Mechanisms operated by endothelin ETA and ETB receptors in the trigeminal ganglion contribute to orofacial thermal hyperalgesia induced by infraorbital nerve constriction in rats

Endothelins, acting through specific endothelin ET_A and/or ET_B receptors, participate in nociceptive processing in models of cancer, inflammatory and neuropathic pain. The present study investigated which cell types express endothelin receptors in the trigeminal ganglion, and the contribution of mechanisms mediated by endothelin ET_A and ET_B receptors to orofacial heat hyperalgesia induced by unilateral constriction of the infraorbital nerve (CION). Both receptor types were identified by immunohistochemistry in the trigeminal ganglion, ET_A receptors on small-sized non-myelinated and myelinated A-fibers and ET_B receptors on both satellite glial cells and small-sized non-myelinated neuronal cells. CION promoted ipsilateral orofacial heat hyperalgesia which lasted from Day 2 until Day 10 after surgery. Ongoing CION-induced heat hyperalgesia (on Day 4) was reduced transiently, but significantly, by systemic or local treatment with antagonists of endothelin ET_A receptors (atrasentan, 10 mg/kg, i.v.; or BQ-123, 10 nmol/lip), endothelin ET_B receptors (A-192621, 20 mg/kg, i.v.; or BQ-788, 10 nmol/ lip), or of both ET_A/ET_B receptors (bosentan, 10 mg/kg, i.v.; or BQ-123 plus BQ-788, each at 10 nmol/lip). On the other hand, CION-induced heat hyperalgesia was transiently abolished over the first 90 min following i.p. injection of morphine hydrochloride (2.5 mg/kg), but fully resistant to reversal by indomethacin (4 mg/kg, i.p.) or celecoxib (10 mg/kg, i.p.). Thus, heat hyperalgesia induced by CION is maintained, in part, by peripheral signaling mechanisms operated by ET_A and ET_B receptors. Endothelin receptors might represent promising therapeutic targets for the control of trigeminal neuropathic pain.     

Antigen-induced inflammatory mechanical hypernociception in mice is mediated by IL-18

There is pre-clinical evidence that therapies targeting IL-18 might be beneficial in controlling arthropathies, which are accompanied by hypernociception (nociceptor sensitization). In the present study, we addressed the hypernociceptive role of IL-18 in a model of antigen-induced inflammation in mice and its mechanisms. In naïve mice, the intraplantar injection of IL-18 induced dose- and time-dependent mechanical hypernociception, which was inhibited in IFN-γ deficient (−/−) mice, and by the pre-treatment with bosentan (dual endothelin [ET] receptor antagonist), BQ123 (ET_A receptor antagonist) or indomethacin (cyclooxygenase inhibitor). IL-18 hypernociception was unaffected in TNFR1^−/− mice or by the pre-treatment with sIL-15Rα (soluble form of IL-15 receptor), BQ788 (ET_B receptor antagonist) or guanethidine (sympathetic blocker). The ovalbumin (OVA) challenge-induced mechanical hypernociception in immunized mice was inhibited by the pre-treatment with anti-IL-18 antibody or in IL-18^−/− mice. Furthermore, IL-18 induced significant IFN-γ production in the paw skin of naïve mice. The OVA challenge-induced IFN-γ and ET-1 productions were inhibited in IL-18^−/− immunized mice, as well as ET-1 production in IFN-γ^−/− immunized mice. In addition, significant PGE₂ production was detected after IL-18 or ET-1 (via ET_A receptors) injection in naïve mice. Taken together with previous data, these results suggest that IL-18 plays a significant role in antigen-induced inflammatory hypernociception via the production of IFN-γ, ET-1 and PGE₂. Thus, IL-18 and IL-18-downstream mediators demonstrated herein might constitute targets to inhibit antigen-induced inflammatory pain.     

