Differential effects of GABAB receptor subtypes, {gamma}-hydroxybutyric Acid, and Baclofen on EEG activity and sleep regulation.

The role of GABA(B) receptors in sleep is still poorly understood. GHB (γ-hydroxybutyric acid) targets these receptors and is the only drug approved to treat the sleep disorder narcolepsy. GABA(B) receptors are obligate dimers comprised of the GABA(B2) subunit and either one of the two GABA(B1) subunit isoforms, GABA(B1a) and GABA(B1b). To better understand the role of GABA(B) receptors in sleep regulation, we performed electroencephalogram (EEG) recordings in mice devoid of functional GABA(B) receptors (1(-/-) and 2(-/-)) or lacking one of the subunit 1 isoforms (1a(-/-) and 1b(-/-)). The distribution of sleep over the day was profoundly altered in 1(-/-) and 2(-/-) mice, suggesting a role for GABA(B) receptors in the circadian organization of sleep. Several other sleep and EEG phenotypes pointed to a more prominent role for GABA(B1a) compared with the GABA(B1b) isoform. Moreover, we found that GABA(B1a) protects against the spontaneous seizure activity observed in 1(-/-) and 2(-/-) mice. We also evaluated the effects of the GHB-prodrug GBL (γ-butyrolactone) and of baclofen (BAC), a high-affinity GABA(B) receptor agonist. Both drugs induced a state distinct from physiological sleep that was not observed in 1(-/-) and 2(-/-) mice. Subsequent sleep was not affected by GBL whereas BAC was followed by a delayed hypersomnia even in 1(-/-) and 2(-/-) mice. The differential effects of GBL and BAC might be attributed to differences in GABA(B)-receptor affinity. These results also indicate that all GBL effects are mediated through GABA(B) receptors, although these receptors do not seem to be involved in mediating the BAC-induced hypersomnia.

DuP 532, an angiotensin II receptor antagonist: first administration and comparison with losartan.

We investigated the tolerability and angiotensin II antagonist activity of oral DuP 532 in healthy male subjects. DuP 532 (1 to 200 mg) was well tolerated, with no effect on blood pressure or heart rate. Compared with losartan (100 mg), DuP 532 (200 mg) was a weak antagonist of pressor responses to intravenous angiotensin II. Maximum inhibition of diastolic pressor response was 86% (95% confidence interval [CI], 84%, 88%) approximately 4.6 hours after losartan and 48% (95% CI, 38%, 56%) 8.7 hours after DuP 532. Twenty-four hours after dosing, inhibition by losartan and DuP 532 was similar (40% to 45%). DUP 532 is extensively bound in human plasma, with an in vitro free fraction of 0.06. Although DuP 532 and EXP3174 (losartan's active metabolite) have similar AT1-receptor potency, and plasma concentrations of DuP 532 were much greater than losartan/EXP3174, the level of antagonism was much less for DuP 532. These results indicate that multiple factors determine the in vivo potency of angiotensin II antagonists, including affinity for and distribution to the receptor as modulated by plasma binding.

Structural determinants involved in the activation and regulation of G protein-coupled receptors: lessons from the alpha1-adrenegic receptor subtypes.

The aim of a large number of studies on G protein-coupled receptors was centered on understanding the structural basis of their main functional properties. Here, we will briefly review the results obtained on the alpha1-adrenergic receptor subtypes belonging to the rhodopsin-like family of receptors. These findings contribute, on the one hand, to further understand the molecular basis of adrenergic transmission and, on the other, to provide some generalities on the structure-functional relationship of G protein-coupled receptors.

Drug concentration response relationships in normal volunteers after oral administration of losartan, an angiotensin II receptor antagonist.

The aim of this study was to investigate the relationships between plasma concentrations of losartan, an orally active angiotensin II inhibitor, its active metabolite EXP3174, and angiotensin II blockade. Six healthy subjects received single oral doses of 40, 80, or 120 mg losartan and placebo at 1-week intervals in a crossover study. Angiotensin II blockade was assessed by the blood pressure response to exogenous angiotensin II before and after losartan administration. EXP3174 reached higher plasma concentrations and was eliminated more slowly than its parent compound; its levels paralleled the profile of angiotensin II blockade closer than losartan. Inhibition of the pressure response was dose dependent. The Hill-shaped relationship between response and EXP3174 concentration (or time-integrated variables) approached a plateau with 80 mg. The dose-dependent increase in plasma renin and angiotensin II exhibited a considerable individual scatter. We conclude that losartan produces a dose-dependent, effective angiotensin II blockade that is largely determined by the active metabolite EXP3174.

