Involvement of ETA and ETB receptors in the activation of phospholipase D by endothelins in cultured rat cortical astrocytes

1. This study was performed to characterize the receptor subtypes involved in the endothelin stimulation of phospholipase D (PLD) in rat cortical astrocytes in primary culture. PLD activity was determined by measuring the formation of [³²P]phosphatidylbutanol in [³²P]orthophosphate prelabelled cells stimulated in the presence of 25 mM butanol.
2. The agonists endothelin-1 (ET-1), endothelin-3 (ET-3), sarafotoxin 6c (S6c) and IRL 1620 elicited PLD activation in a concentration-dependent manner. The potencies of ET-1, ET-3 and S6c were similar. The maximal effects evoked by the ET_B-preferring agonists, ET-3, S6c and IRL 1620, were significantly lower than the maximal response to the non-selective agonist ET-1.
3. The response to 1 nM ET-1 was inhibited by increasing concentrations of the ET_A receptor antagonist BQ-123 in a biphasic manner. A high potency component of the inhibition curve (24.2±3.5% of the ET-1 response) was defined at low (up to 1 μM) concentrations of BQ-123, yielding an estimated K_i value for BQ-123 of 21.3±2.5 nM. In addition, the presence of 1 μM BQ-123 significantly reduced the maximal response to ET-1 but did not change the pD₂ value.
4. Increasing concentrations of the ET_B selective antagonist BQ-788 inhibited the S6c response with a K_i of 17.8±0.8 nM. BQ-788 also inhibited the effect of ET-1, although, in this case, two components were defined, accounting for approximately 50% of the response, and showing K_i values of 20.9±5.1 nM and 439±110 nM, respectively. The ET-1 concentration-response curve was shifted to the right by 1 μM BQ-788, also revealing two components. Only one of them, corresponding to 69.8±4.4% of the response, was sensitive to BQ-788 which showed a K_i value of 28.8±8.9 nM.
5. Rapid desensitization was achieved by preincubation with ET-1 or S6c. In cells pretreated with S6c neither ET-3 nor S6c activated PLD, but ET-1 still induced approximately 40% of the response shown by non-desensitised cells. This remaining response was insensitive to BQ-788, but fully inhibited by BQ-123.
6. In conclusion, endothelins activate PLD in rat cortical astrocytes acting through both ET_A and ET_B receptors, and this response desensitizes rapidly in an apparently homologous fashion. The percentage contribution of ET_A and ET_B receptors to the ET-1 response was found to be approximately 20% and 80%, respectively, when ET_B receptors were not blocked, and 30–50% and 50–70%, respectively, when ET_B receptors were inhibited or desensitized. These results may be relevant to the study of a possible role of PLD in the proliferative effects shown by endothelins on cultured and reactive astrocytes.

     

Essential role for endothelin ETB receptors in fever induced by LPS (E. coli) in rats

1. The influence of endothelin receptor antagonists on febrile responses to E. coli lipopolysaccharide (LPS), interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α) and endothelin-1 (ET-1) was assessed in conscious rats.
2. Intravenous (i.v.) LPS (5.0 μg kg⁻¹) markedly increased rectal temperature to a peak of 1.30°C over baseline at 2.5 h. Pretreatment with the mixed endothelin ET_A/ET_B receptor antagonist bosentan (10 mg kg⁻¹, i.v.) or the selective endothelin ET_B receptor antagonist BQ-788 (N-cis-2,6-dimethyl-piperidinocarbonyl-L-γ-methylleucyl-D-1-methoxycarboyl-D-norleucine; 3 pmol, into a lateral cerebral ventricle–i.c.v.) reduced the peak response to LPS to 0.90 and 0.75°C, respectively. The selective endothelin ET_A receptor antagonist BQ-123 (cyclo[D-Trp-D-Asp-Pro-D-Val-Leu]; 3 pmol, i.c.v.) was ineffective.
3. Increases in temperature caused by IL-1β (180 fmol, i.c.v.), TNF-α (14.4 pmol, i.c.v.) or IL-1β (150 pmol kg⁻¹, i.v.) were unaffected by BQ-788 (3 pmol, i.c.v.).
4. Central injection of endothelin-1 (0.1 to 3 fmol, i.c.v.) caused slowly-developing and long-lasting increases in rectal temperature (starting 2 h after administration and peaking at 4–6 h between 0.90 and 1.15°C) which were not clearly dose-dependent. The response to endothelin-1 (1 fmol, i.c.v.) was prevented by BQ-788, but not by BQ-123 (each at 3 pmol, i.c.v.). Intraperitoneal pretreatment with the cyclo-oxygenase inhibitor indomethacin (2 mg kg⁻¹), which partially reduced LPS-induced fever, did not modify the hyperthermic response to endothelin-1 (3 fmol, i.c.v.).
5. Therefore, central endothelin(s) participates importantly in the development of LPS-induced fever, via activation of a prostanoid-independent endothelin ET_B receptor-mediated mechanism possibly not situated downstream from IL-1β or TNF-α in the fever cascade.