Effects of a new magnesium-rich artificial cerebrospinal fluid on contractile 5-hydroxytryptamine and endothelin receptors in rat cerebral arteries

ABSTRACT

Objectives Cerebral vasospasm after subarachnoid haemorrhage (SAH) is associated with cerebrovascular contractile receptor upregulation resulted from haemolysis in the subarachnoid space. This study developed a new magnesium-rich artificial cerebrospinal fluid (MACSF) formula and investigated its effects on receptor-mediated contraction in rat basilar arteries.

Methods Clear and haemorrhagic cerebrospinal fluid (CSF) were collected from patients with hydrocephalus or SAH. MACSF was freshly prepared using clinical intravenous injections. Rat basilar arteries were segmented and incubated with clear CSF, haemorrhagic CSF or MACSF. The contractile responses were studied by myograph. The messenger ribonucleic acid (mRNA) and protein expression of 5-hydroxytryptamine 1B (5-HT_1B), endothelin subtype B (ET_B) and endothelin subtype A (ET_A) receptors were evaluated by real-time polymerase chain reaction (PCR) and Western blot analyses.

Results Haemorrhagic CSF exposure shifted the contractile curves induced by 5-hydroxytryptamine (5-HT), sarafotoxins 6c (S6c) and endothelin-1 (ET-1) leftward with increased maximal contraction values. Furthermore, mRNA and protein expression were markedly elevated for 5-HT_1B, ET_B and ET_A receptors on arteries exposed to haemorrhagic CSF. However, the contractile responses to 5-HT, S6c or ET-1 and expression of 5-HT_1B, ET_B and ET_A receptors in rat cerebral arteries exposed to MACSF remained unaffected compared to those exposed to clear CSF. Besides, unlike normal saline which can inactive in-vitro vessels, MACSF can maintain their physiological activity.

Conclusion Haemorrhagic CSF induces upregulation of 5-HT_1B, ET_B and ET_A receptors in rat cerebral arteries. However, MACSF can maintain in-vitro rat basilar arteries in good physiological activity and normal expression of contractile 5-HT and ET receptors.     

ETA and ETB receptor antagonists synergistically increase extracellular endothelin-1 levels in primary rat astrocyte cultures

Astrocytes produce and bind endothelins (ETs), suggesting that these cells have ET autoregulatory and eliminatory functions. To further investigate these functions in primary rat astrocytes, ET-1 levels in the cell culture media (RIA/HPLC) and intracellular content of ET-1 mRNA (RT PCR) were measured under basal and stimulated (thrombin, 2.2 U/ml) conditions in the presence and absence of ET_A and ET_B selective antagonists (BQ123 or LU135252, and BQ788, respectively). Neither basal nor stimulated ET-1 levels in astrocyte media were influenced by ET_A or ET_B antagonists alone, but were significantly increased by a combination of both. ir ET-3 levels were not affected by antagonist treatment. Exogenous ET-1, added to the cultures, was rapidly cleared from the supernatant; this clearance was markedly inhibited by a combination of BQ123 and BQ788. ET-1 mRNA levels were not altered by any treatment. To conclude, in primary rat astrocyte cultures, extracellular ET-1 is cleared by binding to ET-receptors, apparently involving both, ET_A and ET_B sites. Thus, a blockade of the astrocytic ET eliminatory function as a consequence of the in vivo application of non-selective ET receptor antagonists may lead to increased extracellular ET levels in the brain.     

In vivo neurochemical evidence that stimulation of accumbal GABAA and GABAB receptors each reduce acetylcholine efflux without affecting dopamine efflux in the nucleus accumbens of freely moving rats

