, n=6).
3. Solubilized (0.4% Triton X-100) 5-HT₃ receptors were affinity purified using Affi-Gel 15 coupled to the high affinity 5-HT₃ receptor ligand GR119566X. Radioligand binding studies indicated that the pharmacological profile of the affinity purified 5-HT₃ receptor, assessed using ligands with a range of affinities spanning 3 orders of magnitude, was similar to that in both crude homogenates (r=0.85) and solubilized 5-HT₃ receptor sites (r=0.85) from pig brain. The specific activity for the purified 5-HT₃ receptor overlapped the theoretical specific activity of the receptor (B_max=3.27±1.41 and 5.35±2.33 nmol mg⁻¹ protein, assessed by saturation and competition studies respectively, mean±s.e.mean, n=3–4), which indicated a 60 000–100 000 fold purification of the membrane bound receptor.
4. Under non-reducing conditions, samples of the affinity purified protein failed to enter a 10% separating gel in SDS–PAGE analysis, indicating a molecular mass for the receptor complex of >200 kDa. Further investigation of the non-reduced purified protein with a 7.5% separating gel gave a mass for the complex of ∼279 kDa. Under reducing conditions, SDS–PAGE analysis of the affinity purified 5-HT₃ receptor resulted in 3–6 silver stained bands at apparent molecular masses of 37, 44–50, 52, 57–61, 63 and 65–71 kDa (n=12). Unlike protein bands at 45, 50, 60 and 66 kDa, the bands corresponding to proteins of 52, 57, 63 and 71 kDa consistently gave no reaction with an antiserum specific for the cloned A subunit of the 5-HT₃ receptor in both a modified dot blot procedure and a Western blot procedure (n=2–5).
5. We conclude that we have purified the 5-HT₃ receptor from pig brain to homogeneity and suggest this may contain non-5-HT₃-A receptor subunit(s).

     

In vitro and in vivo characterization of NK3 receptors in the rabbit eye by use of selective non-peptide NK3 receptor antagonists