     

Elevated plasma endothelin-1 level in streptozotocin-induced diabetic rats and responsiveness of the mesenteric arterial bed to endothelin-1

1. Both the plasma endothelin-1 (ET-1) levels and the plasma glucose levels were markedly elevated in streptozotocin (STZ)-induced diabetic rats.
2. The maximum contractile response of the mesenteric arterial bed to ET-1 was significantly reduced, and the vasodilatation induced by the ET_B-receptor agonist IRL-1620 in the mesenteric arterial bed was significantly reduced in STZ-induced diabetic rats.
3. ET-1 (10⁻⁸ M) caused a transient vasodilatation followed by a marked vasoconstriction in methoxamine-preconstricted mesenteric arterial beds. The ET-1-induced vasodilatation was significantly larger in beds from diabetic rats than in those from age-matched controls. By contrast, the ET-1-induced vasoconstriction was significantly smaller in STZ-induced diabetic rats than in the controls.
4. Both removal of the endothelium with Triton X-100 and preincubation with BQ-788 (10⁻⁶ M) (ET_B-receptor antagonist) abolished the ET-1-induced vasodilatation. Preincubation with BQ-485 (10⁻⁶ M) or BQ-123 (3×10⁻⁶) (ET_A-receptor antagonist) significantly augmented the ET-1-induced vasodilatation in control mesenteric arterial beds, but not that in beds from diabetic rats.
5. These results demonstrate that marked increases not only in plasma glucose, but also in plasma ET-1 occur in STZ-induced diabetic rats. We suggest that the decreased contractile response and the increased vasodilator response of the mesenteric arterial bed to ET-1 may both be due to desensitization of ET_A receptors, though ET_B receptors may also be desensitized. This desensitization may result from the elevation of the plasma ET-1 levels seen in STZ-induced diabetic rats.

     

Characterization of the endothelin receptor subtype mediating epithelium-derived relaxant nitric oxide release from guinea-pig trachea

1. The endothelin (ET) receptor subtype that mediates niric oxide (NO)-dependent airway relaxation in tracheal tube preparations precontracted with carbachol and pretreated with indomethacin was investigated. The release of NO induced by ET from guinea-pig trachea using a recently developed porphyrinic microsensor was also measured.
2. ET-1 (1 pM–100 nM) contracted tracheal tube preparations pretreated with the NO-synthase inhibitor, L-NMMA, and relaxed, in an epithelium-dependent manner, preparations pretreated with the inactive enantiomer D-NMMA. The effect of L-NMMA was reversed by L-Arg, but not by D-Arg.
3. The selective ET_B receptor agonists, IRL 1620 or sarafotoxin S6c, both (1 pM–100 nM) contracted tracheal tube preparations in a similar manner either after treatment with D-NMMA or with L-NMMA. In the presence of the ET_A receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (10 μM), ET-1 administration resulted in a contraction that was similar after either L-NMMA or D-NMMA. In the presence of the ET_B receptor antagonist, BQ788 (1 μM), ET-1 relaxed and contracted tracheas pretreated with D-NMMA and L-NMMA, respectively.
4. Exposure of tracheal segments to ET-1 (1–1000 nM) caused a concentration-dependent increase in NO release that was reduced by L-NMMA. IRL1620 (1 μM) did not cause any significant NO release. {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (10 μM), but not, BQ788 (1 μM), inhibited the NO release induced by ET-1.
5. These results demonstrate that in the isolated guinea-pig trachea activation of ET_B receptors results in a contractile response, whereas activation of ET_A receptors cause both a contraction, and an epithelium-dependent relaxation that is mediated by NO release.

     

Role of endothelin receptors,calcium and nitric oxide in the potentiation by endothelin-1 of the sympathetic contraction of rabbit ear artery during cooling

1. To examine further the potentiation by endothelin-1 on the vascular response to sympathetic stimulation, we studied the isometric response of isolated segments, 2 mm long, from the rabbit central ear artery to electrical field stimulation (1–8 Hz), under different conditions, at 37°C and during cooling (30°C).
2. Electrical stimulation produced frequency-dependent contraction, which was reduced (about 63% for 8 Hz) during cooling. At 30°C, but not at 37°C, endothelin-1 (1, 3 and 10 nM) potentiated the contraction to electrical stimulation in a dose-dependent way (from 43±7% to 190±25% for 8 Hz).
3. This potentiation by endothelin-1 was reduced by the antagonist for endothelin ET_A receptors BQ-123 (10 μM) but not by the antagonist for endothelin ET_B receptors BQ-788 (10 μM). The agonist for endothelin ET_B receptors IRL-1620 (0.1 μM) did not modify the contraction to electrical stimulation.
4. The blocker of L-type Ca²⁺ channels verapamil (10 μM l⁻¹) reduced (about 72% for 8 Hz) and the unspecific blocker of Ca²⁺-channels NiCl₂ (1 mM) practically abolished (about 98%), the potentiating effects of endothelin-1 found at 30°C.
5. Inhibition of nitric oxide synthesis with N^G-nitro-L-arginine (L-NOARG, 0.1 mM) increased the contraction to electrical stimulation at 30°C more than at 37°C (for 8 Hz, this increment was 297±118% at 30°C, and 66±15% at 37°C). Endothelium removal increased the contraction to electrical stimulation at 30°C (about 91% for 8 Hz) but not at 37°C. Both L-NOARG and endothelium removal abolished the potentiating effects of endothelin-1 on the response to electrical stimulation found at 30°C.
6. These results in the rabbit ear artery suggest that during cooling, endothelin-1 potentiates the contraction to sympathetic stimulation, which could be mediated at least in part by increasing Ca²⁺ entry after activation of endothelin ET_A receptors. This potentiating effect of endothelin-1 may require the presence of an inhibitory tone due to endothelial nitric oxide.

