Nitric oxide modulation of transcellular biosynthesis of cys-leukotrienes in rabbit leukocyte-perfused heart

1. We have studied the role of nitric oxide (NO) in the regulation of the transcellular biosynthesis of sulphidopeptide leukotrienes (cys-LT) generated upon neutrophil-vascular wall interactions and their functional consequences, in the spontaneously beating, cell-perfused, heart of the rabbit.
2. Hearts were perfused under recirculating conditions (50 ml) with 5×10⁶ purified human neutrophils (PMNL), and challenged with 0.5 μM A-23187 for 30 min. Coronary perfusion pressure (CPP) and left-ventricular end-diastolic pressure (LVEDP) were monitored. Cys-LT formation was measured by reversed phase high performance liquid chromatography (h.p.l.c.) and u.v. spectral analysis. Myeloperoxidase (MPO) enzyme activity, assayed in aliquots of the recirculating buffer, was used as a marker of PMNL adhesion to the coronary endothelium.
3. Basal CPP and LVEDP values averaged 45±1.4 mmHg and 5±0.1 mmHg, respectively; A-23187 triggered an increase in CPP (134±9 mmHg, at 30 min) which was significantly attenuated by pretreatment with L-arginine, 100 μM (90±3 mmHg, at 30 min). Pretreatment with N^G-monomethyl-L-arginine, 10 μM (L-NMMA), induced a marked increase in CPP (290±40 mmHg, at 20 min) and in LVEDP (47±16 mmHg), so pronounced that it caused cardiac arrest in systole in 5 out of 6 hearts and these were prevented by L-arginine, 100 μM (CPP 115±10 mmHg, LVEDP 6±1.1 mmHg, at 30 min).
4. The increase in CPP was accompanied by the release of cys-LT in the circulating buffer, which was reduced significantly by L-arginine. Pretreatment with L-NMMA, caused a marked rise in cys-LT concentrations which was prevented by L-arginine.
5. Neither L-arginine nor L-NMMA affected directly the A-23187-induced arachidonic acid (AA) metabolism in isolated PMNL alone.
6. Pretreatment with L-NMMA caused a prompt drop in myeloperoxidase (MPO) activity, suggesting rapid adhesion of PMNL to the coronary wall; this effect was significantly blunted by L-arginine.
7. This study suggests that NO provides cardioprotection in an organ model of transcellular metabolism of cys-LT by preventing PMNL adhesion to the coronary intima.

     

Effects of excitatory and inhibitory neurotransmission on motor patterns of human sigmoid colon in vitro

Background and purpose:

To characterize the in vitro motor patterns and the neurotransmitters released by enteric motor neurons (EMNs) in the human sigmoid colon.

Experimental approach:

Sigmoid circular strips were studied in organ baths. EMNs were stimulated by electrical field stimulation (EFS) and through nicotinic ACh receptors.

Key results:

Strips developed weak spontaneous rhythmic contractions (3.67±0.49 g, 2.54±0.15 min) unaffected by the neurotoxin tetrodotoxin (TTX; 1 μM). EFS induced strong contractions during (on, 56%) or after electrical stimulus (off, 44%), both abolished by TTX. Nicotine (1–100 μM) inhibited spontaneous contractions. Latency of off-contractions and nicotine responses were reduced by N^G-nitro-L-arginine (1 mM) and blocked after further addition of apamin (1 μM) or the P2Y₁ receptor antagonist MRS 2179 (10 μM) and were unaffected by the P2X antagonist NF279 (10 μM) or α-chymotrypsin (10 U mL⁻¹). Amplitude of on- and off-contractions was reduced by atropine (1 μM) and the selective NK₂ receptor antagonist Bz-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH₂ (1 μM). MRS 2179 reduced the amplitude of EFS on- and off-contractions without altering direct muscular contractions induced by ACh (1 nM–1 mM) or substance P (1 nM–10 μM).

Conclusions and implications:

Latency of EFS-induced off-contractions and inhibition of spontaneous motility by nicotine are caused by stimulation of inhibitory EMNs coreleasing NO and a purine acting at muscular P2Y₁ receptors through apamin-sensitive K⁺ channels. EFS-induced on- and off-contractions are caused by stimulation of excitatory EMNs coreleasing ACh and tachykinins acting on muscular muscarinic and NK₂ receptors. Prejunctional P2Y₁ receptors might modulate the activity of excitatory EMNs. P2Y₁ and NK₂ receptors might be therapeutic targets for colonic motor disorders.     

A role for nitroxyl (HNO) as an endothelium-derived relaxing and hyperpolarizing factor in resistance arteries

Background and purpose:

Nitroxyl (HNO) is emerging as an important regulator of vascular tone as it is potentially produced endogenously and dilates conduit and resistance arteries. This study investigates the contribution of endogenous HNO to endothelium-dependent relaxation and hyperpolarization in resistance arteries.

Experimental approach:

Rat and mouse mesenteric arteries were mounted in small vessel myographs for isometric force and smooth muscle membrane potential recording.

