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.

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.

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.

Regional hemodynamic effects of the AT1 receptor antagonist CV-11974 in conscious renal hypertensive rats.

Angiotensin II subtype 1 (AT1) receptor antagonists reduce mean arterial pressure in various experimental models of hypertension, including two-kidney, one clip (2K1C) renal hypertension. However, the regional hemodynamic mechanisms underlying the hypotensive effect of AT1 receptor antagonists in 2K1C rats under dynamic conditions have not been documented. Therefore, in the present study we determined the hemodynamic profile of the AT1 receptor antagonist CV-11974 in conscious 2K1C rats and sham-operated control rats. Approximately 4 weeks after clipping, rats underwent a further two-stage operation for implantation of Doppler flow probes on the contralateral (left) renal artery, superior mesenteric artery, and distal aorta as well as for the implantation of intravascular catheters. At least 24 hours after the last operation continuous recordings were made of mean arterial pressure; heart rate; and renal, mesenteric, and hindquarters flows and conductances (Doppler shift/mean arterial pressure) in response to three doses of CV-11974 (0.01, 0.1, and 1.0 mg/kg i.v.). CV-11974 caused a small hypotensive effect (decrease of approximately 15 mm Hg) in the sham group, but regional flows and vascular conductances did not change. By contrast, in 2K1C rats CV-11974 caused dose-dependent hypotension that was maximal (-19 +/- 6, -41 +/- 4, and -51 +/- 8 mm Hg, respectively) after 6 hours. These changes were associated with generalized vasodilatation (increased conductance) in all three vascular beds, although there were subtle differences with the different CV-11974 doses.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Effect of acute and chronic treatment with the angiotensin II subtype 1 receptor antagonist EXP 3174 on baroreflex function in conscious spontaneously hypertensive rats.

OBJECTIVE: To determine whether the angiotensin II subtype 1 (AT1) receptor antagonist EXP 3174 can modify the baroreceptor-heart rate reflex in conscious rats. DESIGN: EXP 3174 was given acutely, both peripherally and centrally, as well as chronically to spontaneously hypertensive rats (SHR), and baroreflex function was then examined. METHODS: Baroreceptor reflex activity was assessed by constructing sigmoidal mean arterial pressure (MAP)-heart rate curves after injection of pressor (phenylephrine) and depressor (sodium nitroprusside) agents. Baroreflex testing was performed before and after intravenous (1 mg/kg) and intracerebroventricular (1 microgram) administration of EXP 3174 in separate groups of conscious SHR and Wistar-Kyoto (WKY) rats. EXP 3174 was also given subcutaneously for 15 days (5 mg/kg per day) in SHR, and baroreceptor reflex activity was assessed approximately 24 h later. RESULTS: EXP 3174 lowered MAP after acute peripheral administration in SHR and, to a lesser extent, WKY rats. MAP was not altered after central administration of EXP 3174 in either group. Baroreceptor reflex function was not significantly modified after acute peripheral or central injection of the AT1 receptor antagonist in SHR or WKY rats. In contrast, chronic treatment of SHR with EXP 3174 markedly decreased MAP compared with vehicle-treated SHR. Moreover, the MAP-heart rate curve was shifted leftwards together with an enhancement of reflex bradycardia, such that baroreflex function was restored to the level observed in untreated WKY rats. CONCLUSION: Chronic, but not acute, administration of EXP 3174 normalized baroreflex function in SHR. This facilitation of baroreflex function might contribute to the greater antihypertensive effect observed after chronic administration of EXP 3174 in SHR.

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.

Impaired arterial baroreceptor reflex and cardiopulmonary vagal reflex in conscious spontaneously hypertensive rats.

The activity of baroreceptor reflexes and cardiopulmonary reflexes was examined in conscious spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) rats. The baroreceptor heart rate reflex, elicited by phenylephrine- and nitroprusside-induced changes in blood pressure, had a reduced range and lower heart rate plateau in SHR than in WKY rats, which suggests impaired vagal control of the heart rate in SHR. Cardiopulmonary receptor reflex activity was assessed by intravenous injections of phenyldiguanide which evoke the Bezold-Jarisch reflex. Phenyldiguanide elicited dose-dependent bradycardic and hypotensive responses in WKY rats, but these were significantly attenuated in SHR. This is the first demonstration of impaired Bezold-Jarisch responses in conscious SHR and provides evidence of both impaired vagally mediated arterial baroreceptor activity and impaired cardiopulmonary receptor activity in this rat strain.