     

Pharmacological characterization of endothelin-induced rat pulmonary arterial dilatation

1. The aim of study was to characterize endothelin (ET)-induced vasodilatation in isolated extrapulmonary rat arteries (EPA) and in intrapulmonary arteries (IPA) preconstricted with 1 μM phenylephrine.
2. The ET-3 (1 nM–100 nM)- and ET-1 (10 nM–100 nM)-induced transient vasodilatations in EPA were more potent than those in IPA. The vasodilatation induced by ET-3 (100 nM) was larger than that induced by ET-1 (100 nM).
3. Both the ET_B antagonist, BQ788 (3 μM) and or endothelium denudation, but not the ET_A antagonist, BQ123 (3 μM), abolished the vasodilatation induced by ET-1 or ET-3 (100 nM each) in EPA and in IPA. The ATP-sensitive K+channel blocker, glibenclamide (20 μM) and the nitric oxide synthase inhibitor, N^G-monomethyl-L-arginine (L-NMMA, 1 mM) suppressed the ET-induced vasodilatation in EPA and in IPA.
4. We conclude that the vasodilatation induced by endothelins is markedly reduced in rat isolated IPA, and suggest that the endothelial ET_B-mediated vasodilatation varies depending on rat pulmonary arterial regions. Furthermore, ET_B-mediated vasodilatation involves activation of ATP-sensitive K⁺ channels and of nitric oxide synthase in rat isolated EPA and IPA.

     

Desensitization of ETA endothelin receptor-mediated negative chronotropic response in right atria–species difference and intracellular mechanisms

1. Desensitization of ET_A endothelin receptor (ET_AR) was compared between the rat and guinea-pig with regard to negative chronotropic response (NC) in the right atria (RA).
2. ET-1 (100 nM) produced distinct NC in the presence of BQ788 (300 nM), and positive chronotropic response (PC) in the presence of BQ123 (1 μM) in both species, showing that ET_AR and ET_B endothelin receptor (ET_BR) mediate NC and PC, respectively.
3. Repetitive applications of ET-1 (50 nM) desensitized PC, and the second application only induced a strong NC in both species. Later applications of ET-1 produced virtually no response in the rat RA, whereas they produced BQ123-sensitive NCs repetitively in guinea-pig RA, exhibiting marked species difference in desensitization of ET_AR-mediated NC.
4. Pretreatment with staurosporine (100 nM) prevented desensitization of ET_AR in the rat RA altogether. However, phorbol 12-myristate 13-acetate (PMA, 300 nM) failed to induce, but rather hampered, desensitization of ET_AR.
5. Partial amino acid sequencing of ET_ARs, spanning from the 2nd through the 4th intracellular loops, revealed that all the potential Ser/Thr phosphorylation sites, including a protein kinase C (PKC) site, are conserved among guinea-pigs, rats, rabbits, bovines and humans.
6. In guinea pig RA, pretreatment with okadaic acid (1 μg ml⁻¹) and PMA did not facilitate desensitization of ET_AR whereas these agents successfully desensitized ET_AR during combined stimulation of β-adrenoceptor and ET_AR by isoproterenol (300 nM) and ET-1 (100 nM).
7. These results suggest that species differences in desensitization of ET_AR are not caused by differences in the site(s) of, but caused by differences in the environment for phosphorylation of the receptor. Desensitization of ET_AR appears to require phosphorylation of the receptor by PKC as well as a kinase stimulated by β-adrenoceptor activation.

     

Endothelin converting enzyme (ECE) activity in human vascular smooth muscle

1. We have characterized the human smooth muscle endothelin converting enzyme (ECE) present in the media of the endothelium-denuded human umbilical vein preparation.
2. Endothelin-1 (ET-1) and ET-2 were potent constrictors of umbilical vein with EC₅₀ values of 9.2 nM and 29.6 nM, respectively. ET-1 was at least 30 times more potent than ET-3 suggesting the presence of constrictor ET_A receptors. Little or no response was obtained to the ET_B-selective agonist sarafotoxin 6c. These data suggest that endothelin-mediated vasoconstriction is via ET_A receptors in this preparation.
3. Autoradiographical visualization of endothelin receptors with subtype selective ligands confirmed the predominance of the ET_A receptor in the media of umbilical vein. High density of binding was obtained with the ET_A selective [¹²⁵I]-PD151242, with much lower levels detected with the ET_B selective [¹²⁵I]-BQ3020.
4. Big ET-1 (EC₅₀=42.7 nM) and big ET-2_(1-38) (EC₅₀=99.0 nM) were less potent than ET-1 and ET-2, respectively. Big ET-2_(1-38) was more potent than its isoform big ET-2_(1-37) with concentration–response curves to big ET-2_(1-37) incomplete at 300 nM. No response was obtained to big ET-3 at concentrations up to 700 nM. The C-terminal fragments, big ET-1_(22-38) and big ET-2_(22-38) were inactive.
5. Responses to ET-1 were unaffected by either the neutral endopeptidase (NEP) inhibitor thiorphan (10⁻⁵ M) or by the dual NEP/ECE inhibitor phosphoramidon (10⁻⁵ M). Big ET-1 was also unaffected by thiorphan but antagonized in a concentration-dependent manner by phosphoramidon (10⁻⁵ M and 10⁻⁴ M).
6. Addition of all four big endothelin peptides to human umbilical vein preparations resulted in detectable amounts of ET-IR in the bathing medium. Therefore, although big ET-3 was functionally inactive this reflects the low potency of ET-3 at the ET_A receptor rather than the lack of ability of this smooth muscle ECE to convert big ET-3 to ET-3.
7. To conclude we have demonstrated the presence of a phosphoramidon-sensitive ECE on the smooth muscle layer of the human umbilical vein which can convert big ET-1, big ET-2_(1-37), big ET-2_(1-38) and big ET-3 to their mature biologically active forms. The precise subcellular localization of this enzyme and its physiological relevance remains to be determined.

