A novel agent with histone deacetylase inhibitory activity attenuates neointimal hyperplasia.

PURPOSE: Neointimal hyperplasia (NIH), a pathophysiological event identified in bypass graft and stent re-stenosis, is characterised by aberrant vascular smooth muscle cell (VSMC) migration and proliferation. Recent evidence identifies histone deacetylase modulation as a regulator of VSMC proliferation and migration and a potential therapeutic target in the treatment of NIH. The purpose of our study was to determine the in vitro and in vivo potential of a novel agent, MCT-3, to modulate VSMC migration, proliferation and NIH. METHODS: In vitro VSMC studies utilized reverse transcriptase and real time Q-PCR gene expression analysis, western blot, elisa assay and cellular proliferation and migration scratch assay's. In vivo studies utilized the partial carotid artery ligation model of NIH together with immunohistochemistry in FVB/N mice. RESULTS: MCT-3 treatment induced histone H3 and H4 acetylation and inhibited VSMC migration and proliferation in vitro and significantly attenuated NIH in vivo. MCT-3-mediated regulation of orphan nuclear receptor NUR77, Plasminogen Activator Inhibitor Type-1 (PAI-1) and cyclin dependent kinase inhibitors (CDKI) p21(CIP1/WAF1) and p27(KIP1) expression was also identified. CONCLUSIONS: Together these observations identify a novel agent, MCT-3, with histone deacetylase inhibitory activity, able to inhibit NIH and identify a potential molecular mechanism responsible for these effects. Additional pre-clinical studies may be warranted to determine the potential clinical utility of this compound.

Vasoactive role for angiotensin II type 2 receptors in human radial artery.

Angotensin II type 2 receptors are believed to counter the effects of the angiotensin type 1 receptors and there is no data relating to the co-localisation of either receptor in human diseased arteries. We sought to determine whether AT2R counter the effects of AT1R and immunolocalise both receptors to cells in human diseased arteries. Human radial arteries (RA, n=11) were placed in organ bath chambers and preincubated with the AT2R antagonist PD123319 for twenty minutes before an angiotensin II dose response curve. Immunohistochemistry was performed to identify receptors and pathology was quantified by image analysis software. We observed both receptors in human arteries. Angiogenic blood vessels within occluded arteries expressed both receptors. PD123319 impaired angiotensin II mediated vasoconstriction by 20 percent (n=5, p less than 0.05), however in other arteries, PD123319 exacerbated angiotensin II-mediated vasoconstriction by 60 percent (n=6, p less than 0.01), respectively. We conclude that inhibition of AT2R can enhance or reduce angiotensin II-mediated vasoconstriction. These data indicate that the role of AT2R in human diseased arteries is divergent although the AT2R-mediated vasorelaxation prevails.

Differential mechanisms of ang (1-7)-mediated vasodepressor effect in adult and aged candesartan-treated rats.

Angiotensin (1-7) (Ang (1-7)) causes vasodilator effects in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) via angiotensin type 2 receptors (AT(2)R). However, the role of vascular AT(2)R in aging is not known. Therefore, we examined the effect of aging on Ang (1-7)-mediated vasodepressor effects and vascular angiotensin receptor localization in aging. Blood pressure was measured in conscious adult (~17 weeks) and aged (~19 months) normotensive rats that received drug combinations in a randomised fashion over a 4-day protocol: (i) Ang (1-7) alone, (ii) AT(1)R antagonist, candesartan, alone, (iii) Ang (1-7) and candesartan, or (iv) Ang-(1-7), candesartan, and the AT(2)R antagonist, PD123319. In a separate group of animals, the specific MasR antagonist, A779, was administered in place of PD123319. Receptor localisation was also assessed in aortic sections from adult and aged WKY rats by immunofluorescence. Ang (1-7) reduced blood pressure (~15 mmHg) in adult normotensive rats although this effect was dependant on the background dose of candesartan. This depressor effect was reversed by AT(2)R blockade. In aged rats, the depressor effect of Ang (1-7) was evident but was now inhibited by either AT(2)R blockade or MasR blockade. At the same time, AT(2)R, MasR, and ACE2 immunoreactivity was markedly elevated in aortic sections from aged animals. These results indicate that the Ang (1-7)-mediated depressor effect was preserved in aged animals. Whereas Ang (1-7) effects were mediated exclusively via stimulation of AT(2)R in adult WKY, with aging the vasodepressor effect of Ang (1-7) involved both AT(2)R and MasR.

Stimulation of angiotensin AT2 receptors by the non-peptide agonist, Compound 21, evokes vasodepressor effects in conscious spontaneously hypertensive rats.