Key results:

Vasorelaxation to the HNO donor, Angeli''s salt, was attenuated in both species by the soluble guanylate cyclase inhibitor (ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one), the voltage-dependent K⁺ channel inhibitor, 4-aminopyridine (4-AP) and the HNO scavenger, l-cysteine. In mouse mesenteric arteries, nitric oxide (NO) synthase inhibition (with l-NAME, N^ω-Nitro-L-arginine methyl ester) markedly attenuated acetylcholine (ACh)-mediated relaxation. Scavenging the uncharged form of NO (NO^•) with hydroxocobalamin (HXC) or HNO with l-cysteine, or 4-AP decreased the sensitivity to ACh, and a combination of HXC and l-cysteine reduced ACh-mediated relaxation, as did l-NAME alone. ACh-induced hyperpolarizations were significantly attenuated by 4-AP alone and in combination with l-NAME. In rat mesenteric arteries, blocking the effects of endothelium-derived hyperpolarizing factor (EDHF) (charybdotoxin and apamin) decreased ACh-mediated relaxation 10-fold and unmasked a NO-dependent component, mediated equally by HNO and NO^•, as HXC and l-cysteine in combination now abolished vasorelaxation to ACh. Furthermore, ACh-evoked hyperpolarizations, resistant to EDHF inhibition, were virtually abolished by 4-AP.

Conclusions and implications:

The factors contributing to vasorelaxation in mouse and rat mesenteric arteries are NO^• = HNO > EDHF and EDHF > HNO = NO^• respectively. This study identified HNO as an endothelium-derived relaxing and hyperpolarizing factor in resistance vessels.British Journal of Pharmacology (2009) 157, 540–550; doi:10.1111/j.1476-5381.2009.00150.x; published online 26 March 2009This article is commented on by Martin, pp. 537–539 of this issue and is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009     

Myogenic nitric oxide synthase activity in canine lower oesophageal sphincter: morphological and functional evidence

1. Studies on canine lower oesophageal sphincter (LOS) evaluated the existence and function of a myogenic, nitric oxide synthase (NOS) by use of immunocytochemistry for NOS isozymes, NADPH-d histochemistry, [³H]-L-arginine to [³H]-L-citrulline transformation. In addition, functional studies in the muscle bath were performed.
2. Smooth muscle bundles or freshly isolated smooth muscle cells of LOS were NADPH-d reactive but did not recognize some antibodies against neural, endothelial or inducible NOS. NADPH-d reactivity and immunoreactivity to a neural NOS antibody were colocalized in LOS enteric nerves. Muscle plasma membrane-enriched fractions from fresh and cultured LOS cells converted [³H]-L-arginine to [³H]-L-citrulline; activity was mostly Ca²⁺/calmodulin-dependent.
3. N-Nitro-L-arginine (L-NOARG) persistently increased tone (blocked by L-arginine) in muscle strips despite blockade of nerve function. Nifedipine prevented or abolished L-NOARG-induced, but not carbachol-induced, contraction showing that tone increase by L-NOARG required functional L-Ca channels.
4. Membrane-bound, myogenic NOS in canine LOS may release NO continuously when Ca²⁺ entry through L-Ca channels occurs under physiological conditions and thereby modulate tone in LOS.

     

N-arachidonoyl glycine, an endogenous lipid that acts as a vasorelaxant via nitric oxide and large conductance calcium-activated potassium channels

Background and purpose:

N-arachidonoyl glycine (NAGly) is an endogenous lipid that is structurally similar to the endocannabinoid, N-arachidonoyl ethanolamide (anandamide). While NAGly does not activate cannabinoid receptors, it exerts cannabimimetic effects in pain regulation. Here, we have determined if NAGly, like anandamide, modulates vascular tone.

Experimental approach:

In rat isolated small mesenteric arteries, the relaxant responses to NAGly were characterized. Effects of N-arachidonoyl serine and N-arachidonoyl γ-aminobutyric acid were also examined.

Key results:

In endothelium-intact arteries, NAGly-induced relaxation (pEC_50%= 5.7 ± 0.2; relaxation at 30 µM = 98 ± 1%) was attenuated by l-NAME (a nitric oxide synthase inhibitor) or iberiotoxin [selective blocker of large conductance Ca²⁺-activated K⁺ channels (BK_Ca)], and abolished by high extracellular K⁺ concentration. Endothelial removal reduced the potency of NAGly, and the resultant relaxation was inhibited by iberiotoxin, but not l-NAME. NAGly responses were sensitive to the novel cannabinoid receptor antagonist O-1918 independently of endothelial integrity, whereas pertussis toxin, which uncouples G_i/o proteins, attenuated NAGly relaxation only in endothelium-intact arteries. Treatments with antagonists for CB₁, CB₂ and TRPV1 receptors, or inhibitors of fatty acid amide hydrolase and COX had no effect. The two other arachidonoyl amino acids also induced iberiotoxin- and L-NAME-sensitive relaxations.

Conclusion and implications:

NAGly acts as a vasorelaxant predominantly via activation of BK_Ca in rat small mesenteric arteries. We suggest that NAGly activates an unknown G_i/o-coupled receptor, stimulating endothelial release of nitric oxide which in turn activates BK_Ca in the smooth muscle. In addition, NAGly might also activate BK_Ca through G_i/o- and nitric oxide-independent mechanisms.     