     

Pharmacological characterization of endothelin-induced contraction in the guinea-pig oesophageal muscularis mucosae

1. In the oesophageal muscularis mucosae, we examined the effects of endothelin-1 (ET-1), endothelin-2 (ET-2), endothelin-3 (ET-3) and sarafotoxin S6c (SX6c) as agonists, and {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317, BQ-123 and RES-701-1 as endothelin receptor antagonists.
2. All of the endothelins produced tonic contractions which were frequently superimposed on rhythmic motility in a concentration-dependent manner. The order of potency (−log EC₅₀) was ET-1 (8.61)=SX6c (8.65)>ET-2 (8.40)>ET-3 (8.18).
3. {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (1–3 μM) and BQ-123 (1 μM) caused parallel rightward shifts of the concentration-response curve to ET-1, but at higher concentrations caused no further shift. RES-701-1 (3 μM) caused a rightward shift of the concentration-response curve to ET-1, while RES-701-1 (10 μM) had no additional effect. RES-701-1 (0.1–1 μM) concentration-dependently caused a rightward shift of the concentration-response curve to SX6c. The contraction to ET-1 (10 nM) in preparations desensitized to the actions of SX6c was greatly inhibited by pretreatment with {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (10 μM).
4. Modulation of the Ca²⁺ concentration in the Krebs solution caused the concentration-response curve to ET-1 or SX6c to shift to the right and downward as external Ca²⁺ concentrations decreased. Verapamil (30 μM) abolished rhythmic motility induced by ET-1 or SX6c. Ni²⁺ (0.1 mM) weakly inhibited ET-1- or SX6c-induced tonic contraction. SK&F 96365 (60 μM) completely inhibited ET-1-induced contractions.
5. We conclude that there are two types of ET-receptors, excitatory ET_A- and ET_B-receptors in the oesophageal muscularis mucosae. These receptors mediate tonic contractions predominantly by opening receptor-operated Ca²⁺ channels (ROCs) and partly by opening T-type Ca²⁺ channels, and mediate rhythmic motility by opening L-type Ca²⁺ channels.

     

Endothelin receptors mediating contraction of rat and human pulmonary resistance arteries: effect of chronic hypoxia in the rat

1. We examined the endothelin (ET) receptors mediating contractions to ET-1, ET-3 and sarafotoxin S6c (SX6c) in rat pulmonary resistance arteries by use of peptide and non-peptide ET receptor antagonists. Changes induced by pulmonary hypertension were examined in the chronically hypoxic rat. The effect of the mixed ET_A/ET_B receptor antagonist SB 209670 on endothelin-mediated contraction was also examined in human pulmonary resistance arteries.
2. In rat vessels, the order of potency for the endothelin agonists was SX6c=ET-3>ET-1 (pEC₅₀ values in control rats: 9.12±0.10, 8.76±0.14 and 8.12±0.04, respectively). Maximum contractions induced by ET-3 and ET-1 were increased in vessels from chronically hypoxic rats.
3. The ET_A receptor antagonist FR 139317 (1 μM) had no effect on the potency of ET-1 in any vessel studied but abolished the increased response to ET-1 in the chronically hypoxic vessels. The ET_A receptor antagonist BMS 182874 (1 μM) increased the potency of ET-1 in control rat vessels without effecting potency in the pulmonary hypertensive rat vessels.
4. Bosentan (non-peptide mixed ET_A/ET_B receptor antagonist) increased the potency of ET-1 in control rat vessels but was without effect in the pulmonary hypertensive rat vessels. Bosentan (1 μM) inhibited responses to SX6c in control and chronically hypoxic rat vessels with pK_b values of 5.84 and 6.11, respectively. The ET_B receptor antagonist BQ-788 (1 μM) did not inhibit responses to ET-1 in any vessel tested but did inhibit responses to both SX6c and ET-3 (pK_b values in control and chronically hypoxic rat vessels respectively: SX6c 7.15 and 7.22; ET-3: 6.68 and 6.89). BQ-788 (1 μM) added with BMS 182874 (10 μM) did not inhibit responses to ET-1 in control vessels but caused a significant inhibition of responses to ET-1 in chronically hypoxic preparations.
5. SB 209670 inhibited responses to ET-1 in both control and chronically hypoxic vessels with pK_b values of 7.36 and 7.39, respectively. SB 209670 (0.1 and 1 μM) virtually abolished responses to ET-1 in the human pulmonary resistance artery.
6. In conclusion, in rat pulmonary resistance arteries, vasoconstrictions induced by ET-1, SX6c and ET-3 are mediated predominantly by activation of an ET_B–like receptor. However, lack of effect of some antagonists on ET-1 induced vasoconstriction suggests that ET-1 stimulates an atypical ET_B receptor. The increase in potency of ET-1 in the presence of some antagonists suggests the presence of an inhibitory ET_A-like receptor. The influence of this is reduced, or absent, in the chronically hypoxic rats. Increased responses to ET-1 are observed in the chronically hypoxic rat and may be mediated by increased activation of ET_A receptors. SB 209670 is unique in its potency against responses to ET-1 in both control and chronically hypoxic rats, as well as human, isolated pulmonary resistance arteries.