BACKGROUND AND PURPOSE: Angiotensin type 2 receptor (AT(2) receptor) stimulation evokes vasodilator effects in vitro and in vivo that oppose the vasoconstrictor effects of angiotensin type 1 receptors (AT(1) receptors). Recently, a novel non-peptide AT(2) receptor agonist, Compound 21, was described, which exhibited high AT(2) receptor selectivity. EXPERIMENTAL APPROACH: Functional cardiovascular effects of the drug candidate Compound 21 were assessed, using mouse isolated aorta and rat mesenteric arteries in vitro and in conscious spontaneously hypertensive rats (SHR). KEY RESULTS: Compound 21 evoked dose-dependent vasorelaxations in aortic and mesenteric vessels, abolished by the AT(2) receptor antagonist, PD123319. In vivo, Compound 21 administered alone, at doses ranging from 50 to 1000 ng.kg(-1).min(-1) over 4 h did not decrease blood pressure in conscious normotensive Wistar-Kyoto rats or SHR. However, when given in combination with the AT(1) receptor antagonist, candesartan, Compound 21 (300 ng.kg(-1).min(-1)) lowered blood pressure in SHR only. Further analysis in separate groups of conscious SHR revealed that, at a sixfold lower dose, Compound 21 (50 ng.kg(-1).min(-1)) still evoked a significant depressor response in adult SHR ( approximately 30 mmHg) when combined with different doses of candesartan (0.01 or 0.1 mg.kg(-1)). Moreover, the Compound 21-evoked depressor effect was abolished when co-infused (50 microg.kg(-1).min(-1) for 2 h) with the AT(2) receptor antagonist PD123319. CONCLUSION AND IMPLICATIONS: Collectively, our results indicate that acute administration of Compound 21 evoked blood pressure reductions via AT(2) receptor stimulation. Thus Compound 21 can be considered an excellent drug candidate for further study of AT(2) receptor function in cardiovascular disease.

Minocycline treatment attenuates microglia activation and non-angiotensin II [125I] CGP42112 binding in brainstem following nodose ganglionectomy.

We have previously shown that following unilateral nodose ganglionectomy, [125I] CGP42112 binds to a non-angiotensin II (Ang II) related binding site in rat dorsal motor nucleus of the vagus nerve, ambiguus nucleus and nucleus of the solitary tract. Furthermore, this up-regulated binding site localizes with activated microglia. Given that some tetracyclines may inhibit microglia activation in brain, we examined the effect of minocycline treatment on the binding of [125I] CGP42112 and [3H] PK11195 (an established radioligand for microglia), as well as OX-42 immunoreactivity (an immunomarker for activated microglia), following nodose ganglionectomy. Male Wistar Kyoto rats underwent unilateral nodose ganglionectomy or sham operation and were treated with saline or minocycline (50 mg/kg i.p.) 12 h before surgery and twice daily after surgery (each 50mg/kg i.p.) for 3 days. Subsequent to nodose ganglionectomy, [125I] CGP42112 binding (insensitive to PD123319 or Ang II) was increased approximately two-fold in the ipsilateral nucleus of the solitary tract and was also induced in the ipsilateral dorsal motor nucleus of the vagus nerve and ambiguus nucleus of saline-treated rats. Treatment with minocycline reduced this non-angiotensin II [125I] CGP42112 binding (40-50% reduction) in the nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve and ambiguus nucleus. Analogous experiments using [3H] PK11195 also revealed up-regulated binding in the ipsilateral nucleus of the solitary tract ( approximately 205%), dorsal motor nucleus of the vagus nerve (approximately 80%) and ambiguus nucleus (approximately 210%) of saline-treated rats following nodose ganglionectomy, which was reduced by 40-100% with minocycline treatment. Immunoreactivity to OX-42 confirmed an increase in microglia activation and accumulation of macrophages in these brain stem nuclei following nodose ganglionectomy, which was also attenuated following treatment with minocycline. These data demonstrate that non-Ang II [125I] CGP42112 binding following nodose ganglionectomy is attenuated by minocycline treatment. This minocycline-induced effect was associated with reduced activation of microglia and an apparent reduction in the number of macrophages in the abovementioned nuclei. This evidence suggests that a non-Ang II [125I] CGP42112 binding site is located on, or associated with, activated microglia and macrophages, providing a useful tool with which to quantitate the neuroprotective effects of centrally acting anti-inflammatory compounds.

Non-angiotensin II [(125)I] CGP42112 binding is a sensitive marker of neuronal injury in brainstem following unilateral nodose ganglionectomy: comparison with markers for activated microglia.