Role of endothelin receptor subtypes in volume-stimulated ANF secretion.

The role of endothelin (ET) receptors was tested in volume-stimulated atrial natriuretic factor (ANF) secretion in conscious rats. Mean ANF responses to slow infusions (3 x 3.3 ml/8 min) were dose dependently reduced (P < 0.05) by bosentan (nonselective ET-receptor antagonist) from 64.1 +/- 18.1 (SE) pg/ml (control) to 52.6 +/- 16.1 (0.033 mg bosentan/rat), 16.1 +/- 7.6 (0. 33 mg/rat), and 11.6 +/- 6.5 pg/ml (3.3 mg/rat). The ET-A-receptor antagonist BQ-123 (1 mg/rat) had no effect relative to DMSO controls, whereas the putative ET-B antagonist IRL-1038 (0.1 mg/rat) abolished the response. In a second protocol, BQ-123 (>/=0.5 mg/rat) nonsignificantly reduced the peak ANF response (106.1 +/- 23.0 pg/ml) to 74.0 +/- 20.5 pg/ml for slow infusions (3.5 ml/8.5 min) but reduced the peak response (425.3 +/- 58.1 pg/ml) for fast infusions (6.6 ml/1 min) by 49.9% (P < 0.001) and for 340 pmoles ET-1 (328.8 +/- 69.5 pg/ml) by 83.5% (P < 0.0001). BQ-123 abolished the ET-1-induced increase in arterial pressure (21.8 +/- 5.2 mmHg at 1 min). Changes in central venous pressure were similar for DMSO and BQ-123 (slow: 0.91 and 1.14 mmHg; fast: 4.50 and 4.13 mmHg). The results suggest 1) ET-B receptors mainly mediate the ANF secretion to slow volume expansions of <1.6%/min; and 2) ET-A receptors mainly mediate the ANF response to acute volume overloads.

Pharmacology of valsartan, an angiotensin II receptor antagonist.

Valsartan is the second orally-active, non-peptide angiotensin II receptor blocker to reach the market in Europe and the USA for the treatment of hypertension. Preclinical studies have demonstrated that this blocker is specific for the AT(1) receptor and has no affinity for the angiotensin II AT(2) receptor. Experimentally, valsartan dose-dependently inhibits the vasoconstriction induced by angiotensin II and lowers blood pressure in renin-dependent models of hypertension. Pharmacologically, oral valsartan is characterised by a low bioavailability but a rapid absorption and distribution with a half-life in keeping with once-daily administration. Thus, after oral administration, the maximal plasma concentration is reached 2 h after dosing and the elimination half-life is about 6 h. Clinically, several dose-finding and comparative studies have demonstrated that valsartan is an effective and well-tolerated antihypertensive drug in patients with mild to moderate hypertension. Valsartan has also been shown to be effective in severe hypertension. Valsartan is at least as effective as ACE inhibitors, diuretics, beta-blockers and calcium antagonists. However, none of the side-effects observed with these latter agents, including cough and lower limb oedema, has been observed with the administration of valsartan. Three large clinical trials are now underway to demonstrate whether valsartan can reduce morbidity and mortality: one in hypertensives with a high cardiovascular risk profile (VALUE), one in patients with heart failure previously treated with an angiotensin-converting enzyme inhibitor (VAL-HeFT) and one in post-myocardial infarct patients (VALIANT). These studies will further define the place of valsartan beyond the treatment of hypertension.

Telmisartan: a different angiotensin II receptor blocker protecting a different population?