Slow delayed rectifier K+ current block by HMR 1556 increases dispersion of repolarization and promotes Torsades de Pointes in rabbit ventricles

Background and purpose:

The slow delayed rectifier K⁺ current (I_Ks) contributes to ventricular repolarization when the action potential (AP) is prolonged. I_Ks block during drug-induced AP prolongation may promote Torsades de Pointes (TdP), but whether this is due to additional AP prolongation is uncertain.

Experimental approach:

In bradycardic perfused rabbit ventricles, the incidence of spontaneous TdP, monophasic AP duration at 90% repolarization (MAPD₉₀) and ECG interval between the peak and the end of T wave (T_peak−end) (index of dispersion of repolarization) were measured after the administration of veratridine (125 nM, slows Na⁺ channel inactivation), dofetilide (7.5 or 10 nM, a rapid delayed rectifier blocker) and HMR 1556 (HMR, 100 nM, an I_Ks blocker), alone or in combinations (n=6 each).

Key results:

HMR did not prolong MAPD₉₀, whereas veratridine or 7.5 nM dofetilide prolonged MAPD₉₀ (P<0.01) without inducing TdP. Veratridine+7.5 nM dofetilide additively prolonged MAPD₉₀ (P<0.05), induced 4±6 TdP per heart and prolonged T_peak−end by 12±10 ms. Subsequent addition of HMR did not further prolonged MAPD₉₀, but increased the number of TdP to 22±18 per heart and increased T_peak−end by 39±21 ms (P<0.05). Increasing dofetilide concentration from 7.5 to 10 nM (added to veratridine) produced a longer MAPD₉₀, but fewer TdP (5±5 per heart) and less T_peak−end prolongation (17±8 ms) compared to the veratridine+7.5 nM dofetilide+HMR group (P<0.05).

Conclusions and implications:

Adding I_Ks block markedly increases TdP incidence in hearts predisposed to TdP development by increasing the dispersion of repolarization, but without additional AP prolongation.     

Oleanolic acid induces relaxation and calcium-independent release of endothelium-derived nitric oxide

Background and purpose:

The present study investigated the mechanisms by which oleanolic acid, a component of olive oil, increases release of nitric oxide (NO).

Experimental approach:

Measurements of isometric tension, NO concentration, or endothelial cell calcium were made in rat isolated mesenteric arteries. Immunoblotting for endothelial NOS (eNOS) and Akt kinase were performed in primary cultures of human umbilical vein endothelial cells (HUVECs).

Key results:

Oleanolic acid (3–30 μM) evoked endothelium-dependent relaxations in noradrenaline-contracted rat superior and small mesenteric arteries. In rat superior mesenteric arteries, oleanolic acid induced simultaneous increases in NO concentration and relaxation, and these responses were inhibited by an inhibitor of NOS, asymmetric dimethyl-L-arginine (300 μM) and by the NO scavenger, oxyhaemoglobin (10 μM). Oleanolic acid-evoked NO increases were not reduced in Ca²⁺-free solution and in the presence of an inhibitor of endoplasmic reticulum calcium-ATPase, thapsigargin (1 μM). Oleanolic acid evoked relaxation without changes in endothelial cell calcium, but decreased smooth muscle calcium in arterial segments. Oleanolic acid failed to increase calcium in HUVECs, but increased time-dependently phosphorylation of Akt kinase at Serine⁴⁷³ (Akt-Ser⁴⁷³) and eNOS at Serine¹¹⁷⁷ (eNOS-Ser¹¹⁷⁷), which was attenuated by inhibitors of phosphoinositide-3-kinase.

Conclusions and implications:

This study provides direct evidence that a component of olive oil, oleanolic acid, activated endothelium-dependent release of NO and decreased smooth muscle cell calcium followed by relaxation. The oleanolic acid-evoked endothelium-derived NO release was independent of endothelial cell calcium and involved phosphoinositide-3-kinase-dependent phosphorylation of Akt-Ser⁴⁷³ followed by phosphorylation of eNOS-Ser¹¹⁷⁷.     

Response of normoxic pulmonary arteries of the rat in the resting and contracted state to NO synthase blockade

1. The pulmonary vasculature is normally in a low resting state of tone. It has been hypothesized that this basal tone is actively maintained by the continuous release of a vasodilator in the resting state. However, evidence for basal release of nitric oxide (NO) is inconclusive.
2. We studied the release of NO in arteries from the pulmonary circulation of male Wistar-Kyoto rats by examining the effects of the L-arginine analogue N^G-nitro-L-arginine methyl ester (L-NAME) on resting pulmonary arteries and on vessels pre-contracted with prostaglandin F_2α (PGF_2α).
3. Rats (n=21) were killed by an overdose with pentobarbitone. Pulmonary arteries were dissected (mean internal diameter 459±11 μm) and mounted in a small vessel wire myograph. Resting tensions were set to simulate transmural pressures of 17.5 mmHg.
4. L-NAME (100 μM) was found to produce a contraction of 0.64±0.09 mN mm⁻¹ in resting pulmonary arteries when added alone to the myograph bath. This contraction was not produced following removal of the endothelium. Vessel contraction to PGF_2α (100 μM) was found to be significantly greater when carried out in the presence of L-NAME (100 μM)–1.37±0.15 mN mm⁻¹ compared with 1.96±0.17 mN mm⁻¹. Dilatation following acetylcholine (ACh) (1 μM) was abolished in the presence of L-NAME (100 μM).
5. Rat pulmonary artery contraction in response to the addition of L-NAME and the absence of contraction upon removal of the endothelium provides supportive evidence of the active release of nitric oxide for the maintenance of resting tone.