     

Development of endothelin receptors in perinatal rabbit pulmonary resistance arteries

1. Contractile responses to endothelin-1 (ET-1) and sarafotoxin S6c (S6c) were studied in pulmonary resistance arteries (∼320 μm i.d.) from fetal, 0–24 h, 4 day and 7 day rabbits. The effects of the ET_A-selective antagonist {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317, the selective ET_B receptor antagonist BQ-788 and the non-selective ET_A/ET_B receptor antagonist SB 209670, on these responses, were determined. Acetylcholine-induced vasodilation and noradrenaline-evoked contractions were also examined.
2. ET-1 potency was in the following order (pEC₅₀ values): fetal (8.7) = 0–24 h (8.8) = 4 day (8.6) > 7 day (8.0). The order of potency for S6c was 7 days (11.1) = 4 days (10.8) >0–24 h (9.7) > fetal (8.6). Hence, S6c and ET-1 were equipotent in the fetus but S6c was increasingly more potent than ET-1 with increasing age, being some 1000 times more potent by 7 days. By 7 days, responses to ET-1 were also resistant to both {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 and BQ-788. {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 inhibited responses to ET-1 in vessels from 0–24 h and 4 day, but not fetal, rabbits (pK_b: 6.4 in 4 day rabbits). BQ-788 inhibited responses to ET-1 at all age points except for 7 days (pK_b: 6.7 at 0–24 h; 6.2 at 4 days). BQ-788 inhibited responses to S6c at all age points (pK_b: 8.5 at 4 days). SB 209670 inhibited responses to ET-1 and S6c at 0–24 h and 4 days (pK_b for ET-1: 8.3 and 8.0 respectively; pK_b for S6c: 9.2 and 10.2 respectively).
3. Acetylcholine (1 μM) induced vasodilation at all age points (inhibited by 100 μM L-N^ω-nitroarginine methylester) although the degree of vasodilation was significantly reduced (∼75%) at 0–24 h. Noradrenaline induced contraction at all age points except 7 days and its response was significantly enhanced at 0–24 h.
4. Over the first week of life, the potency of S6c increases whilst that to ET-1 decreases suggesting differential development of responses to ET-1 and S6c and heterogeneity of ET_A- or `ET_B-like'' receptor-mediated responses. There is no synergism between ET_A and ET_B receptors at birth but this is established by 7 days. Immediately after birth rabbit Pulmonary Resistance Arteries are hyperresponsive to ET-1 and noradrenaline but exhibit impaired nitric-oxide dependent vasodilation.

     

Pharmacologically distinct GABAB receptors that mediate inhibition of GABA and glutamate release in human neocortex

1. The release of endogenous γ-aminobutyric acid (GABA) and glutamic acid in the human brain has been investigated in synaptosomal preparations from fresh neocortical samples obtained from patients undergoing neurosurgery to reach deeply located tumours.
2. The basal outflows of GABA and glutamate from superfused synaptosomes were largely increased during depolarization with 15 mM KCl. The K⁺-evoked overflows of both amino acids were almost totally dependent on the presence of Ca²⁺ in the superfusion medium.
3. The GABA_B receptor agonist (−)-baclofen (1, 3 or 10 μM) inhibited the overflows of GABA and glutamate in a concentration-dependent manner. The inhibition caused by 10 μM of the agonist ranged from 45–50%.
4. The effect of three selective GABA_B receptor antagonists on the inhibition of the K⁺-evoked GABA and glutamate overflows elicited by 10 μM (−)-baclofen was investigated. Phaclofen antagonized (by about 50% at 100 μM; almost totally at 300 μM) the effect of (−)-baclofen on GABA overflow but did not modify the inhibition of glutamate release. The effect of (−)-baclofen on the K⁺-evoked GABA overflow was unaffected by 3-amino-propyl (diethoxymethyl)phosphinic acid (CGP 35348; 10 or 100 μM); however, CGP 35348 (10 or 100 μM) antagonized (−)-baclofen (complete blockade at 100 μM) at the heteroreceptors on glutamatergic terminals. Finally, [3-[[(3,4-dichlorophenyl) methyl]amino]propyl] (diethoxymethyl) phosphinic aid (CGP 52432), 1 μM, blocked the GABA_B autoreceptor, but was ineffective at the heteroreceptors. The selectivity of CGP 52432 was lost at 30 μM, as the compound, at this concentration, inhibited completely the (−)-baclofen effect both on GABA and glutamate release.
5. It is concluded that GABA and glutamate release evoked by depolarization of human neocortex nerve terminals can be affected differentially through pharmacologically distinct GABA_B receptors.

     

Renal vascular effects of the selective endothelin receptor antagonists in anaesthetized rats

1. Endothelin (ET) is a potent vasoconstrictor peptide which has been shown to have an important role in the regulation of systemic and renal haemodynamics. In order to elucidate the role of endogenous ET in the kidney, we examined the effects of ET receptor antagonists on systemic and renal vasculature in normotensive anaesthetized rats.
2. Intravenous injection of a selective ET_A receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (0.5 μmol kg⁻¹, for 20 min) induced a very small fall in blood pressure. Similarly, a non-selective ET_A/ET_B receptor antagonist, TAK-044 (12.5 μmol kg⁻¹, for 20 min) slightly decreased blood pressure. A selective ET_B receptor antagonist, BQ-788 (0.5 μmol kg⁻¹, for 20 min) had no effect on blood pressure.
3. {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 and TAK-044 did not affect renal blood flow or calculated renal vascular resistance. In contrast, BQ-788 significantly reduced renal blood flow by 18.2±2.4% and increased renal vascular resistance. Furthermore, the renal vascular action of BQ-788 was not observed when combined with {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317.
4. Pretreatment with a nitric oxide (NO) synthase inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME, 37 μmol kg⁻¹, i.v.) and a cyclo-oxygenase inhibitor ibuprofen (44 μmol kg⁻¹, i.v.) completely abolished the BQ-788-mediated renal vasoconstriction.
5. These results indicate that activation of ET_B receptors by endogenous ET acts as a physiological brake for the ET_A-mediated renal vasoconstriction; this effect appears to be mediated by stimulation of NO and/or vasodilator prostaglandin(s) release.