1. Inhibition of NK₃ receptor agonist-induced contraction in the rabbit isolated iris sphincter muscle was used to assess the in vitro functional activity of three 2-phenyl-4-quinolinecarboxamides, members of a novel class of potent and selective non-peptide NK₃ receptor antagonists. In addition, an in vivo correlate of this in vitro response, namely NK₃ receptor agonist-induced miosis in conscious rabbits, was characterized with some of these antagonists.
2. In vitro senktide (succinyl-[Asp⁹,MePhe⁸]-substance P (6-11) and [MePhe⁷]-neurokinin B ([MePhe⁷]-NKB) were potent contractile agents in the rabbit iris sphincter muscle but exhibited quite different profiles. Senktide produced monophasic log concentration-effect curves with a mean pD₂=9.03±0.06 and mean n_H=1.2±0.02 (n=14). In contrast, [MePhe⁷]-NKB produced shallow log concentration-effect curves which often appeared biphasic (n_H=0.54±0.04, n=8), preventing the accurate determination of pD₂ values.
3. The contractile responses to the NK₃ receptor agonist senktide were antagonized in a surmountable and concentration-dependent manner by SB 223412 ((−)-(S)-N-(α-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-carboxamide; 3–30 nM, pA₂=8.4, slope=1.8±0.3, n=4), SB 222200 ((−)-(S)-N-(α-ethylbenzyl)-3-methyl-2-phenylquinoline-4-carboxamide; 30–300 nM, pA₂=7.9, slope=1.4±0.06, n=4) and SB 218795 ((−)-(R)-N-(α-methoxycarbonylbenzyl)-2-phenylquinoline-4-carboxamide; 0.3 and 3 μM apparent pK_B=7.4±0.06, n=6).
4. Contractile responses to the NK₃ receptor agonist [MePhe⁷]-NKB in the rabbit iris sphincter muscle were unaffected by SB 218795 (0.3 and 3 μM, n=8). In contrast, SB 223412 (30 and 300 μM, n=4) and SB 222200 (0.3 and 3 μM, n=4) inhibited responses to low concentrations (⩽1 nM), to a greater extent than higher concentrations (>1 nM) of [MePhe⁷]-NKB. Furthermore, log concentration-effect curves to [MePhe⁷]-NKB became steeper and monophasic in the presence of each antagonist.
5. SB 218795 (3 μM, n=4) had no effect on contractions induced by transmural nerve stimulation (2 Hz) or substance P, exemplifying the selectivity of this class of antagonist for functional NK₃ receptors over NK₁ receptors in the rabbit.
6. In vivo, senktide (1, 10 and 25 μg i.v., i.e. 1.2, 11.9 and 29.7 nmol, respectively) induced concentration-dependent bilateral miosis in conscious rabbits (maximum pupillary constriction=4.25±0.25 mm; basal pupillary diameter 7.75±0.48 mm; n=4). The onset of miosis was within 2–5 min of application of senktide and responses lasted up to 30 min. Responses to two i.v. administrations of 25 μg senktide given 30 min apart revealed no evidence of tachyphylaxis. Topical administration of atropine (1%) to the eye enhanced pupillary responses to 25 μg senktide. This was probably due to the mydriatic effect of atropine since it significantly increased baseline pupillary diameter from 7.0±0.4 mm to 9.0±0.7 mm (n=4), thereby increasing the maximum capacity for miosis. Senktide-induced miosis was inhibited by SB 222200 (1 and 2 mg kg⁻¹, i.v., i.e. 2.63 and 5.26 μmol kg⁻¹; maximum inhibition 100%; n=3–4), SB 223412 (0.5 and 1 mg kg⁻¹, i.v., i.e. 1.31 and 2.61 μmol kg⁻¹; maximum inhibition 100%; n=3), SB 218795 (0.5 and 1 mg kg−1, i.v., i.e. 1.26 and 2.52 μmol kg−1; maximum inhibition 78%; n=3), and the structurally distinct NK₃ receptor antagonist SR 142801 ((S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl)-4-phenylepipiperidin-4-yl)-N-methylacetamide; 1.5 mg kg⁻¹, i.v., i.e. 2.47 μmol kg⁻¹, maximum inhibition 92%; n=3).
7. Topical administration of senktide (25 μg; 29.7 nmol) to the eye induced unilateral miosis in the treated eye only. At this dose there was no significant difference (P<0.05) between pupillary constriction obtained by topical or i.v. senktide, and topically administered atropine had no significant effect on responses to topical senktide (n=4).
8. [MePhe⁷]-NKB (125, 250 and 500 μg, i.v., i.e. 98.31, 196.62 and 393.24 nmol, respectively) also induced bilateral miosis in conscious rabbits (maximum pupillary constriction=4.13±0.30 mm; n=4), but in contrast to in vitro studies this agonist was approximately 100 fold less potent than senktide. [MePhe⁷]-NKB-induced miosis was inhibited by SB 222200 (5 mg kg⁻¹, i.v., i.e. 13.14 μmol kg⁻¹; maximum inhibition 69%; n=3).
9. In summary, SB 223412, SB 222200 and SB 218795 are potent and selective antagonists of NK₃ receptor-mediated contraction in the rabbit isolated iris sphincter muscle. In addition, NK₃ receptor agonist-induced miosis in conscious rabbits is a good in vivo correlate of the in vitro rabbit iris sphincter muscle preparation and appears to be a useful model for characterizing the pharmacodynamic profile and efficacy of structurally distinct NK₃ receptor antagonists, such as SB 222200, SB 223412, SB 218795 and SR 142801.