Previously we reported that a non-angiotensin II [(125)I] CGP42112 binding site is up-regulated in rat brainstem nuclei as a result of unilateral nodose ganglionectomy. In the present study, we compared non-angiotensin II [(125)I] CGP42112 binding with microglia/macrophage activation following nodose ganglionectomy, using both in vitro autoradiography and immunohistochemistry. Specific [(125)I] CGP42112 binding was observed in the nucleus of the solitary tract (NTS) and revealed an AT(2) receptor component as well as a non-angiotensin II receptor component. Subsequent to unilateral nodose ganglionectomy, [(125)I] CGP42112 binding in the ipsilateral NTS was increased approximately two-fold and was also induced in the ipsilateral dorsal motor nucleus (DMX) and the nucleus ambiguus (n.amb). This non-angiotensin II [(125)I] CGP42112 binding site was displaced by CGP42112 but not other ligands. Increased [(3)H] PK11195 binding (a known marker of reactive gliosis) was also observed in the same brainstem nuclei as non-angiotensin II [(125)I] CGP42112 binding after nodose ganglionectomy. The similarity in binding patterns between [(125)I] CGP42112 and [(3)H] PK11195 was shown to be primarily due to retrograde degeneration in the ipsilateral NTS, DMX and n.amb, as both radioligands were localized to similar cellular targets within the interstial space and over cellular debris. Immunohistochemical data confirmed reactive gliosis within the ipsilateral NTS, DMX and n.amb, following nodose ganglionectomy, which was predominantly characterized by an increase in OX-42 immunoreactivity (a marker for activated microglia/macrophages), with only a small increase in glial fibrillary acidic protein immunoreactivity (a marker of astrogliosis) detected. These data demonstrate for the first time that non-angiotensin II [(125)I] CGP42112 binding is associated with activated microglia, as well as macrophages, following unilateral nodose ganglionectomy. Furthermore, these studies also demonstrate the potential use of non-angiotensin II [(125)I] CGP42112 binding as a marker for quantitating inflammatory events which occur as a result of damage to the CNS.

Persistent cardiovascular effects of chronic renin-angiotensin system inhibition following withdrawal in adult spontaneously hypertensive rats.

OBJECTIVES: It is generally accepted that short-term (4 weeks) inhibition of the renin-angiotensin system (RAS) of young spontaneously hypertensive rats (SHR) in their prehypertensive phase confers long-lasting protection from fully hypertensive levels in adulthood. However, there is very little data pertaining to the effects of such treatment in adult SHR with established hypertension. Therefore, we determined the relative effects of angiotensin converting enzyme (ACE) inhibition (perindopril), AT1 receptor blockade (candesartan cilexetil) and RAS-independent vasodilatation (hydralazine) and their withdrawal in adult SHR, on blood pressure measured by radiotelemetry, as well as on cardiac and vascular structure. METHODS: Adult male SHR were instrumented with radiotelemetry probes to measure blood pressure and heart rate continuously. SHR were given either vehicle, perindopril (1 mg/kg per day), candesartan cilexetil (2 mg/ kg per day) or hydralazine (30 mg/kg per day) at equieffective depressor doses for 4 weeks (treatment study). Separate groups of animals were also given identical treatments but were then monitored for a further 8 weeks after drug withdrawal (withdrawal study). An indirect in-vivo assessment of whole body vascular hypertrophy (mean arterial pressure during maximum vasoconstriction) was made during and after drug withdrawal, as was the pressor activity evoked by angiotensin I and angiotensin II. The effect of antihypertensive treatment on microalbuminuria was also assessed during and after drug withdrawal. Finally, left ventricular: body weight (Iv: bw) and mesenteric media: lumen ratios were determined either immediately after 4-week treatment (treatment study) or 8 weeks later (withdrawal study). RESULTS: Perindopril persistently lowered blood pressure in adult SHR whereas blood pressure returned to vehicle levels within approximately 4 and 15 days after withdrawal of hydralazine and candesartan cilexetil, respectively. Cardiac hypertrophy was reduced by all three treatments, but to a lesser extent by hydralazine (treatment study), and this regression of cardiac hypertrophy persisted only with both types of RAS inhibition (withdrawal study). Vascular hypertrophy, measured indirectly and directly, was also reduced by all three treatments, with perindopril and candesartan cilexetil causing hypotrophic and eutrophic remodelling, respectively (treatment study), although these changes were generally not maintained after drug withdrawal (withdrawal study). Angiotensin I-induced pressor responses were equally inhibited during treatment with either candesaran cilexetil or perindopril (and were unaffected by hydralazine) but normalized rapidly in both groups (within approximately 2-4 days) after withdrawal of RAS inhibition. In addition, there was a small age-related increase in microalbuminuria over the study period, which was not significantly affected by any treatment. CONCLUSIONS: Following 4-week treatment, candesartan cilexetil, perindopril and hydralazine caused similar antihypertensive effects; however, only perindopril persistently reduced blood pressure following drug withdrawal. Both types of RAS inhibition and hydralazine caused marked cardiac and vascular remodelling during treatment, whereas only the RAS inhibitors persistently regressed cardiac hypertrophy 8 weeks later. Collectively, these results indicate the importance of the RAS for the maintenance of hypertension and cardiovascular hypertrophy in adult SHR, as well as identifying differential effects of ACE inhibition and AT1 receptor blockade on persistent blood pressure reduction.