     

Potentiation by vasopressin of adrenergic vasoconstriction in the rat isolated mesenteric artery

1. The aim of the present study was to investigate in rat mesenteric artery rings whether low concentrations of vasopressin could modify the contractile responses to noradrenaline and electrical stimulation of perivascular nerves.
2. Vasopressin (10⁻¹⁰–10⁻⁷ M) caused concentration-dependent contractions (pD₂=8.36±0.09). The V₁-receptor antagonist d(CH₂)₅Tyr(Me)AVP (10⁻⁹–10⁻⁸ M) produced parallel rightward shifts of the control curve for vasopressin. Schild analysis yielded a pA₂ value of 9.83 with a slope of 1.10±0.14.
3. Vasopressin (3×10 ⁻¹⁰ and 10⁻⁹ M) caused concentration-dependent potentiation of the contractions elicited by electrical stimulation (2–8 Hz; 0.2 ms duration for 30 s) and produced leftward shifts of the concentration-response curve for noradrenaline. The V₁-receptor antagonist induced concentration-dependent inhibitions of potentiation induced by vasopressin. The selective V₁-receptor agonist [Phe^*, Orn⁸]-vasotocin (3×10 ⁻¹⁰ and 10⁻⁹ M) induced potentiation of electrical stimulation-evoked responses which was also inhibited in the presence of the V₁ antagonist (10⁻⁸ M). In contrast, the V₂-receptor agonist deamino-8-D-arginine vasopressin (desmopressin 10⁻⁸–10⁻⁷ M) did not modify the electrical stimulation-induced responses and the V₂-receptor antagonist [d(CH₂)₅, D-Ile^*, Ile⁴, Arg⁸]-vasopressin (10⁻⁸–10⁻⁷ M) did not affect the potentiation evoked by vasopressin.
4. In artery rings contracted by 10⁻⁶ M noradrenaline in the presence of 10⁻⁶ M guanethidine and 10⁻⁶ M atropine, electrical stimulation (2, 4 and 8 Hz) produced frequency-dependent relaxations which were unaffected by 10⁻⁹ M vasopressin but abolished by 10⁻⁶ M tetrodotoxin.
5. Vasopressin also potentiated contractions elicited by KCl and contractions induced by addition of CaCl₂ to KCl depolarized vessels. The augmenting effects were inhibited by the V₁ antagonist.
6. In the presence of the calcium antagonist nifedipine (10⁻⁶ M), vasopressin failed to enhance the contractile responses to electrical stimulation, noradrenaline and KCl.
7. The results demonstrate that low concentrations of vasopressin strongly potentiate the contractions to adrenergic stimulation and KCl depolarization. This effect appears to be mediated by V₁ receptor stimulation which brings about an increase in calcium entry through dihydropyridine-sensitive calcium channels.

     

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.

     

Endothelin receptor subtypes and their functional relevance in human small coronary arteries

1. The potent constrictor peptide endothelin (ET) has been implicated in various cardiovascular disorders including myocardial infarction and atherosclerosis. We have investigated the nature of ET receptor subtypes present on human small coronary arteries.
2. Small coronary arteries were mounted in a wire-myograph for in vitro pharmacology. To investigate the ET receptor subtypes present in different segments of the coronary vascular tree, arteries were grouped according to internal diameter. Responses in arteries with small internal diameters (mean 316.7±7.9 μm; Group B) were compared to those in larger arteries (mean 586.2±23.1 μm; Group A).
3. ET-1 consistently and potently contracted arteries from Group A and B, with EC₅₀ values of 1.7 (0.9–3.2) nM (n=15) and 2.3 (1.4–4.2) nM (n=14), respectively. No correlation was observed between ET-1 potency and internal diameter. The response to ET-1 was potently antagonized by the selective ET_A receptor antagonist PD156707 in both Group A and Group B, yielding pA₂ values of 8.60±0.12 (n=4–6) and 8.38±0.17 (n=4–6), respectively. Slopes from Schild regression were not significantly different from unity.
4. In contrast to ET-1, individual responses to ET-3 were variable. While all arteries from Group A responded to ET-3 (EC₅₀∼69 (23–210) nM) (n=12), no response was obtained in 5 of the 14 tested in Group B. Of those responding, many failed to reach a maximum at concentrations up to 1 μM. ET-1 was more potent than ET-3 in all arteries tested. A biphasic ET-3 response was observed in 8 arteries suggesting that a small ET_B population was also present in some patients. The selective ET_B receptor agonist sarafotoxin S6c had little or no effect up to 10 nM (n=4–6).
5. Responses to ET-1 and ET-3 were unaffected by removal of the endothelium in arteries from both groups suggesting a lack of functional, relaxant ET_B receptors on endothelial cells (n=5).
6. Using autoradiography, specific high density binding of the non-selective, ET_A/ET_B ligand [¹²⁵I]-ET-1 and selective ET_A ligand [¹²⁵I]-PD151242 was detected on the vascular smooth muscle layer of small intramyocardial coronary arteries (n=5). In contrast, little or no binding of the selective ET_B receptor ligand [¹²⁵I]-BQ3020 was observed (n=5). Similarly, [¹²⁵I]-ET-1 binding to vascular smooth muscle was absent in the presence of the selective ET_A receptor antagonist PD156707.
7. We conclude that human small epi- and intramyocardial coronary arteries express predominantly ET_A receptors and it is these receptors which mediate ET-induced contractions. A constrictor ET_B receptor population may exist in some patients. However, these receptors may have a limited role as contractions to ET-1 can be blocked fully by the selective ET_A receptor antagonist PD156707.