Cholinergic neurons in the nucleus accumbens contain GABA_A and GABA_B receptors that are thought to inhibit neural activity. We analyzed the roles of GABA_A and GABA_B receptors in regulating accumbal acetylcholine efflux of freely moving rats using in vivo microdialysis. The effects of GABA receptor ligands on the accumbal dopamine efflux were also analyzed because accumbal cholinergic and dopaminergic neurons could mutually interact. Drugs were applied intracerebrally through the dialysis probe. Doses of compounds indicate total amount administered (mol) during 30–60 min infusions. To monitor basal acetylcholine, a low concentration of physostigmine (50 nM) was added to the perfusate. GABA_A receptor agonist muscimol (3 and 30 pmol) induced a dose‐related decrease in accumbal acetylcholine. GABA_B receptor agonist baclofen (30 and 300 pmol) also produced a dose‐related decrease in acetylcholine. GABA_A receptor antagonist bicuculline (60 pmol) which failed to alter baseline acetylcholine counteracted the muscimol (30 pmol)‐induced decrease in acetylcholine. GABA_B receptor antagonist 2‐hydroxysaclofen (12 nmol) which failed to change baseline acetylcholine, counteracted the baclofen (300 pmol)‐induced decrease in acetylcholine. Neither muscimol (30 pmol) nor baclofen (300 pmol) which reduced accumbal acetylcholine altered baseline accumbal dopamine. Neither bicuculline (60 pmol) nor 2‐hydroxysaclofen (12 nmol) also affected the baseline dopamine. These results show that GABA_A and GABA_B receptors each exert inhibitory roles in the regulation of accumbal cholinergic neural activity. The present results also provides in vivo neurochemical evidence that stimulation of GABA_A and GABA_B receptors each reduce acetylcholine efflux without affecting dopamine efflux in the nucleus accumbens of freely moving rats.     

Endothelin A receptors mediate relaxation of guinea pig internal anal sphincter through cGMP pathway

Background Endothelin (ET) modulates motility of the internal anal sphincter through unclear receptor subtypes. Methods We measured relaxation of guinea pig internal anal sphincter strips caused by ET‐related peptides and binding of ¹²⁵I‐ET‐1 to cell membranes prepared from the internal anal sphincter muscle. Visualization of ¹²⁵I‐ET‐1 binding sites in tissue was performed by autoradiography. Key Results In the guinea pig internal anal sphincter, ET‐1 caused a marked relaxation insensitive to tetrodotoxin, atropine, or ω‐conotoxin GVIA. ET‐2 was as potent as ET‐1. ET‐3 caused a mild relaxation. The relative potencies for ETs to cause relaxation were ET‐1 = ET‐2 > ET‐3. The ET‐1‐induced relaxation was inhibited by BQ‐123, an ET_A antagonist, but not by BQ‐788, an ET_B antagonist. These indicate that ET_A receptors mediate the relaxation. The relaxant response of ET‐1 was attenuated by LY 83583, KT 5823, Rp‐8CPT‐cGMPS, tetraethyl ammonium, 4‐aminopyridine and N(omega)‐nitro‐l‐arginine, but not significantly affected by N^G‐nitro‐l‐arginine methyl ester, N^G‐methyl‐l‐arginine, charybdotoxin, apamin, KT 5720, and Rp‐cAMPS. These suggest the involvement of cyclic guanosine 3′,5′‐cyclic monophosphate (cGMP), and potassium channels. Autoradiography localized ¹²⁵I‐ET‐1 binding to the internal anal sphincter. Binding of ¹²⁵I‐ET‐1 to the cell membranes prepared from the internal anal sphincter revealed the presence of two subtypes of ET receptors, ET_A and ET_B receptors. Conclusions & Inferences Taken together, these results demonstrate that ET_A receptors mediate relaxation of guinea pig internal anal sphincter through the cGMP pathway.     

Dopamine D1and D2Receptor-Mediated Acute and Long-Lasting Behavioral Effects of Glial Cell Line-Derived Neurotrophic Factor Administered into the Striatum