     

YS 51, 1-(beta-naphtylmethyl)-6,7-dihydroxy-1,2,3,4,-tetrahydroisoquinoline, protects endothelial cells against hydrogen peroxide-induced injury via carbon monoxide derived from heme oxygenase-1

Oxidative stress plays an important role in the pathophysiology of several vascular diseases such as atherosclerosis, and great attention has been placed on the protective role of heme oxygenase-1 (HO-1) for vasculature against oxidant-induced injury. We tested whether the protective effects of YS 51, 1-(β-naphtyl-methyl)-6,7-dihydroxy-1,2,3,4,-tetrahydroisoquinoline, against hydrogen peroxide (H₂O₂)-induced cell injury is associated with HO-1 activity in bovine aortic endothelial cells (BAEC). YS 51 increased HO-1 expression and activity in concentration-dependent manners (10-100 μM) and time-dependent manners (1, 3, 6, 18 h), which were correlated well with its protective effect against H₂O₂-induced injury. Zinc protoporphyrin IX (ZnPP IX), a HO inhibitor, significantly inhibited the effect of YS 51 (50 μM). In contrast, [Ru(CO)₃(Cl)₂]₂ (CORM-2, a CO releasing molecule) but not bilirubin protected against H₂O₂-induced injury. Oxyhemoglobin (HbO₂) used as a CO scavenger significantly inhibited the protective effect of both YS 51 and CORM-2. Furthermore, both YS 51 and CORM-2 significantly reduced H₂O₂-induced intracellular reactive oxygen species (ROS) production; however, this was counteracted by ZnPP IX, HbO₂ and deferoxamine. We found evidence for the involvement of PI3/Akt kinase and ERK1/2 pathways in HO-1 induction by YS-51. Taken together, we conclude that CO is, at least, responsible for the YS 51-mediated protective action of endothelial cells against oxidant stress via HO-1 gene induction, involving the activation of the PI3/Akt and ERK1/2 kinase pathways. Thus, YS 51 may be useful in oxidative stress-induced vascular disorders.     

Carbon monoxide is a major contributor to the regulation of vascular tone in aortas expressing high levels of haeme oxygenase-1

1. The contribution of haeme oxygenase-derived carbon monoxide (CO) to the regulation of vascular tone in thoracic aorta was investigated following induction of the inducible isoform of haeme oxygenase (HO-1).
2. Isometric smooth muscle contractions were recorded in isolated rat aortic ring preparations. Rings were incubated in the presence of the nitric oxide (NO) donor S-nitroso-N-acetyl penicillamine (SNAP, 500 μM) for 1 h, then repetitively washed and maintained for a further 4 h prior to producing a concentration-response curve to phenylephrine (PE, 1–3000 nM).
3. Treatment with SNAP resulted in increased mRNA and protein expression of aortic HO-1 and was associated with a significant suppression of the contractile response to PE (P<0.05 vs control). Immunohistochemical staining procedures revealed marked HO-1 expression in the endothelial layer and, to a lesser extent, in smooth muscle cells.
4. Induction of HO-1 in SNAP-treated rings was associated with a higher ¹⁴CO release compared to control, as measured by scintillation counting after incubation of aortas with [2-¹⁴C]-L-glycine, the precursor of haeme. Guanosine 3′,5′-monophosphate (cyclic GMP) content was also greatly enhanced in aortas expressing high levels of HO-1.
5. Incubation of aortic rings with the NO synthase inhibitor, N^G-monomethyl-L-arginine (100 μM), significantly (P<0.05) increased the contractile response to PE in controls but failed to restore PE-mediated contractility in SNAP-treated rings. In contrast, the selective inhibitor of haeme oxygenase, tin protoporphyrin IX (SnPP-IX, 10 μM), restored the pressor response to PE in SNAP-treated rings whilst markedly reducing CO and cyclic GMP production.
6. We conclude that up-regulation of the HO-1/CO pathway significantly contributes to the suppression of aortic contractility to PE. This effect appears to be mediated by the elevation of cyclic GMP levels and can be reversed by inhibition of the haeme oxygenase pathway.