     

Symptomatic and neuroprotective effects following activation of nigral group III metabotropic glutamate receptors in rodent models of Parkinson's disease

Background and purpose:

Increased glutamatergic innervation of the substantia nigra pars reticulata (SNpr) and pars compacta (SNpc) may contribute to the motor deficits and neurodegeneration, respectively, in Parkinson''s disease (PD). This study aimed to establish whether activation of pre-synaptic group III metabotropic glutamate (mGlu) receptors reduced glutamate release in the SN, and provided symptomatic or neuroprotective relief in animal models of PD.

Experimental approach:

Broad-spectrum group III mGlu receptor agonists, O-phospho-l-serine (l-SOP) and l-2-amino-4-phosphonobutyrate (l-AP4), were assessed for their ability to inhibit KCl-evoked [³H]-d-aspartate release in rat nigral prisms or inhibit KCl-evoked endogenous glutamate release in the SNpr in vivo using microdialysis. Reversal of akinesia in reserpine-treated rats was assessed following intranigral injection of l-SOP and l-AP4. Finally, the neuroprotective effect of 7 days'' supra-nigral treatment with l-AP4 was examined in 6-hydroxydopamine (6-OHDA)-lesioned rats.

Key results:

l-SOP and l-AP4 inhibited [³H]-d-aspartate release by 33 and 44% respectively. These effects were blocked by the selective group III mGlu antagonist (RS)-α-cyclopropyl-4-phosphonophenylglycine (CPPG). l-SOP also reduced glutamate release in the SNpr in vivo by 48%. Injection of l-SOP and l-AP4 into the SNpr reversed reserpine-induced akinesia. Following administration above the SNpc, l-AP4 provided neurochemical, histological and functional protection against 6-OHDA lesion of the nigrostriatal tract. Pretreatment with CPPG inhibited these effects.

Conclusions and implications:

These findings highlight group III mGlu receptors in the SN as potential targets for providing both symptomatic and neuroprotective relief in PD, and indicate that inhibition of glutamate release in the SN may underlie these effects.     

Clozapine, but not haloperidol, enhances glial D-serine and L-glutamate release in rat frontal cortex and primary cultured astrocytes

BACKGROUND AND PURPOSE

Deficient transmission at the glutamate NMDA receptor is considered a key component of the pathophysiology of schizophrenia. However, the effects of antipsychotic drugs on the release of the endogenous NMDA receptor partial agonist, d-serine, remain to be clarified.

EXPERIMENTAL APPROACH

We determined the interaction between antipsychotic drugs (clozapine and haloperidol) and transmission-modulating toxins (tetanus toxin, fluorocitrate, tetrodotoxin) on the release of L-glutamate and d-serine in the medial prefrontal cortex (mPFC) of freely moving rats, using microdialysis, and primary cultures of astrocytes using extreme high-pressure liquid chromatography.

KEY RESULTS

Release of L-glutamate and d-serine in the mPFC and in cultured astrocytes was inhibited by tetanus toxin (a synaptobrevin inhibitor) and fluorocitrate (a glial toxin), whereas tetrodotoxin (a voltage-sensitive Na⁺ blocker) inhibited depolarization-induced L-glutamate release in the mPFC without affecting that of d-serine. Clozapine (1 and 5 mg·kg⁻¹), but not haloperidol (0.5 and 1 mg·kg⁻¹), dose-dependently increased L-glutamate and d-serine release from both astrocytes and mPFC. Clozapine-induced release of L-glutamate and d-serine was also reduced by tetanus toxin and fluorocitrate. Tetrodotoxin reduced clozapine-induced mPFC L-glutamate release but not that of d-serine. Clozapine-induced L-glutamate release preceded clozapine-induced d-serine release. MK-801 (a NMDA receptor antagonist) inhibited the delayed clozapine-induced L-glutamate release without affecting that of d-serine.

CONCLUSIONS AND IMPLICATIONS

Clozapine predominantly activated glial exocytosis of d-serine, and this clozapine-induced d-serine release subsequently enhances neuronal L-glutamate release via NMDA receptor activation. The enhanced d-serine associated glial transmission seems a novel mechanism of action of clozapine but not haloperidol.     

Metabolic responses to BRL37344 and clenbuterol in soleus muscle and C2C12 cells via different atypical pharmacologies and beta2-adrenoceptor mechanisms

Background and purpose:

Picomolar concentrations of the β₃-adrenoceptor agonist BRL37344 stimulate 2-deoxyglucose uptake in soleus muscle via undefined receptors. Higher concentrations alter uptake, apparently via β₂-adrenoceptors. Effects of BRL37344 and β₂-adrenoceptor agonists are compared.