To determine the differences in behavioral effects between intrastriatal and intracerebroventricular glial cell-derived neurotrophic factor (GDNF) administration, spontaneous locomotor activity was measured after intrastriatal or intracerebroventricular injection of GDNF (10 μg) in normal adult rats with implanted guide cannulae. In addition, the distribution of GDNF after intracerebral injection was studied immunohistochemically. Intrastriatal administration of GDNF significantly increased rearing behavior 3–4 h after injection. Increases in all three aspects of locomotor activity (motility, locomotion, and rearing) were most pronounced 3 days after intrastriatal injection, and they lasted for several days. This hyperactivity was blocked by the selective dopamine D₁receptor antagonist SCH22390 and by the selective D₂receptor antagonist raclopride at doses of the dopamine receptor antagonists, which by themselves did not affect spontaneous locomotor activity. These results suggest that GDNF has both acute and long-lasting pharmacological effects on dopamine neurons in adult animals and stimulates locomotor activity by activating both dopamine D₁and D₂receptors. On the other hand, intracerebroventricular administration of the same dose of GDNF failed to increase locomotor activity at any time during the test period (12 days). The immunohistochemical study demonstrated widespread distribution of GDNF in the entire body of the striatum within 24 h after intrastriatal injection. It also revealed deep penetration of GDNF from the ventricular space into the brain parenchyma after intracerebroventricular injection. GDNF-immunoreactive neuronal cell bodies were seen in the ipsilateral substantia nigra pars compacta most frequently 6 h after intrastriatal injection. The number of such cell bodies after intracerebroventricular administration, on the other hand, was much lower than that seen after intrastriatal administration. Taken together, these data suggest that intrastriatal administration of GDNF is an effective approach for affecting DA transmission. Long-lasting behavior effects are mediated via dopamine D1 and D2 receptors. Higher doses of GDNF would probably be needed using the intracerebroventricular route as compared to intraparenchymal delivery to exert effects on the nigrostriatal system in Parkinson's disease patients.     

Endothelin-1 induces LIMK2-mediated programmed necrotic neuronal death independent of NOS activity

Background

Recently, we have reported that LIM kinase 2 (LIMK2) involves programmed necrotic neuronal deaths induced by aberrant cyclin D1 expression following status epilepticus (SE). Up-regulation of LIMK2 expression induces neuronal necrosis by impairment of dynamin-related protein 1 (DRP1)-mediated mitochondrial fission. However, we could not elucidate the upstream effecter for LIMK2-mediated neuronal death. Thus, we investigated the role of endothelin-1 (ET-1) in LIMK2-mediated neuronal necrosis, since ET-1 involves neuronal death via various pathways.

Results

Following SE, ET-1 concentration and its mRNA were significantly increased in the hippocampus with up-regulation of ET_B receptor expression. BQ788 (an ET_B receptor antagonist) effectively attenuated SE-induced neuronal damage as well as reduction in LIMK2 mRNA/protein expression. In addition, BQ788 alleviated up-regulation of Rho kinase 1 (ROCK1) expression and impairment of DRP1-mediated mitochondrial fission in CA1 neurons following SE. BQ788 also attenuated neuronal death and up-regulation of LIMK2 expression induced by exogenous ET-1 injection.

Conclusion

These findings suggest that ET-1 may be one of the upstream effectors for programmed neuronal necrosis through abnormal LIMK2 over-expression by ROCK1.

     

GABAB receptors in the dorsomedial hypothalamus and heart rate in anesthetized rats

Previous studies have shown that: (1) activation of neurons in the dorsomedial hypothalamus (DMH) of the rat by blockade of local GABA_A receptors with bicuculline methiodide (BMI) elicits cardiovascular changes resembling those seen in experimental stress, including marked sympathetically-mediated tachycardia, and (2) inhibition of neurons in the same region by local microinjection of the GABA_A receptor agonist muscimol can virtually abolish stress-induced tachycardia. This study examined the possibility that GABA_B receptors exist in the neural circuitry of the DMH, and that stimulation of these receptors might suppress the cardiovascular response to local disinhibition with BMI. Microinjection of BMI 10 pmol into the DMH in urethane-anesthetized rats resulted in marked tachycardia with little or no effect on arterial pressure. Simultaneous injection of the GABA_B receptor agonist baclofen at doses of 2.5, 5.0 and 10 pmol produced dose-related suppression of BMI induced tachycardia. Coinjection of the GABA_B receptor antagonist 2-hydroxysaclofen 100 or 200 pmol had no significant effect on the heart rate response to BMI, but reversed the suppression elicited in the presence of baclofen. These findings indicate that (1) functional GABA_B receptors exist in the DMH, and (2) stimulation of these receptors inhibits the tachycardia resulting from blockade of local GABA_A receptors.     