     

Structural determinants for binding to CGRP receptors expressed by human SK-N-MC and Col 29 cells: studies with chimeric and other peptides

1. Structure-activity relationships for the binding of human α-calcitonin gene-related peptide 8–37 (hαCGRP_8–37) have been investigated at the CGRP receptors expressed by human SK-N-MC (neuroblastoma) and Col 29 (colonic epithelia) cells by radioligand binding assays and functional assays (hαCGRP stimulation of adenylate cyclase).
2. On SK-N-MC cells the potency order was hαCGRP_8–37>hαCGRP_19–37=AC187>rat amylin_8–37> hα[Tyr⁰]-CGRP_28–37 (apparent pKBs of 7.49±0.25, 5.89±0.20, 6.18±0.19, 5.85±0.19 and 5.25±0.07). The SK-N-MC receptor appeared CGRP₁-like.
3. On Col 29 cells, only hαCGRP_8–37 of the above compounds was able to antagonize the actions of hαCGRP (apparent pKB=6.48±0.28). Its receptor appeared CGRP₂-like.
4. hα[Ala^11,18]-CGRP_8–37, where the amphipathic nature of the N-terminal α-helix has been reduced, bound to SK-N-MC cells a 100 fold less strongly than hαCGRP_8–37.
5. On SK-N-MC cells, hαCGRP_8–18, _28–37 (M433) and mastoparan-hαCGRP_28–37 (M432) had apparent pKBs of 6.64±0.16 and 6.42±0.26, suggesting that residues 19–27 play a minor role in binding. The physico-chemical properties of residues 8–18 may be more important than any specific side-chain interactions.
6. M433 was almost as potent as hαCGRP_8–37 on Col 29 cells (apparent pKB=6.17±0.20). Other antagonists were inactive.

     

Characterization of [125I]-PD164333, an ETA selective non-peptide radiolabelled antagonist,in normal and diseased human tissues

1. We have synthesized a new low molecular weight, non-peptide radioligand, [¹²⁵I]-PD164333, an analogue of the orally active butenolide antagonists of the endothelin ET_A receptor.
2. Analysis of saturation binding assays demonstrated that [¹²⁵I]-PD164333 bound with high affinity to a single population of receptors (n⩾3 individuals ±s.e.mean) in human aorta (K_D=0.26±0.08 nM; B_max=8.8±3.95 fmol mg^-1 protein), left ventricle from the heart (K_D=0.16±0.02 nM; B_max=34.2± 3.02 fmol mg^-1 protein) and kidney (K_D=1.24±0.16 nM; B_max=125.3±35.07 fmol mg^-1 protein). In each case Hill slopes were close to unity.
3. In kinetic experiments, the binding of [¹²⁵I]-PD164333 to ET_A receptors in sections of heart was time-dependent and rapid at 23°C. The data were fitted to a one site model, with an association rate constant (K₁ of 2.66±0.213×10⁸ M^-1 min^-1, and a half-time for association of 11 min. The binding was reversible at 23°C: analysis of the data indicated [¹²⁵I]-PD164333 dissociated from a single site, with a dissociation rate constant of 0.0031±0.0004 min^-1, a half-time for dissociation of 216 min and a K_D calculated from these kinetic data of 0.01 nM.
4. Unlabelled PD164333 inhibited the binding of [¹²⁵I]-ET-1 to left ventricle (which expresses both subtypes) in a biphasic manner with a K_DET_A of 0.99±0.32 nM and K_DET_B of 2.41±0.22 μM, giving a selectivity of 2500 fold. ET_A-selective ligands competed monophasically for [¹²⁵I]-PD164333 binding in left ventricle, a one site fit was preferred to a two site model giving similar nanomolar affinities: BQ123, K_D=3.93 ±0.18 nM; {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 K_D=3.53±0.69 nM. In contrast, the ET_B selective agonists, BQ3020 and sarafotoxin S6c (1 μM) did not inhibit binding.
5. In human isolated saphenous vein, unlabelled PD164333 was a functional antagonist, producing parallel rightward shifts of the endothelin-1 (ET-1) concentration-response curve (pA₂=8.84) and a slope of unity.
6. In the human brain, autoradiography revealed high levels of [¹²⁵I]-PD164333 binding to the pial arteries of the cerebral cortex and to the numerous smaller intercerebral vessels penetrating the underlying grey and white matter. Conduit and resistance vessels contributing to the control of blood pressure from the heart, kidney, lungs and adrenal also displayed high densities of binding. In diseased vessels, binding of [¹²⁵I]-PD164333 was confined to the medial layer of both coronary arteries with advanced atherosclerotic lesions or occluded saphenous vein grafts. In contrast, little or no binding was detected in the proliferated smooth muscle of the intimal layer or occluded lesion.
7. These results show [¹²⁵I]-PD164333 is a specific, high affinity, reversible non-peptide radioligand for human ET_A receptors, which will facilitate the further characterization of this subtype, in vitro and in vivo.