Experimental approach:

Mouse soleus muscles were incubated with 2-deoxy[1-¹⁴C]-glucose, [1-¹⁴C]-palmitate or [2-¹⁴C]-pyruvate, and BRL37344, β₂-adrenoceptor agonists and selective β-adrenoceptor antagonists. Formation of 2-deoxy[1-¹⁴C]-glucose-6-phosphate or ¹⁴CO₂ was measured. 2-Deoxy[1-¹⁴C]-glucose uptake and β-adrenoceptor mRNA were measured in C2C12 cells.

Key results:

10 pM BRL37344, 10 pM clenbuterol and 100 pM salbutamol stimulated 2-deoxyglucose uptake in soleus muscle by 33–54%. The effect of BRL37344 was prevented by 1 μM atenolol but not by 300 nM CGP20712A or IC3118551, or 1 μM SR59230A; that of clenbuterol was prevented by ICI118551 but not atenolol. 10 nM BRL37344 st4mulated 2-deoxyglucose uptake, whereas 100 nM clenbuterol and salbutamol inhibited uptake. These effects were blocked by ICI118551. Similar results were obtained in C2C12 cells, in which only β₂-adrenoceptor mRNA could be detected by RT-PCR. 10 nM BRL37344 and 10 pM clenbuterol stimulated muscle palmitate oxidation. In the presence of palmitate, BRL37344 no longer stimulated 2-deoxyglucose uptake and the effect of clenbuterol was not significant.

Conclusions and implications:

Stimulation of glucose uptake by 10 pM BRL37344 and clenbuterol involves different atypical pharmacologies. Nanomolar concentrations of BRL37344 and clenbuterol, probably acting via β₂-adrenoceptors, have opposite effects on glucose uptake. The agonists preferentially stimulate fat rather than carbohydrate oxidation, but stimulation of endogenous fat oxidation cannot explain why 100 nM clenbuterol inhibited 2-deoxyglucose uptake.     

Aminoguanidine – effects on endoneurial vasoactive nitric oxide and on motor nerve conduction velocity in control and streptozotocin-diabetic rats

1. The effects of aminoguanidine (AG) treatment on reductions in motor nerve conduction velocity (MNCV) and sciatic nerve blood flow, indexed by laser Doppler flux (LDF), were investigated in rats with experimental diabetes (streptozotocin-induced; 8–10 weeks duration). The contribution of endoneurial vasoactive nitric oxide to the LDF of these animals was also investigated by the direct micro-injection of N^G-nitro-L-arginine methyl ester (L-NAME; 1 nmol in 1 μl), followed by L-arginine (100 nmol in 1 μl), into the sciatic nerve endoneurium.
2. The MNCV (m s⁻¹, mean±1 s.d.) of diabetic rats (38.2±1.5) was lower (P<0.01) than that of age-matched controls (47.2±4.2). AG treatment (50 mg kg⁻¹ day⁻¹, i.p.) attenuated the diabetes-induced deficits in MNCV (43.4±5.9; P<0.01), but had no effect in controls (48.8±3.8) or, if administered via drinking water (1 g l⁻¹), diabetics (37.4±4.1).
3. L-NAME markedly reduced the resting LDF (arbitrary units; mean±s.e.mean) of controls (209±13 to 120±18; P<0.005), an effect reversed by subsequent L-arginine (to 206±27). In diabetic rats the LDF reduction following L-NAME was much smaller (111±11 to 84±6; P<0.05), but the change with L-arginine was significantly increased (to 145±12; P<0.001).
4. AG treatment increased the resting LDF of control (265±34) and diabetic rats (133±14 for daily injection and 119±13 for drinking water). The responses to L-NAME and L-arginine were not changed markedly by AG treatment. However, L-arginine appeared to be less effective.
5. In conclusion, these data suggest that AG treatment may affect nitric oxide production in the vasa nervorum of peripheral nerves. However, the effects of AG-treatment are not consistent with the prevention of a diabetes-associated reduction in endoneurial nitric oxide production. The mechanisms by which AG attenuates nerve conduction slowing in streptozotocin-diabetic rats therefore remain unclear.

     

The induction of nitric oxide-mediated relaxation of human isolated pulmonary arteries by PACAP

1. The effects of pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) were analysed in human isolated circular segments of pulmonary arteries. Guinea-pig pulmonary arteries were used for comparison. The responses obtained were analysed in relation to the vascular endothelium and the nitric oxide (NO) synthase inhibitor N^G-monomethyl L-arginine (L-NMMA).
2. PACAP and VIP induced concentration-dependent relaxations of precontracted pulmonary arteries. The maximal dilator response (I_max,%) and the potency (pEC₅₀ value) were the same for both peptides, and there were no differences in the effects obtained on human and guinea-pig segments. PACAP and VIP were both more potent that acetylcholine (ACh).
3. Removal of the vascular endothelium abolished the PACAP induced dilator response in pulmonary arteries from both species. The VIP induced dilatation was unaffected, whereas the response to ACh was abolished. L-NMMA given before PACAP inhibited the dilatation. Furthermore, L-NMMA also reversed the dilatation already induced by PACAP and excess concentrations of L-arginine restored the dilator response of the L-NMMA treated arteries.
4. PACAP is a potent dilator of human pulmonary arteries. Although the dilator effect seems to be similar in amplitude to the one induced by VIP, the present results suggest differences in the underlying mechanisms of action (endothelium-dependency) between the two peptides.