Characterisation of vasopressin V(1A), angiotensin AT(1) and AT(2) receptor distribution and density in normotensive and hypertensive rat brain stem and kidney: effects of restraint stress.

In the present study, we have examined neurochemical correlates that may be involved in the differential cardiovascular responses observed in normotensive and hypertensive rats during stress. Using a restraint stress paradigm, both normotensive Wistar Kyoto (WKY) and Spontaneously Hypertensive rats (SHR) underwent acute (1 h restraint in a perspex tube), chronic (1 h restraint for ten consecutive days) or no restraint (control) stress. Following cessation of restraint, rats were processed by incubating sections of brain stem and kidney with [125I]-HO-LVA (0.03 nM) or [125I]Sar(1)Ile(8)-AngiotensinII (0.5 nM), in the presence of PD123319 (10 microM) or losartan (10 microM), to determine the distribution and density of vasopressin V(1A), angiotensin AT(1) and AT(2) receptors, respectively. Analysis of autoradiograms indicated changes in the density of radioligand binding in acutely and chronically-stressed rats, as compared to controls. For example, V(1A) binding in the medial nucleus tractus solitarius (SolM) decreased in the WKY but increased in the SHR. AT(1) binding in SolM did not significantly change in the WKY but decreased in the SHR with repeated restraint. In kidney slices, AT(1) binding decreased with stress in the WKY (-17%) but increased in SHR (+10-15%). AT(2) binding in the kidney showed a pattern similar to that of AT(1) binding in SHR, but not WKY. Graded increases in V(1A) binding were measured in kidney medulla and cortex of both strains (+50-60% with chronic restraint). These results suggest that physiological adaptation to restraint is associated with specific changes in V(1A), AT(1) and AT(2) receptor density within brain nuclei and kidney.

Restraint stress : differential cardiovascular responses in Wistar-Kyoto and spontaneously hypertensive rats.

With the use of a restraint stress paradigm, both normotensive Wistar-Kyoto (WKY ) rats and spontaneously hypertensive rats (SHR) underwent acute (1-hour restraint in a Perspex tube), chronic (1-hour restraint for 10 consecutive days), or no-restraint (control) stress. Rats experiencing chronic restraint were previously implanted with telemetric probes to measure heart rate and blood pressure. Basal (prestress session) cardiovascular variables did not change during the course of the study (SHR: mean arterial pressure 129+/-1 mm Hg, heart rate 288+/-4 bpm; WKY rats: mean arterial pressure 103+/-1 mm Hg, heart rate 285+/-3 bpm). Restraint caused tachycardia and pressor responses in the WKY rats and SHR, but these effects were greater in the hypertensive strain. The duration of restraint-induced tachycardia did not change in the WKY rats between acute and chronic stress; however, a graded reduction in the duration of restraint-induced tachycardia occurred in the SHR, decreasing to WKY rat levels by day 7 of the 10-day regimen. These data indicate that although the WKY rats can effectively "cope" within a single period of restraint, the coping mechanism is apparently impaired in the SHR compared with the WKY rats. A reduced capacity to cope with processive stressors may thus have an affect on cardiovascular regulation and represent an additional risk factor in hypertension.

AT(2) receptor stimulation enhances antihypertensive effect of AT(1) receptor antagonist in hypertensive rats.

In the present study, we investigated the role of the angiotensin type 2 (AT(2)) receptor in the regulation of blood pressure in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). We tested the hypothesis that AT(2) receptor activation may contribute to the antihypertensive effects of angiotensin type 1 (AT(1)) receptor antagonists. Mean arterial pressure (MAP) and heart rate were measured over a 4-day protocol in various groups of rats that received the following drug combinations: the AT(1) receptor antagonist candesartan (0.01 or 0.1 mg/kg IV) alone, the AT(2) receptor agonist CGP42112 (1 microg/kg per minute) alone, and candesartan plus CGP42112. In both SHR and WKY, 4-hour infusions of saline and CGP42112 alone did not alter MAP. In WKY, both doses of candesartan alone caused small decreases in MAP, which were similar when combined with CGP42112. In SHR, candesartan (0.1 mg/kg) caused an immediate, marked decrease in MAP, which was unaffected when combined with CGP42112. By contrast, in separate SHR, a 10-fold lower dose of candesartan (0.01 mg/kg) caused a slower-onset depressor response, which was enhanced when combined with CGP42112. The involvement of AT(2) receptors was confirmed in another group of SHR, since this facilitation of the antihypertensive effect of candesartan by CGP42112 was abolished by the coinfusion of the AT(2) receptor antagonist PD123319 (50 microg/kg per minute) with the candesartan/CGP42112 combination. Collectively, these data suggest that in SHR, AT(2) receptor activation can facilitate the initial depressor response caused by an AT(1) receptor antagonist.