     

Pharmacological characterization of muscarinic receptors in rabbit isolated iris sphincter muscle and urinary bladder smooth muscle

1. The pharmacological characteristics of muscarinic receptors in the rabbit iris sphincter muscle were studied and compared to M₃ receptors in rabbit urinary bladder smooth muscle.
2. (+)-Cis-dioxolane induced concentration-dependent contractions of the iris sphincter muscle (pEC₅₀=6.41±0.10, E_max=181±17 mg, n=38) and urinary bladder smooth muscle (pEC₅₀=6.97±0.04, E_max=4.28±0.25 g, n=54). These contractions were competitively antagonized by a range of muscarinic receptor antagonists (pK_B values are given for the iris sphincter muscle and the bladder smooth muscle, respectively): atropine (9.30±0.07 and 9.40±0.04), AQ-RA 741 (6.35±0.04 and 6.88±0.03), darifenacin (9.56±0.05 and 9.12±0.05), methoctramine (5.75±0.07 and 5.81+0.06), oxybutynin (8.10±0.09 and 8.59±0.06), pirenzepine (6.79±0.05 and 6.89±0.04), secoverine (7.54±0.05 and 7.66±0.05), p-F-HHSiD (7.55±0.09 and 7.50±0.05) and zamifenacin (8.69±0.10 and 8.36±0.06). A significant correlation between the pK_B values in the bladder and the pK_B values in the iris was obtained.
3. In both tissues, the pK_B values correlated most favorably with pK_i values for these compounds at human recombinant muscarinic m3 receptors. A reasonable correlation was also noted at human recombinant muscarinic m5 receptors given the poor discriminative ability of ligands between m3 and m5 receptors.
4. Overall, the data from this study suggest that the muscarinic receptors mediating contraction of the rabbit iris sphincter muscle and urinary bladder smooth muscle are similar and equate most closely with the pharmacologically-defined muscarinic M₃ receptor.

     

Recombinant saphenous vein 5-HT1B receptors of the rabbit: comparative pharmacology with human 5-HT1B receptors

1. The rabbit recombinant saphenous vein 5-hydroxytryptamine_1B (rb 5-HT_1B) receptor stably transfected in rat C6-glial cells was characterized by measuring adenosine 3′:5′-cyclic monophosphate (cyclic AMP) formation upon exposure to various 5-HT receptor ligands. The effects of agonists and antagonists were compared with their effects determined previously at the human cloned 5-HT_1B (h 5-HT_1B) receptor under similar experimental conditions.
2. Intact C6-glial cells expressing rb 5-HT_1B receptors exhibited [³H]-5-carboxamidotryptamine (5-CT) binding sites with a K_d of 0.80±0.13 nM and a B_max between 225 to 570 fmol mg⁻¹ protein. The binding affinities of a series of 5-HT receptor ligands determined in a membrane preparation with [³H]-5-CT or [³H]-N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)benzamide (GR 125,743) were similar. With the exception of ketanserin, ligand affinities were comparable to those determined at the cloned h 5-HT_1B receptor site.
3. rb 5-HT_1B receptors were negatively coupled to cyclic AMP formation upon stimulation with 5-HT agonists. Of the several 5-HT agonists tested, 5-CT was the most potent, the potency rank order being: 5-CT>5-HT>zolmitriptan>naratriptan>rizatriptan>sumatriptan>R(+)-8-(hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The maximal responses of these agonists were similar to those induced by 5-HT. The potency of these agonists showed a positive correlation (r²=0.87; P<0.002) with their potency at the cloned h 5-HT_1B receptor subtype.
4. 2′-Methyl-4-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide (GR 127,935), methiothepin and ketanserin each behaved as silent, competitive antagonists at rb 5-HT_1B receptors; pK_B values were 8.41, 8.32 and 7.05, respectively when naratriptan was used as an agonist. These estimates accorded with their binding affinities and the potencies found on 5-HT and/or sumatriptan-mediated contraction of isolated rabbit saphenous vein segments.
5. In conclusion, the recombinant saphenous vein 5-HT_1B receptor of the rabbit shares important pharmacological similarities with the cloned h 5-HT_1B receptor. However, ketanserin is a more potent antagonist of rb 5-HT_1B receptors.