The ONgoing Telmisartan Alone and in combination with Ramipril Global Endpoint Trial (ONTARGET()) showed that the angiotensin II receptor blocker (ARB) telmisartan was as protective as the reference-standard ramipril in a broad cross-section of patients at increased cardiovascular risk, but was better tolerated. Telmisartan has a unique profile among ARBs, with a high affinity for the angiotensin II type 1 receptor, a long duration of receptor binding, a high lipophilicity and a long plasma half life. This leads to sustained and powerful blood pressure lowering when compared with the first marketed ARBs, such as losartan and valsartan. Some pharmacological properties of telmisartan clearly distinguish it from other members of the ARB class and may contribute to the clinical effects seen with telmisartan. A class effect for ARBs cannot be assumed. To date, telmisartan is the only ARB that has been shown to reduce cardiovascular risk in at-risk cardiovascular patients.

Human fetal malformations associated with the use of an angiotensin II receptor antagonist: Case Report

Introduction: The potential risks related to drug exposure during pregnancy represent a vast chapter in modern obstetrics and data regarding the safety of antihypertensive drugs during pregnancy are relatively scarce. Case report: A 37-year-old patient discovered her fifth pregnancy at our hospital after 26 weeks and 4 days of gestation. She reported a history of hypertension and was currently being treated with Losartan. Hospitalization was recommended for the patient and further evaluation of fetal vitality was performed. On the fourth day an ultrasound was performed, resulting in a severe oligohydramnios, fetal centralization and abnormal ductus venosus. After 36 hours, the newborn died. Pathologic evaluation: At autopsy, the skullcap had large fontanels and deficient ossification. The kidneys were slightly enlarged. A microscopic examination detected underdevelopment of the tubules and the presence of some dilated lumens. Immunohistochemical detection of epithelial membrane antigen was positive. Immunoreactivity of CD 15 was also assayed to characterize the proximal tubules, and lumen collapse was observed in some regions. Discussion: Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor antagonists (ARAs) are among the most widely prescribed drugs for hypertension. They are often used by hypertensive women who are considering become pregnant. While their fetal toxicity in the second or third trimesters has been documented, their teratogenic effect during the first trimester has only recently been demonstrated. Conclusion: Constant awareness by physicians and patients should be encouraged, particularly in regard to the prescription of antihypertensive drugs in women of childbearing age who are or intend to become pregnant.

Role of Nuclear Angiotensin-II Receptor Mediated Signalling in Cardiovascular Remodelling