     

Further insights into the anti-aggregating activity of NMDA in human platelets

1. In the present study the effect of N-methyl-D-aspartate (NMDA) on thromboxane B₂ synthesis and on [Ca²⁺]_i was studied in human platelets.
2. NMDA (10⁻⁷ M) completely inhibited the synthesis of thromboxane B₂ from exogenous arachidonic acid (AA), while it did not interfere with the aggregating effect of the thromboxane A₂ receptor agonist U-46619.
3. NMDA (0.1 μM–10 μM) dose-dependently increased intracellular calcium in washed platelets pre-loaded with fura 2 AM, and this effect was not additive with that of AA.
4. NMDA shifted the dose-response curve of AA to the right. At the highest AA concentrations platelet aggregation was not inhibited.
5. The antiaggregating effect of NMDA was not antagonized by N^G-monomethyl-L-arginine (L-NMMA), a nitric oxide synthase (NOS) inhibitor.
6. Finally, NMDA (0.01 nM–100 nM) associated with either aspirin or indomethacin significantly potentiated the antiaggregating activity of both cyclo-oxygenase inhibitors.
7. It was concluded that NMDA is a potent inhibitor of platelet aggregation and thromboxane B₂ synthesis in human platelet rich plasma (PRP).

     

Antagonist properties of (?)-pindolol and WAY 100635 at somatodendritic and postsynaptic 5-HT1A receptors in the rat brain

1. The aim of the present work was to characterize the 5-hydroxytryptamine_1A (5-HT_1A) antagonistic actions of (−)-pindolol and WAY 100635 (N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexane carboxamide). Studies were performed on 5-HT_1A receptors located on 5-hydroxytryptaminergic neurones in the dorsal raphe nucleus (DRN) and on pyramidal cells in the CA1 and CA3 regions of the hippocampus in rat brain slices.
2. Intracellular electrophysiological recording of CA1 pyramidal cells and 5-hydroxytryptaminergic DRN neurones showed that the 5-HT_1A receptor agonist 5-carboxamidotryptamine (5-CT) evoked in both cell types a concentration-dependent cell membrane hyperpolarization and a decrease in cell input resistance. On its own, (−)-pindolol did not modify the cell membrane potential and resistance at concentrations up to 10 μM, but it antagonized the 5-CT effects in a concentration-dependent manner. Similar antagonism of 5-CT effects was observed in the CA3 hippocampal region. (−)-Pindolol also prevented the 5-HT_1A receptor-mediated hyperpolarization of CA1 pyramidal cells due to 5-HT (15 μM). In contrast, the 5-HT-induced depolarization mediated by presumed 5-HT₄ receptors persisted in the presence of 3 μM (−)-pindolol.
3. In the hippocampus, (−)-pindolol completely prevented the hyperpolarization of CA1 pyramidal cells by 100 nM 5-CT (IC₅₀=92 nM; apparent K_B=20.1 nM), and of CA3 neurones by 300 nM 5-CT (IC₅₀=522 nM; apparent K_B=115.1 nM). The block by (−)-pindolol was surmounted by increasing the concentration of 5-CT, indicating a reversible and competitive antagonistic action.
4. Extracellular recording of the firing rate of 5-hydroxytryptaminergic neurones in the DRN showed that (−)-pindolol blocked, in a concentration-dependent manner, the decrease in firing elicited by 100 nM 5-CT (IC₅₀=598 nM; apparent K_B=131.7 nM) or 100 nM ipsapirone (IC₅₀=132.5 nM; apparent K_B=124.9 nM). The effect of (−)-pindolol was surmountable by increasing the concentration of the agonist. Intracellular recording experiments showed that 10 μM (−)-pindolol were required to antagonize completely the hyperpolarizing effect of 100 nM 5-CT.
5. In vivo labelling of brain 5-HT_1A receptors by i.v. administration of [³H]-WAY 100635 ([O-methyl-³H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl-N-(2-pyridyl)cyclo-hexane-carboxamide) was used to assess their occupancy following in vivo treatment with (−)-pindolol. (−)-Pindolol (15 mg kg⁻¹) injected i.p. either subchronically (2 day-treatment before i.v. injection of [³H]-WAY 100635) or acutely (20 min before i.v. injection of [³H]-WAY 100635) markedly reduced [³H]-WAY 100635 accumulation in all 5-HT_1A receptor-containing brain areas. In particular, no differences were observed in the capacity of (−)-pindolol to prevent [³H]-WAY 100635 accumulation in the DRN and the CA1 and CA3 hippocampal areas.
6. Intracellular electrophysiological recording of 5-hydroxytryptaminergic DRN neurones showed that WAY 100635 prevented the hyperpolarizing effect of 100 nM 5-CT in a concentration-dependent manner (IC₅₀=4.9 nM, apparent K_B=0.25 nM). In CA1 pyramidal cells, hyperpolarization induced by 50 nM 5-CT was also antagonized by WAY 100635 (IC₅₀=0.80 nM, apparent K_B=0.28 nM).