Cardiovascular effects of angiotensin-(1-7) in conscious spontaneously hypertensive rats.

In the present study, we reassessed whether angiotensin (Ang)-(1-7) can exert short- and long-term cardiovascular effects because there has been a resurgence of interest in this N-terminal heptapeptide fragment of Ang II. In particular, we studied 3 aspects relating to the reported cardiovascular effects of Ang-(1-7): does this peptide (1) potentiate the hypotensive effect of bradykinin in normotensive Wistar-Kyoto rats and spontaneously hypertensive rats (SHR), (2) cause a depressor effect after long-term treatment in SHR, and (3) contribute to the antihypertensive effects of angiotensin-converting enzyme inhibitors? In the first series of experiments, Ang-(1-7) failed to enhance the dose-related hypotensive responses evoked by bradykinin in SHR (n=11) and Wistar-Kyoto (n=5) rats. In the second series of experiments, a 7-day intravenous infusion of Ang-(1-7) (24 microg x kg(-1) x h(-1)) decreased blood pressure in SHR (n=12) on days 4 and 5, although this effect waned despite continual Ang-(1-7) infusion. However, a new finding was that the Ang-(1-7) antagonist A-779 (24 microg x kg(-1) x h(-1) for 7 days) attenuated the depressor effect of Ang-(1-7) when given concurrently in a separate group of SHR (n=8). In the third series of novel experiments, the angiotensin-converting enzyme inhibitor perindopril was given in drinking water for 7 days (0.3 mg. kg(-1) x day(-1)), either alone (n=6) or combined with an intravenous infusion of A-779 (24 microg x kg(-1) x h(-1) for 7 days, n=8). Although this dose of A-779 attenuated the depressor effect of Ang-(1-7), it did not alter the antihypertensive effect caused by perindopril. Thus, the present results contrast with a number of previous studies and argue against Ang-(1-7) playing a major role in blood pressure regulation.

Differential haemodynamic effects of endothelin receptor antagonist, SB 209670, in conscious hypertensive and normotensive rats.

Endothelin has been implicated in the pathogenesis and/or maintenance of hypertension. Endothelin receptor antagonists lower blood pressure in the spontaneously hypertensive rat (SHR), but the regional haemodynamic effects of such drugs in the SHR remain unknown. The aim of this study was to examine the regional haemodynamic effects of the endothelin receptor antagonist, (+/-)-(1S,2R,3S)-3-(2-carboxymethoxy-4-methoxyphenyl)-1-(3, 4-methylenedioxyphenyl)-5-(prop-1-yloxy)-indane-2-carboxylic acid (SB 209670), in SHR and Wistar-Kyoto (WKY) rats. Rats underwent a two-stage operation for implantation of Doppler flow probes and intravascular catheters. Recordings were made of mean arterial pressure (MAP), heart rate (HR) and renal (Ren), mesenteric (Mes) and hindquarters (HQ) blood flows and conductances (Cond). SHR and WKY received 4.5 h infusions of saline or SB 209670 (5 mg/kg priming dose+1 or 5 mg/kg/h, i.v.). SB 209670 lowered blood pressure in both SHR (-23+/-2 mm Hg) and WKY rats (-13+/-1 mm Hg). In addition, a lower dose infusion of SB 209670 also had an antihypertensive effect in SHR (-15+/-5 mm Hg). In SHRs which received the higher dose of antagonist, Ren, Mes and HQ Cond were significantly increased as was the HQ Cond in a low-dose group. In WKY rats, SB 209670 decreased Ren blood flow whilst increasing Mes and HQ blood flows and Cond. SB 209670 also attenuated the regional vasoconstrictor effects of endothelin-1, except in the Mes circulation in SHR. This study illustrates that SB 209670 causes differential haemodynamic effects in SHR and WKY rats. In SHR, the antihypertensive effect of SB 209670 was accompanied by a generalised vasodilatation in the Ren, Mes and HQ vascular beds. In WKY rats, the hypotensive effect of SB 209670 was accompanied by Mes and HQ vasodilatation, but with Ren vasoconstriction. Thus, endothelin is involved in the maintenance of blood pressure and vascular tone in both SHR and WKY rats, but the haemodynamic profiles of these effects differ between the two strains.