Le remodelage cardiaque est le processus par lequel la structure ou la fonction cardiaque change en réponse à un déséquilibre pathophysiologique tel qu'une maladie cardiaque, un contexte d'arythmie prolongée ou une modification de l'équilibre hormonal. Le système rénine-angiotensine (SRA) est un système hormonal largement étudié et il est impliqué dans de nombreuses activités associées au remodelage cardiovasculaire. L’existence d'un système circulatoire couplé à un système de tissus locaux est une représentation classique, cependant de nouvelles données suggèrent un SRA indépendant et fonctionnellement actif à l'échelle cellulaire. La compréhension de l'activité intracellulaire du SRA pourrait mener à de nouvelles pistes thérapeutiques qui pourraient prévenir un remodelage cardiovasculaire défavorable. L'objectif de cette thèse était d'élucider le rôle du SRA intracellulaire dans les cellules cardiaques. Récemment, les récepteurs couplés aux protéines G (RCPG), les protéines G et leurs effecteurs ont été détectés sur des membranes intracellulaires, y compris sur la membrane nucléaire, et les concepts de RCPG intracellulaires fonctionnels sont en voie d'être acceptés comme une réalité. Nous avons dès lors fait l'hypothèse que la signalisation du SRA délimitant le noyau était impliquée dans le contrôle de l'expression des gènes cardiaques. Nous avons démontré la présence de récepteurs d'angiotensine de type-1 (AT1R) et de type-2 (AT2R) nucléaires dans les cardiomyocytes ventriculaires adultes et dans une fraction nucléaire purifiée de tissu cardiaque. Des quantités d'Ang II ont été détectées dans du lysat de cardiomyocytes et des microinjections d'Ang-II-FITC ont donné lieu à des liaisons préférentielles aux sites nucléaires. L'analyse transcriptionnelle prouve que la synthèse d'ARN de novo dans des noyaux isolés stimulés à l'Ang-II, et l'expression des ARNm de NF-κB étaient beaucoup plus importants lorsque les noyaux étaient exposés à de l'Ang II par rapport aux cardiomyocytes intacts. La stimulation des AT1R nucléaires a engendré une mobilisation de Ca2+ via les récepteurs de l'inositol trisphosphate (IP3R), et le blocage des IP3R a diminué la réponse transcriptionnelle. Les méthodes disponibles actuellement pour l'étude de la signalisation intracrine sont limitées aux méthodes indirectes. L'un des objectifs de cette thèse était de synthétiser et caractériser des analogues d'Ang-II cellule-perméants afin d’étudier spécifiquement dans les cellules intactes l'activité intracellulaire du SRA. Nous avons synthétisé et caractérisé pharmacologiquement des analogues photosensibles Ang-II encapsulée en incorporant un groupement 4,5-diméthoxy-2-nitrobenzyl (DMNB) photoclivable sur les sites actifs identifiés du peptide. Chacun des trois analogues d'Ang II encapsulée synthétisés et purifiés: [Tyr(DMNB)4]Ang-II, Ang-II-ODMNB et [Tyr(DMNB)4]Ang-II-ODMNB a montré une réduction par un facteur deux ou trois de l'affinité de liaison envers AT1R et AT2R dans les dosages par liaison compétitive et une activité réduite dans la contraction de l'aorte thoracique. La photostimulation de [Tyr(DMNB)4]Ang-II dans des cellules HEK a augmenté la phosphorylation d'ERK1/2 (via AT1R) et la production de cGMP (via AT2R) alors que dans les cardiomyocytes isolés elle générait une augmentation de Ca2+ nucléoplasmique et initiait la synthèse d'ARNr 18S et d'ARNm du NF-κB. Les fibroblastes sont les principaux générateurs de remodelage cardiaque structurel, et les fibroblastes auriculaires sont plus réactifs aux stimuli profibrotiques que les fibroblastes ventriculaires. Nous avons émis l'hypothèse que l’Ang-II intracellulaire et l'activation des AT1R et AT2R nucléaires associés contrôlaient les profils d'expression des gènes des fibroblastes via des systèmes de signalisation distincts et de ce fait jouaient un rôle majeur dans le développement de la fibrose cardiaque. Nous avons remarqué que les fibroblastes auriculaires expriment l’AT1R et l’AT2R nucléaire et l'Ang-II au niveau intracellulaire. L’expression d'AT1R nucléaire a été régulés positivement dans les cas d’insuffisance cardiaque (IC), tandis que l'AT2R nucléaire a été glycosylé post-traductionnellement. La machinerie protéique des protéines G, y compris Gαq/11, Gαi/3, et Gβ, a été observée dans des noyaux isolés de fibroblastes. AT1R et AT2R régulent l'initiation de la transcription du fibroblaste via les voies de transduction de signal d'IP3R et du NO. La photostimulation de [Tyr(DMNB)4]Ang-II dans une culture de fibroblastes auriculaire déclenche la libération de Ca2+ nucléoplasmique, la prolifération, et la synthèse et sécrétion de collagène qui ne sont pas inhibées par les bloqueurs d'AT1R et/ou AT2R extracellulaires.

Muscarinic M1, M3, Nicotinic,GABAA and GABAB Receptor Subtypes Gene Expression in Insulin Induced Hypoglycemic Rat Brain Regions: Functional Regulation through Phospholipase C and CREB Protein

In the present study, a detailed investigation on the alterations of muscarinic M1, M3, α7 nicotinic acetylcholine receptor (α7 nAchR), GABA receptors and its subtypes; GABAAα1 and GABAB in the brain regions of streptozotocin induced diabetic and insulin induced hypoglycemic rats were carried out. Gene expression of acetylcholine esterase (AChE), choline acetyltransferase (ChAT), GAD, GLUT3, Insulin receptor, superoxide dismutase (SOD), Bax protein, Phospholipase C and CREB in hypoglycemic and hyperglycemic rat brain were studied. Muscarinic M1, M3 receptors, AChE, ChAT, GABAAα1, GABAB, GAD, Insulin receptor, SOD, Bax protein and Phospholipase C expression in pancreas was also carried out. The molecular studies on the CNS and PNS damage will elucidate the therapeutic role in the corrective measures of the damage to the brain during hypoglycemia and hyperglycemia.