Systemic hemodynamic and regional circulatory effects of centrally administered endothelin-1 are mediated through ETA receptors

Central endothelin (ET) has been implicated in the regulation of the cardiovascular system. The effect of intracerebroventricular (i.c.v.) administration of ET-1 or IRL 1620 (5, 15 and 45 ng) on the systemic hemodynamics and regional circulation was studied in anesthetized rats using a radioactive microsphere technique. Systemic hemodynamics and regional blood circulation were determined before (baseline) and at 30 min after the injection of each dose of ET-1 or IRL 1620. Administration of saline (5 μl, i.c.v.) did not produce any significant cardiovascular effects. The lower doses of ET-1 (5 and 15 ng) did not produce any significant effect on blood pressure (BP), heart rate (HR), cardiac output (CO), stroke volume (SV), total peripheral resistance (TPR) and regional blood circulation. However, the higher dose (45 ng) produced a transient rise (26%) followed by a sustained fall (48%) in BP. The decrease in BP was accompanied by significant decreases in CO (44%) and SV (39%), while HR and TPR were not affected. ET-1 (45 ng, i.c.v.) also produced a significant reduction in blood flow to the brain (75%), heart (49%), kidneys (66%), GIT (40%), portal system (52%) and musculo-skeletal system (38%), while blood flow to the skin was not affected. To determine pharmacological specificity of the central effects of ET-1, studies were performed in rats pretreated with BQ-123, a specific ET_A receptor antagonist. Pretreatment with BQ-123 (10 μg, i.c.v.), 15 min prior to the administration of ET-1, completely antagonized the systemic hemodynamic as well as the regional circulatory effects of ET-1 (45 ng, i.c.v.). In order to determine whether stimulation of central ET_B receptors produces any cardiovascular effects, studies were performed using IRL 1620, a specific ET_B receptor agonist. Administration of IRL 1620 (5, 15 and 45 ng, i.c.v.) did not produce any effect on systemic hemodynamics and regional blood circulation in rats. It is concluded that ET_A but not ET_B receptors are involved in the central cardiovascular actions of ET.     

A rat model of endothelin-3-induced middle cerebral artery occlusion with controlled reperfusion

Surge hyperemia and mechanical damage to the cerebrovascular endothelium may serve to exacerbate the neuropathological outcome in animal models of focal cerebral ischemia. We have modified an existing model of endothelin-1-induced middle cerebral artery (MCA) occlusion to enable controlled reperfusion without damage to the cerebral vasculature. Endothelin-1 (ET-1) and endothelin-3 (ET-3) were injected via a double-injection cannula into brain parenchyma adjacent to the MCA of anesthetized rats to produce focal cerebral ischemia. ET-1 and ET-3 produced large ischemic lesions that were restricted to those cortical and subcortical structures supplied by the MCA. The volume of ischemic damage produced by 100 pmol of ET-1 and ET-3 was similar. The endothelin-A (ET_A) receptor antagonist FR139317 (3 or 30 nmol) injected 10 min after ET-1 did not significantly alter the volume of damage. By contrast, the lesion produced by ET-3 was completely inhibited by FR139317 at the 10 min time-point. FR139317 partially attenuated the ET-3-induced lesion when administered 30 min post-occlusion, but injection 90 min following ET-3 produced a lesion not different to that produced by ET-3 alone. These findings were supported by laser Doppler flowmetry which determined FR139317 induces reperfusion when injected 10 or 90 min following ET-3. ET-3-induced MCA occlusion is therefore amenable to reversal by the ET_A receptor antagonist FR139317, and this model may offer a means to investigate the neuropathology of reperfusion without the procedure-related artifacts associated with some reperfusion models.     