     

Developmental changes in endothelium-dependent vasodilation and the influence of superoxide anions in perinatal rabbit pulmonary arteries

1. ACh-induced vasodilation was investigated in pulmonary arteries from 8 and 2 day pre-term foetal, neonatal (0–12 h and 4 day old) and adult rabbits. The effects of superoxide anion generation [with hypoxanthine (HX, 0.1 mM)/xanthine oxidase (XO, 15 mu ml⁻¹)], endogenous superoxide dismutase (SOD) inhibition [with the Cu-Zn SOD inhibitor triethylenetetramine (TETA, 1 mM)], endogenous superoxide anion scavenging [by superoxide dismutase (SOD, 50 u ml⁻¹)] and inhibition of endothelial nitric oxide synthase (eNOS) [with, N^ω-nitro-L-arginine methylester (L-NAME, 0.1 mM)], on basal and ACh-induced NO activity were studied by examining phenylephrine-induced contraction and ACh-induced vasodilation respectively.
2. L-NAME and endothelium removal abolished all ACh-induced vasodilation and 1 μM sodium nitroprusside fully dilated all vessels. ACh-induced vasodilation was absent in the 8 day pre-term foetus and 0–12 h neonate but present at all other ages. L-NAME itself contracted 2 day pre-term foetal vessels. At 0–12 h, SOD, but not the phosphodiesterase 5 inhibitor zaprinast (1 μM), uncovered ACh-induced vasodilation. At this age SOD reduced phenylephrine-induced contraction which was not influenced by TETA, L-NAME or HX/XO, and L-NAME itself did not cause contraction. This suggests both ACh-induced and basal NO activity are compromise in these vessels by endogenous superoxide anion production and deficiencies in endogenous SOD activity.
3. In 4 day vessels, but not adult vessels, L-NAME, TETA and HX/XO augmented contractions to phenylephrine, and L-NAME itself induced vasoconstriction, suggesting that basal NO and SOD activities were present by 4 days but were not evident in the adult. ACh-induced NO activity, and the influence of endogenous SOD on this, were present in the adult (and 4 day) vessels as superoxide generation with HX/XO significantly reduced ACh-induced vasodilation and this effect was inhibited by SOD and augmented by TETA.
4. Increased oxygen tensions >500 mmHg attenuated ACh-induced vasodilation in the foetal but not neonatal rabbits. Raising the oxygen tension from ∼20 to ∼120 mmHg revealed ACh-induced vasodilation in the 8 day pre-term vessels.
5. In summary, superoxide anion accumulation combined with deficiencies in SOD activity may transiently compromise basal and ACh-induced NO activity at birth. Experimental oxygen tensions markedly influence ACh-induced vasodilation in foetal rabbit pulmonary arteries.

     

On the selectivity of neuronal NOS inhibitors

Background and Purpose

Isoform-selective inhibitors of NOS enzymes are desirable as research tools and for potential therapeutic purposes. Vinyl-l-N-5-(1-imino-3-butenyl)-l-ornithine (l-VNIO) and N^ω-propyl-l-arginine (NPA) purportedly have good selectivity for neuronal over endothelial NOS under cell-free conditions, as does N-[(3-aminomethyl)benzyl]acetamidine (1400W), which is primarily an inducible NOS inhibitor. Although used in numerous investigations in vitro and in vivo, there have been surprisingly few tests of the potency and selectivity of these compounds in cells. This study addresses this deficiency and evaluates the activity of new and potentially better pyrrolidine-based compounds.

Experimental Approach

The inhibitors were evaluated by measuring their effect on NMDA-evoked cGMP accumulation in rodent hippocampal slices, a response dependent on neuronal NOS, and ACh-evoked cGMP synthesis in aortic rings of the same animals, an endothelial NOS-dependent phenomenon.

Key Results

l-VNIO, NPA and 1400W inhibited responses in both tissues but all showed less than fivefold higher potency in the hippocampus than in the aorta, implying useless selectivity for neuronal over endothelial NOS at the tissue level. In addition, the inhibitors had a 25-fold lower potency in the hippocampus than reported previously, the IC₅₀ values being approximately 1 μM for l-VNIO and NPA, and 150 μM for 1400W. Pyrrolidine-based inhibitors were similarly weak and nonselective.

Conclusion and Implications

The results suggest that l-VNIO, NPA and 1400W, as well as the newer pyrrolidine-type inhibitors, cannot be used as neuronal NOS inhibitors in cells without stringent verification. The identification of inhibitors with useable selectivity in cells and tissues remains an important goal.     