Dopamine Receptor Subtypes,IP3, cAMP and cGMP Functional Regulation in Parkinsonism Induced by Unilateral Infusion of Rotenone in Rats: Recovery with Bone Marrow Cells,Serotonin and GABA Supplementation

In the present study, the effects of 5-HT, GABA and Bone Marrow Cells infused intranigrally to substantia nigra individually and in combinations on unilateral rotenone infused Parkinsonism induced rats. Scatchard analysis of DA, DA D1 and D2 receptors in the corpus striatum, cerebral cortex, cerebellum, brain stem and hippocampus showed a significant increase in the Brain regions of rotenone infused rat compared to control. Real Time PCR amplification of DA D1, D2, Bax and ubiquitin carboxy-terminal hydrolase were up regulated in the brain regions of rotenone infused rats compared to control. Gene expression studies of -Synuclien, cGMP and Cyclic AMP response element-binding protein showed a significant down regulation in Rotenone infused rats compared to control. Behavioural studies were carried out to confirm the biochemical and molecular studies.Our study demonstrated that BMC administration alone cannot reverse the above said molecular changes occurring in PD rat. 5-HT and GABA acting through their specific receptors in combination with bone marrow cells play a crucial role in the functional recovery of PD rats. 5-HT, GABA and Bone marrow cells treated PD rats showed significant reversal to control in DA receptor binding and gene expression. 5-HT and GABA have co-mitogenic property. Proliferation and differentiation of cells re-establishing the connections in Parkinson's disease facilitates the functional recovery. Thus, it is evident that 5-HT and GABA along with BMC to rotenone infused rats renders protection against oxidative, related motor and cognitive deficits which makes them clinically significant for cellbased therapy. The BMC transformed to neurons when co-transplanted with 5-HT and GABA which was confirmed with PKH2GL and nestin. These newly formed neurons have functional significance in the therapeutic recovery of Parkinson’s disease.

Cholinergic receptor subtypes functional regulation in spinal cord injured monoplegic rats

The present study deals with the Cholinergic Receptor subtypes functional regulation in spinal cord injured monoplegic rats: Effect of 5-HT GABA and bone marrow cells.Spinal cord injury causes permanent and irrevocable motor deficits and neurodegeneration. Disruption of the spinal cord leads to diminished transmission of descending control from the brain to motor neurons and ascending sensory information. Behavioural studies showed deficits in motor control and coordination in SCI rats. Cholinergic system plays an important role in SCI, the evaluation of which provides valuable insight on the underlying mechanisms of motor deficit that occur during SCI. The cholinergic transmission was studied by assessing the muscarinic and nicotinic receptors; cholinergic enzymes- ChAT and AChE; second messenger enzyme PLC; transcription factor CREB and second messengers - IP3, cAMP and cGMP. We observed a decrease in the cholinergic transmission in the brain and spinal cord of SCI rats. The disrupted cholinergic system is the indicative of motor deficit and neuronal degeneration in the spinal cord and brain regions. SCI mediated oxidative stress and apoptosis leads to neuronal degeneration in SCI rats. The decreased expression of anti oxidant enzymes – SOD, GPx and neuronal cell survival factors - BDNF, GDNF, IGF-1, Akt and cyclin D2 along with increased expression of apoptotic factors – Bax, caspase-8, TNFa and NF-kB augmented the neuronal degeneration in SCI condition. BMC administration in combination with 5-HT and GABA in SCI rats showed a reversal in the impaired cholinergic neurotransmission and reduced the oxidative stress and apoptosis. It also enhanced the expression of cell survival factors in the spinal cord region. In SCI rats treated with 5-HT and GABA, the transplanted BMC expressed NeuN confirming that 5-HT and GABA induced the differentiation and proliferation of BMC to neurons in the spinal cord. Neurotrophic factors and anti-apoptotic elements in SCI rats treated with 5-HT and GABA along with BMC rendered neuroprotective effects accompanied by improvement in behavioural deficits. This resulted in a significant reversal of altered cholinergic neurotransmission in SCI. The restorative and neuro protective effects of BMC in combination with 5-HT and GABA are of immense therapeutic significance in the clinical management of SCI.