Both ETA and ETB receptors are involved in mitogen‐activated protein kinase activation and DNA synthesis of astrocytes: study using ETB receptor‐deficient rats (aganglionosis rats)

Endothelin (ET) is known to be a potent mitogen in astrocytes. However, the contribution and signalling pathway of ET_A and/or ET_B receptor to the proliferation of astrocytes remain unclear. We investigated ET-induced DNA synthesis in astrocytes using ET_B receptor-deficient mutant rats (aganglionosis rats: sl/sl). Western blotting with anti-ET receptor subtype-specific antibodies and Scatchard analysis of binding revealed that ET_B receptor expression in astrocytes depended on gene dosage (+/+: sl/+: sl/sl = 2: 1: 0), whereas ET_A receptor expression was unchanged among the three genotypes. ET-1 (10 nm ) stimulated [³H]thymidine incorporation and mitogen-activated protein kinase (MAP kinase) activity not only in +/+ via both ET_A and ET_B receptors, but also in sl/sl astrocytes via ET_A receptor with about half the extent of those observed in +/+ astrocytes. Treatment with pertussis toxin (PTX) suppressed the ET-1-induced increases in the incorporation and MAP kinase activity in +/+, but not sl/sl astrocytes, indicating that the ET_B receptor-, but not the ET_A receptor-, mediated pathway to DNA synthesis involves PTX-sensitive G proteins, e.g. G_i and/or G_o (G_i/o). In +/+ astrocytes, ET-1 (1 nm ) stimulated cAMP accumulation, and the ET_B receptor-selective agonist IRL 1620 (1 nm ) suppressed 10 μm forskolin-induced cAMP accumulation, suggesting G_s coupling to the ET_A receptor and G_i/o coupling to the ET_B receptor. On the other hand, ET-1 did not increase cAMP accumulation in sl/sl astrocytes, although ET-1 (1 nm ) suppressed the forskolin-induced response, suggesting G_i/o coupling to the ET_A receptor. Our results suggest the possibility that the selectivity of G protein for ET_A receptor is changed from G_s to G_i/o in ET_B receptor-deficient astrocytes.     

Pharmacological involvement of the calcium channel blocker flunarizine in dopamine transmission at the striatum

Single intrastriatal microinjections of 25, 50 and 100nmol/microl of flunarizine in normal rats produced a dose-dependent turning behavior toward the injected side when they were challenged with apomorphine (1mg/kg, s.c). This effect was seen at 1, 3 and 7 days following administration of the high dose of flunarizine, but had subsided by 24h after administration of the intermediate dose; the low dose was ineffective. However, intrastriatal injection of the high dose of flunarizine resulted in a local lesion and thereafter this dose was not used. A similar dose-response relationship was determined for nifedipine, an L-type calcium channel antagonist. Injection of this antagonist did not result in apomorphine-elicited rotational behavior, reflecting its lack of antidopaminergic action. Intrastriatal injections of haloperidol (5microg/microl), an antagonist of dopamine D(2) receptors, or the sodium channel blocker lidocaine (40microg/microl), were given in order to compare their effects to those observed with flunarizine. Intracerebral injection of haloperidol produced ipsilateral turning in response to systemic administration of apomorphine given 60min after. The same response was obtained with the injection of apomorphine 10min after the injection of intracerebral lidocaine. This effect was no longer apparent 24h after the microinjection of haloperidol and 60min after the injection of lidocaine. In rats rendered hemiparkinsionian by lesioning the nigrostriatal pathway with 6OHDA, intrastriatal microinjection of flunarizine (50nmol/microl) significantly reduced apomorphine (0.2mg/kg, s.c.)-elicited turning behavior towards the non-lesioned side. These results suggest an antidopaminergic effect of flunarizine mediated by antagonistic action of post-synaptic striatal dopamine receptors. However, an action of the drug on sodium channels may not be ruled out. These studies offer additional supporting evidence for the induction or aggravation of extrapyramidal side-effects in patients receiving flunarizine.     