Type I and II metabotropic glutamate receptor agonists and antagonists evoke cardiovascular effects after intrathecal administration in conscious rats.

1. In the present study, the role of metabotropic glutamate receptors (mGluRs) in central cardiovascular regulation in conscious rats was examined. To this end, agonists and antagonists for type I and II mGluRs were administered intrathecally, and the temporal changes in blood pressure and heart rate were recorded. 2. L-glutamate (1 micromol) and the prototypical mGluR agonist (1S,3R)-ACPD (0.1 and 0. 3 micromol) both increased mean arterial pressure (MAP) and heart rate (HR), implicating functional mGluRs in the spinal cord. The type I mGluR agonist DHPG (0.01 - 0.1 micromol) evoked increases in MAP (max=25+/-5 mmHg) and HR (max=88+/-23 beats min-1). The duration of action, but not the maximum effects, were dose-related and ranged from approximately 10 min to <90 min and 1 min to >90 min for MAP and HR, respectively. 3. The type I/II mGluR agonist CCG-1 (0.1 and 0. 3 micromol) caused smaller, variable increases in MAP and HR of intermediate duration (5 - 20 min), whereas the type II MGluR agonist APDC (0.1 and 1.0 micromol) caused marked, but transient (3 - 5 min), pressor and tachycardic responses. The highest doses of DHPG and CCG-1, but not APDC, also evoked behavioural responses similar to a spontaneous nociceptive behavioural effect reported previously. 4. The type I and II mGluR antagonists (AIDA and LY307452, respectively) were also given approximately 5 min before the administration of the respective type I and II mGluR agonists (DHPG and APDC). Both compounds caused pressor and tachycardic responses, with the effect of AIDA, but not LY307452, returning to control levels before mGluR agonist administration. AIDA significantly attenuated the overall cardiovascular effects of DHPG, while LY307452 significantly attenuated the overall cardiovascular effects of APDC. 5. These results indicate that functional type I and II mGluRs exist in the spinal cord, and that their activation evokes prolonged cardiovascular effects.

Type I and II metabotropic glutamate receptors mediate depressor and bradycardic actions in the nucleus of the solitary tract of anaesthetized rats.

The potential role of metabotropic glutamate (mGlu) receptors in cardiovascular function in the nucleus of the solitary tract was examined following the microinjection of a number of selective mGlu receptor compounds into this site of anaesthetized rats. The prototypic mGlu receptor selective agonist 1S,3R-1-amino-cyclopentane dicarboxylate elicited depressor and bradycardic actions following microinjection into the nucleus tractus solitarius, which were similar to those produced by L-glutamate. Similarly, decreases in blood pressure and heart rate were observed upon administration of the type I and II selective mGlu receptor agonists, (R,S)-3,5-dihydroxyphenylglycine (DHPG) and 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (APDC), respectively. These actions of DHPG were selectively attenuated by (+/-)-1-aminoindane-1,5-dicarboxylate, a type I mGlu receptor antagonist, whilst cardiovascular responses to APDC were unaffected by this compound. Interestingly, the proposed type II antagonist, (2S,4S)-2-amino-4-(4,4-diphenylbut-1-yl)-pentane-1,5-doic acid, reduced the cardiovascular responses to intra-nucleus tractus solitarius administration of both APDC and DHPG. The type III mGlu receptor agonist, L-2-amino-4-phosphonobutyrate, however, failed to elicit any cardiovascular actions when microinjected into the nucleus tractus solitarius. These studies provide new evidence for functional type I and II mGlu receptors in modulating cardiovascular responses in the nucleus tractus solitarius.

Vasopressin V2 receptor enhances gain of baroreflex in conscious spontaneously hypertensive rats.

The aim of the present study was to determine the receptor subtype involved in arginine vasopressin (AVP)-induced modulation of baroreflex function in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats using novel nonpeptide AVP V1- and V2-receptor antagonists. Baroreceptor heart rate (HR) reflex was investigated in both SHR and WKY rats which were intravenously administered the selective V1- and V2-receptor antagonists OPC-21268 and OPC-31260, respectively. Baroreflex function was assessed by obtaining alternate pressor and depressor responses to phenylephrine and sodium nitroprusside, respectively, to construct baroreflex curves. In both SHR and WKY rats baroreflex activity was tested before and after intravenous administration of vehicle (20% DMSO), OPC-21268 (10 mg/kg), and OPC-31260 (1 and 10 mg/kg). Vehicle did not significantly alter basal mean arterial pressure (MAP) and HR values or baroreflex function in SHR or WKY rats. The V1-receptor antagonist had no significant effect on resting MAP or HR values or on baroreflex parameters in both groups of rats, although this dose was shown to significantly inhibit the pressor response to AVP (5 ng iv; ANOVA, P < 0.05). In SHR but not WKY rats the V2-receptor antagonist significantly attenuated the gain (or slope) of the baroreflex curve (to 73 +/- 3 and 79 +/- 7% of control for 1 and 10 mg/kg, respectively), although AVP-induced pressor responses were also attenuated with the higher dose of the V2-receptor antagonist. These findings suggest that AVP tonically enhances baroreflex function through a V2 receptor in the SHR.