     

Pharmacological characterization of CGRP receptors mediating relaxation of the rat pulmonary artery and inhibition of twitch responses of the rat vas deferens

1. CGRP receptors mediating vasorelaxation of the rat isolated pulmonary artery and inhibition of contractions of the rat isolated prostatic vas deferens were investigated using CGRP agonists, homologues and the antagonist CGRP_8-37.
2. In the pulmonary artery, human (h)α-CGRP-induced relaxation of phenylephrine-evoked tone was abolished either by removal of the endothelium or by N^G-nitro-L-arginine (10⁻⁵ M). The inhibitory effect of N^G-nitro-L-arginine was stereoselectively reversed by L- but not by D-arginine (10⁻⁴ M). Thus, CGRP acts via nitric oxide released from the endothelium.
3. In the endothelium-intact artery, hα-CGRP, hβ-CGRP and human adrenomedullin (10⁻¹⁰–3×10⁻⁷ M), dose-dependently relaxed the phenylephrine-induced tone with similar potency. Compared with hα-CGRP, rat amylin was around 50 fold less potent, while [Cys(ACM^2,7)] hα-CGRP (10⁻⁷–10⁻⁴ M) was at least 3000 fold less potent. Salmon calcitonin was inactive (up to 10⁻⁴ M).
4. Human α-CGRP_8-37 (3×10⁻⁷–3×10⁻⁶ M) antagonized hα-CGRP (pA₂ 6.9, Schild plot slope 1.2±0.1) and hβ-CGRP (apparent pK_B of 7.1±0.1 for hα-CGRP_8-37 10⁻⁶ M) in the pulmonary artery. Human β-CGRP_8-37 (10⁻⁶ M) antagonized hα-CGRP responses with a similar affinity (apparent pK_B 7.1±0.1). Human adrenomedullin responses were not inhibited by hα-CGRP_8-37 (10⁻⁶ M).
5. In the prostatic vas deferens, hα-CGRP, hβ-CGRP and rat β-CGRP (10⁻¹⁰–3×10⁻⁷ M) concentration-dependently inhibited twitch responses with about equal potency, while rat amylin (10⁻⁸–10⁻⁵ M) was around 10 fold less potent and the linear analogue [Cys(ACM^2,7)] hα-CGRP was at least 3000 fold weaker. Salmon calcitonin was inactive (up to 10⁻⁴ M).
6. The antagonist effect of hα-CGRP_8-37 (10⁻⁵–3×10⁻⁵) in the vas deferens was independent of the agonist, with pA₂ values against hα-CGRP of 6.0 (slope 0.9±0.1), against hβ-CGRP of 5.8 (slope 1.1±0.1), and an apparent pK_B value of 5.8±0.1 against both rat β-CGRP and rat amylin. Human β-CGRP_8-37 (3×10⁻⁵–10⁻⁴ M) competitively antagonized hα-CGRP responses (pA₂ 5.6, slope 1.1±0.2). The inhibitory effect of hα-CGRP on noradrenaline-induced contractions in both the prostatic and epididymal vas deferens was antagonized by hα-CGRP_8-37 (pA₂ 5.8 and 5.8, slope 1.0±0.2 and 1.0±0.3, respectively).
7. The effects of hα-CGRP and hα-CGRP_8-37 in both rat pulmonary artery and vas deferens were not significantly altered by pretreatment with peptidase inhibitors (amastatin, bestatin, captopril, phosphoramidon and thiorphan, all at 10⁻⁶ M). The weak agonist activity of [Cys(ACM^2,7)] hα-CGRP in the vas deferens was not increased by peptidase inhibitors.
8. These data demonstrate that two different CGRP receptors may exist in the rat pulmonary artery and vas deferens, a CGRP₁ receptor subtype in the rat pulmonary artery (CGRP_8-37 pA₂ 6.9), while the lower affinity for CGRP_8-37 (pA₂ 6.0) in the vas deferens is consistent with a CGRP₂ receptor.

     

Pharmacological modulation of GABAA receptor-mediated postsynaptic potentials in the CA1 region of the rat hippocampus

1. It is unclear whether GABA_A receptor-mediated hyperpolarizing and depolarizing synaptic potentials (IPSP_As and DPSP_As, respectively) are evoked by (a) the same populations of GABAergic interneurones and (b) exhibit similar regulation by allosteric modulators of GABA_A receptor function. We have attempted to address these questions by investigating the effects of (a) known agonists for presynaptic receptors on GABAergic terminals, and (b) a range of GABA_A receptor ligands, on each response.
2. The GABA uptake inhibitor NNC 05-711 (10 μM) enhanced whereas bicuculline (10 μM) inhibited both IPSP_As and DPSP_As.
3. (−)-Baclofen (5 μM), [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAGO; 0.5 μM), and carbachol (10 μM) caused substantial depressions (up to 99%) of DPSP_As that were reversed by CGP 55845A (1 μM), naloxone (10 μM) and atropine (5 μM), respectively. In contrast, 2-chloroadenosine (CADO; 10 μM) only slightly depressed DPSP_As. Quantitatively, the effect of each agonist was similar to that reported for IPSP_As.
4. The neurosteroid ORG 21465 (1–10 μM), the anaesthetic propofol (50–500 μM), the barbiturate pentobarbitone (100–300 μM) and zinc (50 μM) all enhanced DPSP_As and IPSP_As.
5. The benzodiazepine (BZ) agonist flunitrazepam (10–50 μM) and inverse agonist DMCM (1 μM) caused a respective enhancement and inhibition of both IPSP_As and DPSP_As. The BZω₁ site agonist zolpidem (10–30 μM) produced similar effects to flunitrazepam.
6. The anticonvulsant loreclezole (1–100 μM) did not affect either response.
7. These data demonstrate that similar populations of inhibitory interneurones can generate both IPSP_As and DPSP_As by activating GABA_A receptors that are subject to similar allosteric modulation.