     

Selectivity of action of 8-alkylamino analogues of N6-cyclopentyladenosine in vivo: haemodynamic versus anti-lipolytic responses in rats

1. A₁ adenosine receptor agonists with reduced intrinsic activity may be therapeutically useful as result of an increased selectivity of action. In this study the tissue selectivity of three 8-alkylamino substituted analogues of N⁶-cyclopentyladenosine (CPA) was investigated for haemodynamic and anti-lipolytic effects using an integrated pharmacokinetic-pharmacodynamic approach.
2. Chronically instrumented male Wistar rats received intravenous infusions of 4.0 mg kg⁻¹ 8-methylaminoCPA (8MCPA), 12.0 mg kg⁻¹ 8-ethylaminoCPA (8ECPA), 20.0 mg kg⁻¹ 8-butylaminoCPA (8BCPA) or vehicle during 15 min. During experimentation, serial arterial blood samples were drawn for the determination of agonist concentrations and plasma non-esterified fatty acid (NEFA) levels. Blood pressure and heart rate were monitored continuously. In addition to the CPA analogues, each rat received a rapid bolus infusion of CPA to determine the maximal effects of the full agonist.
3. The concentration-time profiles of the CPA analogues could be described by a bi-exponential function. Values for clearance, volume of distribution at steady state and elimination half-life were 44±5, 48±6 and 39±2 ml min⁻¹ kg⁻¹, 0.97±0.09, 0.84±0.10 and 1.05±0.07 1 kg⁻¹ and 25±2, 28±2 and 40±2 min for 8MCPA, 8ECPA and 8BCPA, respectively (mean±s.e.mean, n=6–8).
4. Different models were used to derive the concentration-effect relationships for heart rate and NEFA, yielding estimates of potency (EC₅₀) and instrinsic activity (E_max) for both effects of the compounds in vivo. On heart rate the compounds acted as partial agonists, with E_max values of −173±14, −131±11 and −71±6 beats min⁻¹ for 8MCPA, 8ECPA and 8BCPA, respectively. These E_max values were significantly lower than the maximal effect of CPA (−208±8 beats min⁻¹). With regard to the anti-lipolytic effect all three compounds were full agonists and lowered NEFA levels to the same extent as CPA (69%). The estimated E_max values were 63±5, 63±4 and 68±2%, respectively.
5. Furthermore, the compounds were more potent in causing anti-lipolytic than cardiovascular effects. The EC₅₀ values for the NEFA and heart rate lowering effects were 37±15, 68±22 and 659±108 ng ml⁻¹ and 164±22, 341±76 and 975±190 ng ml⁻¹ for 8MCPA, 8ECPA and 8BCPA, respectively (mean±s.e.mean, n=6–8).
6. This study demonstrates that partial agonists for the A₁ adenosine receptor have increased selectivity of action in vivo. The 8-alkylamino analogues of CPA may be useful anti-lipolytics with less pronounced haemodynamic side effects.