A comparison of the development of renal hypertension in male and female rats.

1. The objective of this study was to determine the influence of gender on the development of renal hypertension in Sprague-Dawley rats using the Goldblatt two-kidney, one-clip (2K1C) model. In addition, this study examined the effect of ovariectomy upon the development of hypertension in female rats.2. At 10 weeks of age, male, intact female and ovariectomized female rats underwent clipping of the right renal artery or sham operation. Tail-cuff plethysmography was used to monitor the systolic blood pressure of all animals for 7 weeks post-clipping or sham operation. Rats were sub-grouped according to whether or not they developed hypertension (systolic blood pressure >=150 mmHg).3. Within 2 to 3 weeks of clipping, hypertension was induced in only 53% (n=120) of the intact female 2K1C rats, but in 83% (n=18) of the male and 78% (n=18) of the ovariectomized female rats.4. Seven weeks after right renal artery clipping, plasma renin activity was determined in a subset of each group and was found to be 5-6 fold higher in male (17.29+/-4.04 ng angiotensin I.h-1.ml-1) and ovariectomized female (9.71+/-1.25 ng angiotensin I.h-1.ml-1) hypertensive rats compared with their respective normotensive or sham-operated counterparts (3.39+/-0.58 ng angiotensin I.h-1.ml-1 and 1.60+/-0.41 ng angiotensin I.h-1.ml-1 respectively) (P<0.05, analysis of variance). In contrast, the plasma renin activity measured in intact female hypertensive rats was not significantly different from that measured in the corresponding 2K1C normotensive or sham-operated groups.5. These results indicate that the success rate of inducing renal hypertension in Sprague-Dawley rats is higher in males than in intact females. Furthermore, these results suggest that the induction of 2K1C hypertension may be influenced by ovarian hormones.

Modulation of AT1 receptor-mediated contraction of rat uterine artery by AT2 receptors.

The aim of this study was to characterize the angiotensin II receptors in isolated uterine arteries from non pregnant and pregnant rats, since it has been reported from binding studies that ovine uterine arteries contain AT2 receptors. Uterine arterial segments were obtained from virgin, non-pregnant and late pregnant (18-21 days) Sprague-Dawley rats and mounted in small vessel myographs. Concentration-response curves were constructed to angiotensin II (1 nM-10 microM) in the absence and presence of various angiotensin II receptor subtype selective compounds. These included losartan (AT1 antagonist; 1, 10 and 100 nM), PD 123319 (AT2 antagonist; 1 microM) and CGP 42112 (AT2 agonist; 1 microM). Responses to angiotensin II were measured as increases in force (mN) and expressed as a per cent of the response to a K+ depolarizing solution. Losartan (1, 10 and 100 nM) caused significant concentration-dependent rightward shifts of the angiotensin II concentration-response curve in uterine arteries from non-pregnant and pregnant rats. The pA2 values calculated from these data were 9.8 and 9.2, respectively, although the slope of the Schild plot in the non-pregnant group was less than unity. PD 123319 (1 microM) caused significant 6- and 3 fold leftward shifts of the angiotensin II concentration-response curve in uterine arteries from non-pregnant and pregnant rats, respectively. In vessels from pregnant rats, PD 123319 also significantly increased the maximum response to angiotensin II. CGP 42112 (1 microM) attenuated the response to angiotensin II of uterine arteries from non-pregnant rats. This was reflected by a 14 fold rightward shift of the angiotensin II concentration-response curve and a decrease in the maximum response. In uterine arteries from pregnant rats, CGP 42112 (1 microM) caused a 3 fold rightward shift of the angiotensin II concentration-response curve, but had no effect on the maximum response. PD 123319 (1 microM) and CGP 42112 (1 microM) had no effect on the concentration-response curves to phenylephrine (PE) of uterine arteries from non-pregnant or pregnant rats. In addition, CGP 42112 (1 nM-1 mM) had no vasodilator effect on tissues precontracted with phenylephrine. These results suggest that the contractile responses of the rat uterine artery are mediated by the AT1 receptor. Furthermore, in this vascular preparation, the AT2 receptor appears to inhibit the response mediated by the AT1 receptor, although, this is not uniform between the non-pregnant and pregnant states.