Expression of endothelin receptors and nitric oxide synthase in the brain of stroke-prone spontaneously hypertensive rats with cerebral apoplexy

Endothelin (ET) receptors, ET-1-like immunoreactivity and nitric oxide synthase (NOS) were examined in the brain of stroke-prone spontaneously hypertensive rats (SHRSPs) with cerebral apoplexy. Our receptor autoradiographic method with ¹²⁵I-ET-1 and unlabeled selective ligands for ET receptors revealed de novo expressions of ET_A and ET_B receptors in areas of neural lesions with cerebrovascular damage in SHRSPs. Immunohistochemical staining for ET-1 showed clear ET-1-like immunoreactivity in areas with highly expressed ET receptors. Histochemical studies on astrocytes and microglia suggested that these glial cells, aggregating in lesions, may carry ET receptors, ET-1-like immunoreactivity. Furthermore, NOS detected histochemically using an NADPH-diaphorase staining method was rich on glial cells in damaged areas of the brain in SHRSPs with cerebral apoplexy. Our data suggest the pathophysiological significance of glial ET_A and ET_B receptors, ET-1 and NOS in neural lesions of SHRSPs.     

The antidopaminergic action of S-20098 is mediated by benzodiazepine/GABAA receptors in the striatum

The naphthalenic compound S-20098, which is a melatonergic agonist, inhibits [³H]diazepam binding in striatal membranes. S-20098 also inhibits apomorphine-induced turning in 6-hydroxydopamine lesioned rats. This antidopaminergic effect is blocked by either intraperitoneal injection of the central-type benzodiazepine (BZ) antagonist, flumazenil, or intrastriatal injection of the GABA_A antagonist, bicuculline. These findings indicate that S-20098 can activate central-type BZ receptors, and its antidopaminergic action, like that of melatonin, involves a GABAergic mechanism in the striatum.     

Intrastriatal angiotensin II induces turning behaviour in 6-hydroxydopamine lesioned rats

In rats with unilateral 6-hydroxydopamine lesions in the nigrostriatal pathway, injection of Angiotensin II (2 nmol) into the unlesioned striatum elicited dose-related tight rotations ipsilateral to the lesion. This rotation was suppressed by coadministration of the Angiotensin AT₁ receptor antagonist, losartan (2 nmol), which had no significant effect when injected alone. Preadministration of the dopamine antagonist, haloperidol (2 mg/kg i.p.) completely blocked angiotensin II-induced turning at doses of 0.3–3 nmol, and partially at 10 nmol. These results further confirm the hypothesis that Ang II is intrinsically involved in modulating dopamine release in the striatum, an effect which is mediated predominantly by AT₁ receptors.     

Calcium-mediated endothelin signaling in C6 rat glioma cells

Endothelin induced intracellular Ca(2+)signaling was studied in C6 rat glial cells. Endothelins 1 and 3 increased transiently intracellular Ca(2+)concentration, endothelin 3 being less potent inducer. Dibutyryl-cAMP treated cells responded with less sensitivity. While BQ123, a specific endothelin A receptor antagonist, inhibited both endothelins induced response in proliferating cells, it failed to inhibit in dibutyryl-cAMP treated ones. IRL1620, a specific endothelin B receptor agonist, was devoid of any significant effect. Although re-stimulation by both endothelins after endothelin-1 did not cause any Ca(2+)oscillation, both endothelins evoked new Ca(2+)transient after endothelin-3 stimulation. Our findings suggest that endothelin induced Ca(2+)signaling is mediated probably through the receptor A in proliferating C6 cells. The lack of both BQ123 and IRL 1620 effect in dibutyryl-cAMP treated cells could be caused by an alteration of endothelin A receptor alone, by a change of receptor expression pattern, or by more complex postreceptor mechanism.     

Improvement of cold injury‐induced mouse brain edema by endothelin ETB antagonists is accompanied by decreases in matrixmetalloproteinase 9 and vascular endothelial growth factor‐A