Nitroglycerin-inhibited whole blood aggregation is partially mediated by calcitonin gene-related peptide–a neurogenic mechanism

1. The role of the vasculature and calcitonin gene-related peptide (CGRP) in nitroglycerin (NTG)-mediated platelet inhibition was studied.
2. In vitro incubations of CGRP in whole blood induced a dose-dependent inhibition of platelet aggregation with an IC₅₀ of 62.1 nM.
3. The platelet inhibition induced by CGRP was blocked by co-incubation of 0.53 μM CGRP_8-37, as well as 30 μM N^G-nitro-monomethyl-L-arginine (L-NMMA).
4. In a separate group of experiments, 100 nM NTG in rat whole blood (WB) induced platelet inhibition of 6.0±1.3% (mean±s.d.), which was enhanced to 77.6±3.5% by the addition of rat aortic tissue (AT) (P<0.001). The inclusion of CGRP_8-37 with NTG and AT in WB reduced platelet inhibition to 31.6±6.8% (P<0.01). Incubation of WB and AT with 30 μM L-NMMA reduced NTG-induced inhibition of platelet aggregation to 26.4±4.2% (P<0.001).
5. It is concluded that vascular tissue contributes to the antiplatelet mechanism of action of NTG. Furthermore, NTG apparently evokes the release of CGRP from vascular tissue and this neuropeptide contributes to the antiplatelet actions of NTG.
6. The antiplatelet activity of CGRP in whole blood is mediated primarily through the activation of nitric oxide synthase.

     

Evaluation of sulfonamide detoxification pathways in haematologic malignancy patients prior to intermittent trimethoprim-sulfamethoxazole prophylaxis

AIMS

Patients with haematologic malignancies have a reportedly high incidence of sulfamethoxazole (SMX) hypersensitivity. The objective of this study was to determine whether deficiencies in sulfonamide detoxification pathways, to include glutathione (GSH) and ascorbate (AA), and cytochrome b₅ (b5) and cytochrome b₅ reductase (b5R), were prevalent in these patients. A secondary pilot objective was to determine whether the incidence of drug hypersensitivity following intermittent trimethoprim-SMX (TMP-SMX) prophylaxis approached that reported for high dose daily regimens.

METHODS

Forty adult patients with haematologic malignancies (HM) and 35 healthy adults were studied; an additional 13 HM patients taking ascorbate supplements (HM-AA) were also evaluated. Twenty-two of 40 HM patients were prescribed and were compliant with TMP-SMX 960 mg three to four times weekly.

RESULTS

There were no significant differences between HM and healthy groups in plasma AA (median 37.2 µmvs. 33.9 µm) or red blood cell GSH (1.9 mmvs. 1.8 mm). However, plasma AA was correlated significantly with leucocyte b5/b5R reduction (r = 0.39, P = 0.002). Deficient b5/b5R activities were not found in HM patients. In fact, patients with chronic lymphocytic leukaemia or myeloma had significantly higher median activities (80.7 µmol mg^–1 min⁻¹) than controls (18.9 µmol mg^–1 min⁻¹, P = 0.008). After 3–4 weeks of treatment, no patients developed SMX-specific T cells and only one patient developed rash.

CONCLUSIONS

Deficiencies of blood antioxidants and b5/b5R reduction were not found in this population with haematologic malignancies, and the development of skin rash and drug-specific T cells appeared to be uncommon with intermittent TMP-SMX prophylaxis.     

Bradykinin regulates human colonic ion transport in vitro

Background and purpose:

Kinins are acknowledged as important regulators of intestinal function during inflammation; however, their effects on human intestinal ion transport have not been reported. Here, we used muscle-stripped human colonic tissue and cultured T₈₄-cell monolayers to study bradykinin (BK) actions on human intestinal ion transport.

Experimental approach:

Ion transport was measured as changes in short-circuit current (I_sc) across colonic epithelia mounted in Ussing chambers.

Key results:

In intact tissue, there was a distinct polarity to BK-elicited I_sc responses. Whereas basolateral BK stimulated sustained responses (EC₅₀=0.5±0.1 μM), those to apical BK were more rapid and transient (EC₅₀=4.1±1.2 nM). In T₈₄ cells, responses to both apical and basolateral BK were similar to those seen upon apical addition to intact tissues. Cross-desensitization between apical and basolateral domains was not observed. BK-induced responses were largely due to Cl⁻ secretion as shown by their sensitivity to bumetanide and removal of Cl⁻ from the bathing solution. Studies using selective agonists and antagonists indicate responses to BK are mediated by B₂ receptors. Finally, responses to basolateral BK in intact tissues were inhibited by tetrodotoxin (1 μM), atropine (1 μM), capsaicin (100 μM) and piroxicam (10 μM). BK-stimulated prostaglandin (PG)E₂ release from colonic tissue.

Conclusions:

BK stimulates human colonic Cl⁻ secretion by activation of apical and basolateral B₂ receptors. Responses to apical BK reflect a direct action on epithelial cells, whereas those to basolateral BK are amplified by stimulation of enteric nerves and PG synthesis.     