Effects of the CCK(A) receptor antagonists SR 27897B and PD140548 on baroreflex function in conscious rats.

Since the cardiovascular effects of cholecystokinin (CCK) seem to particularly involve the A ('peripheral') subtype of CCK (CCK[A]) receptor, we examined the actions of two novel, highly selective CCK(A) receptor antagonists, PD140548 (N-alpha-methyl-N[(tricyclo[3.3.1.1(3,7)]dec-2-yloxy)carbony l]-L-tryptophyl]-D-3-(phenylmethyl)-beta-alanine) and SR 27897B (1-[[2-(4-(2-chlorophenyl)thiazol-2-yl)aminocarbonyl]acetic acid) on CCK-induced alterations in blood pressure and heart rate, and on the baroreceptor reflex in the conscious, instrumented rat. CCK (2 microg, i.v.) produced a pressor response and biphasic effects on heart rate involving an initial bradycardia followed by a pronounced tachycardia. Administration of PD140548 (10 mg/kg, i.v.) and SR 27897B (0.6 mg/kg, i.v.) significantly inhibited the pressor effects of CCK (35 and 47%, respectively), whilst reversing the bradycardic responses to a tachycardia. The CCK(A) receptor antagonists had different effects on the baroreceptor heart rate reflex since only PD140548 caused a significant increase in the gain or sensitivity of the reflex. This effect of PD140548 on gain is likely to occur via a central mechanism and may reflect the increased lipophilicity of PD140548 relative to SR 27897B. Overall, these investigations provide new evidence for the involvement of the CCK(A) receptor in cardiovascular regulation.

Role of the brain renin-angiotensin system in the maintenance of blood pressure in conscious spontaneously hypertensive and sinoaortic baroreceptor-denervated rats.

1. Evidence suggesting an involvement of the brain renin-angiotensin system (RAS) in the development/maintenance of hypertension in spontaneously hypertensive rats (SHR) relies, in part, on early experimental data reporting centrally mediated antihypertensive effects of saralasin. However, recent data using non-peptide AT1 receptor antagonists does not always support this theory because these compounds usually do not lower blood pressure when given centrally. 2. In the present study we have re-assessed the central effects of saralasin in conscious SHR as well as in sinoaortic baroreceptor-denervated (SAD) rats. Both of these models exhibit heightened sensitivity to the central pressor effects of angiotensin II (AngII) and, thus, any potential antihypertensive activity would provide functional evidence of activated brain RAS mechanisms in these models. 3. In SHR, saralasin failed to lower mean arterial pressure (MAP) when given intracerebroventricularly (i.c.v.) as bolus or infusion doses that blocked the centrally mediated pressor effect of AngII. 4. In SAD rats, there was a marked impairment of the baroreceptor-heart rate reflex function and enhanced centrally mediated pressor responses to AngII. However, i.c.v. saralasin infusions again did not alter MAP. 5. Collectively, these results suggest that the central RAS is not involved in the maintenance of MAP in SHR and SAD rats, both of which are models exhibiting a functional hyperresponsiveness to AngII.

A simple versatile method for measuring tail cuff systolic blood pressure in conscious rats.

1. The non-invasive measurement of tail cuff systolic blood pressure in conscious rats is routinely used in long-term cardiovascular studies. There are a number of commercially available tail cuff systems, however, these apparatus are generally expensive and are dedicated for single-task operations. In the present study, a simple method for measuring systolic blood pressure, which requires only minor modifications to the existing hardware found in most cardiovascular laboratories, is described. 2. Systolic blood pressure measurements were made in the conventional manner by determining the systolic blood pressure which coincided with the restoration of the caudal artery pulse. This was achieved by using an inexpensive piezo-electric pulse transducer to detect the pulse, and this was coupled to a standard data-acquisition system (MacLab, ADInstruments) normally set up to record blood pressure. This method was compared with another established tail cuff method, as well as with direct intra-arterial recordings. 3. It was found that the results obtained using both tail cuff systems were in good agreement when systolic blood pressure was measured in Wistar-Kyoto rats and spontaneously hypertensive rats. In addition, systolic blood pressure was measured over 4 weeks in 2K1C rats and sham-operated rats, with both tail cuff methods producing similar results, which were not significantly different from direct intra-arterial recordings in the same animals. 4. Thus, in the present study, with only minor modifications, the same equipment was used for both direct and indirect determinations of systolic blood pressure. This situation differs from other conventional tail cuff systems since these items are designed for a single purpose. Therefore, the current method using piezo-electric sensor/MacLab-technology should be viewed as a relatively simple, flexible and cheap alternative method to measure tail cuff systolic blood pressure in conscious rats.