Brain edema is a potentially fatal pathological state that often occurs after brain injuries such as ischemia and trauma. However, therapeutic agents that fundamentally treat brain edema have not yet been established. We previously found that endothelin ET_B receptor antagonists attenuate the formation and maintenance of vasogenic brain edema after cold injury in mice. In this study, the effects of ET_B antagonists on matrixmetalloproteinase (MMP)9 and vascular endothelial growth factor (VEGF)‐A expression were examined in the cold injury model. Cold injury was performed in the left brain of male ddY mice (5–6 weeks old) for the induction of vasogenic edema. Expression of MMP9 and VEGF‐A mRNA in the mouse cerebrum was increased by cold injury. Immunohistochemical observations showed that the MMP9 and VEGF‐A were mainly produced in reactive astrocytes in the damaged cerebrum. Intracerebroventricular administration of BQ788 (10 μg) or IRL‐2500 (10 μg) (selective ET_B antagonists) attenuated brain edema and disruption of the blood–brain barrier after cold injury. BQ788 and IRL‐2500 reversed the cold injury‐induced increases in MMP9 and VEGF‐A expression. The induction of reactive astrocytes producing MMP9 and VEGF‐A in the damaged cerebrum was attenuated by BQ788 and IRL‐2500. These results suggest that attenuations of astrocytic MMP9 and VEGF‐A expression by ET_B antagonists may be involved in the amelioration of vasogenic brain edema.     

GABA modulates baroreflex in the ventral tegmental area in rat

There are some reports demonstrating the cardiovascular functions of the ventral tegmental area (VTA). About 20–30% of the VTA neurons are GABAergic, which might play a role in baroreflex modulation. This study was performed to find the effects of GABA_A, GABA_B receptors and reversible synaptic blockade of the VTA on baroreflex. Drugs were microinjected into the VTA of urethane anesthetized rats, and the maximum change of blood pressure and the gain of the reflex bradycardia in response to intravenous phenylephrine (Phe) injection were compared with the preinjection and the control values. Microinjection of bicuculline methiodide (BMI, 100 pmol/100 nl), a GABA_A antagonist, into the VTA strongly decreased the Phe‐induced hypertension, indicating that GABA itself attenuated the baroreflex. Muscimol, a GABA_A agonist (30 mM, 100 nl), produced no significant changes. Baclofen, a GABA_B receptor agonist (1000 pmole/100 nl), moderately attenuated the baroreflex, however phaclofen, a GABA_B receptor antagonist (1000 pmole/100 nl), had no significant effect. In conclusion, for the first time, we demonstrated that GABA_A receptors of the VTA strongly attenuate and GABA_B receptors of the VTA moderately attenuate baroreflex in rat. Synapse 69:592–599, 2015 . © 2015 Wiley Periodicals, Inc.     

Effects of Adenosine Receptor Antagonists on the In Vivo LPS-Induced Inflammation Model of Parkinson’s Disease

The study shows effects of the nonselective adenosine A₁/A_2A receptor antagonist caffeine and the selective A_2A receptor antagonist KW6002 on LPS-induced changes in the extracellular levels of dopamine (DA), glutamate, adenosine, hydroxyl radical, and A_2A receptor density in the rat striatum. Intrastriatal LPS (10 μg) injection decreased extracellular level of DA and increased the level of adenosine, glutamate, and hydroxyl radical on the ipsilateral side 24 h after LPS administration. Caffeine (10 and 20 mg/kg i.p.) and KW6002 (1.5 and 3 mg/kg i.p.) given once daily for 6 days and on the 7th day 2 h before and 4 h after LPS injection reversed the LPS-induced changes in extracellular levels of DA, adenosine, glutamate, and hydroxyl radical production. Moreover, LPS-induced decrease in the striatal A_2A receptor density was increased by caffeine and KW6002. In order to show the late LPS effect on oxidative damage of DA neurons, the contents of DA, DOPAC, HVA, and hydroxyl radical were determined 72 h after LPS (10 μg) administration into both striata. LPS decreased striatal and substantia nigra content of DA, DOPAC, and HVA while increased striatal but not nigral content of hydroxyl radical. Caffeine (20 mg/kg) and KW60002 (3 mg/kg) given once daily for 6 days and on the 7th day 2 h before and 4 h after intrastriatal injection of LPS normalized the content of DA and its metabolites in both brain regions as well as decreased LPS-induced increase in the striatal level of hydroxyl radical. In conclusion, our data demonstrated antioxidant effects of caffeine and KW6002 in the inflammatory model of PD.