Differential activation of the epithelial and smooth muscle NK1 receptors by synthetic tachykinin agonists in guinea-pig trachea

1. The presence of tachykinin NK₁ receptors have been shown in the epithelium and smooth muscle of guinea-pig airways. Previous data showed that substance P (SP), and the NK₁ receptor agonist, [Sar⁹, Met (O₂)¹¹]-SP, relax guinea-pig tracheal tube preparations by stimulation of epithelial NK₁ receptors and via nitric oxide (NO) release. However, the selective tachykinin NK₁ receptor agonist, septide, was unable to produce this effect. The aim of the present study was to investigate the ability of a series of SP analogues to stimulate NK₁ receptors of guinea-pig airway epithelium.
2. Isometric tension was recorded in isolated tracheal tube preparations in which compounds were administered intraluminally in the presence of phosphoramidon, indomethacin (both 1 μM) and the tachykinin NK₂ receptor antagonist, SR 48,968 ((S)-N-methyl N-(4-acetyl-amino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl)benzamide) (0.1 μM). Cumulative concentration-response curves were obtained in preparations under resting tone or in preparations precontracted with acetylcholine (ACh, 10 μM).
3. Contractile responses to low concentrations (0.1–10 nM) of substance P (SP) and the selective agonist of NK₁ receptors, [Pro⁹]-SP, in non precontracted tracheae were higher in preparations pretreated with the NO-synthase inhibitor, N^G-monomethyl L-arginine (L-NMMA, 100 μM) than in preparations pretreated with its inactive enantiomer D-NMMA (100 μM). Tracheal tube preparations precontracted with ACh and pretreated with D-NMMA were relaxed by low concentrations of SP and [Pro⁹]-SP (0.1–10 nM). In contrast, after pretreatment with L-NMMA, SP and [Pro⁹]-SP contracted tracheae at all the concentrations tested.
4. Concentration-response curves to the NK₁ receptor agonists, SP methyl ester, [Apa^9–10]-SP and [pGlu⁶] SP (6–11) obtained in non-precontracted tracheae were similar in the presence of either D-NMMA or L-NMMA. SP methyl ester, [Apa^9–10]-SP and [pGlu⁶] SP (6–11) did not produce any relaxation, but instead, cause contractions in tracheal tube preparations precontracted with ACh and pretreated with D-NMMA. Concentration-response curves produced by all these agonists were similar in preparations precontracted with ACh and pretreated with L-NMMA or D-NMMA.
5. In guinea-pig tracheal tube preparations two groups of NK₁ receptor agonists can be distinguished: one group, including [Pro⁹]-SP, stimulator epithelial NK₁ receptors, the other group, including SP methyl ester, [Apa^9–10]-SP and [pGlu⁶] SP (6–11), does not. One possible explanation for these findings and for the existence of compounds with a peculiar ‘septide-like'' pharmacological profile in the guinea-pig trachea could be the recently proposed phenomenon referred to as ‘agonist-directed receptor trafficking''.

     

The proton-coupled amino acid transporter, SLC36A1 (hPAT1), transports Gly-Gly, Gly-Sar and other Gly-Gly mimetics

BACKGROUND AND PURPOSE

The intestinal proton-coupled amino acid transporter, SLC36A1, transports zwitterionic α-amino acids and drugs such as vigabatrin, gaboxadol and δ-aminolevulinic acid. We hypothesize that SLC36A1 might also transport some dipeptides. The aim of the present study was to investigate SLC36A1-mediated transport of Gly-Gly and Gly-Gly mimetics, and to investigate Gly-Sar transport via SLC36A1 and the proton-coupled dipeptide/tripeptide transporter, SLC15A1 in Caco-2 cells.

EXPERIMENTAL APPROACH

Transport of a compound via SLC36A1 was determined by its ability to induce an increase in the inward current of two-electrode voltage clamped SLC36A1 cRNA-injected Xenopus laevis oocytes. SLC36A1-mediated l-[³H]Pro uptake in Caco-2 cells was measured in the absence and presence of Gly-Gly or Gly-Sar. In addition, apical [¹⁴C]Gly-Sar uptake was measured in the absence and presence of the SLC36A1 inhibitor 5-hydroxy-l-tryptophan (5-HTP) or the SLC15A1 inhibitor l-4,4′-biphenylalanyl-l-proline (Bip-Pro).

KEY RESULTS

In SLC36A1-expressing oocytes, an inward current was induced by Gly-Sar, Gly-Gly, δ-aminolevulinic acid, β-aminoethylglycine, δ-aminopentanoic acid, GABA, Gly and Pro, whereas Val, Leu, mannitol, 5-HTP and the dipeptides Gly-Ala, Gly-Pro and Gly-Phe did not evoke currents. In Caco-2 cell monolayers, the apical uptake of 30 mM Gly-Sar was inhibited by 20 and 22% in the presence of 5-HTP or Bip-Pro, respectively, and by 48% in the presence of both.

CONCLUSION AND IMPLICATIONS

Our results suggest that whereas Gly-Gly amid bond bioisosteres are widely accepted by the hPAT1 carrier, dipeptides in general are not; and therefore, Gly-Sar might structurally define the size limit of dipeptide transport via SLC36A1.