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.

Lack of a centrally-mediated antihypertensive effect following acute or chronic central treatment with AT1-receptor antagonists in spontaneously hypertensive rats.

1. The role of the central renin-angiotensin system in the pathogenesis of hypertension in spontaneously hypertensive rats (SHR) was examined following acute and chronic intracerebroventricular (i.c.v.) infusions of angiotensin1 (AT1) receptor antagonists. 2. Groups of SHR were chronically instrumented for acute i.c.v. administration of the AT1 receptor antagonists, losartan and CV-11974, on mean arterial blood pressure (MAP) and heart rate (HR). Other groups of SHR also had mini-osmotic pumps implanted for chronic i.c.v. infusion of CV-11974. 3. Initially both young (15-18 weeks, n = 8) and old (25-29 weeks, n = 9) SHR received acute i.c.v. injections of losartan (10 micrograms) while a third group of young SHR received CV-11974 (1 microgram, n = 6). In all three groups of SHR, MAP and HR did not change up to 24 h after antagonist injection. However, changes in MAP and HR in response to i.c.v. angiotensin II (AII, 100 ng) were abolished 15 min after administration of the AT1 receptor antagonists. These responses had returned to control levels after 3 h in both groups given losartan but were still significantly depressed at 24 h in the CV-11974-treated group. By contrast, responses to i.v. AII (25 ng) before and 1 h after administration of AT1 receptor antagonists were not significantly different. 4. For chronic studies, four groups of SHR received chronic i.c.v. infusion of either vehicle (n = 9) or CV-11974 (1, 5 and 100 micrograms kg-1 day-1) (n = 4, 7 and 8 respectively) for 4 days. Baseline cardiovascular parameters were monitored daily together with changes in MAP and HR in response to both i.c.v. and i.v. AII (100 ng and 50 ng respectively) and i.v. phenylephrine (3 micrograms). Responses to i.c.v. carbachol (5 micrograms) were also recorded on day 4 while baroreflex function was assessed between days 1-3. In SHR treated chronically with i.c.v. vehicle or CV-11974, at 1 or 5 micrograms kg-1 day-1, resting MAP and HR did not vary over the four day infusion period. However, SHR treated with 100 micrograms kg-1 day-1 CV-11974 had significantly lower MAP compared to vehicle-treated SHR. While there was some variation in resting HR, there were no differences between the drug-treated and vehicle-treated groups. Pressor responses following i.c.v. AII administration were slightly, but significantly, inhibited on days 3 and 4 in the low dose CV-11974-treated (1 microgram kg-1 day-1) SHR. However, these responses were abolished on all 4 days in the 5 and 100 micrograms kg-1 day-1 CV-11974-treated groups. By contrast, changes in MAP and HR following i.v. AII injection did not vary over the 4 day infusion between SHR treated with the 2 lowest doses of CV-11974 and the vehicle-treated group. However, in the high dose CV-11974-treated SHR (100 micrograms kg-1 day-1), the cardiovascular effects of AII were abolished. In addition, phenylephrine (i.v.) and carbachol (i.c.v.) induced changes in MAP and HR were not significantly different in all four treatment groups. Similarly, baroreflex function was unaffected by i.c.v. infusion of 100 micrograms kg-1 day-1 CV-11974, except for a significant fall in BP50 which paralleled the fall in resting MAP. 5. Collectively, these results indicate that acute and chronic central AT1 receptor antagonism does not lower MAP in conscious SHR in doses which only block central AII-induced pressor activity. Chronic central infusion of CV-11974 at sufficiently high doses will lower MAP, as has been reported by others, but not without the abolition of the peripheral effects of AII. Therefore it is most likely that peripheral AT1 receptor blockade contributes to the hypotensive action of CV-11974 under these conditions.

Postjunctional alpha 2-adrenoceptors mediate venoconstriction in the hindquarters circulation of anaesthetized cats.

1 A study was made of the subtypes of postjunctional alpha-adrenoceptors which mediate arterial and venous constriction in the hindquarters circulation of anaesthetized cats, as measured by changes in perfusion pressure and vena cava blood flow, respectively. 2 It was found that, while noradrenaline caused constriction in both the arterial and venous compartments, methoxamine caused only arterial constriction. Clonidine and B-HT 920 also caused arterial and venous constriction although autodesensitization to both drugs occurred. 3 The ability of either prazosin or yohimbine to antagonize the constrictor effects of noradrenaline was also examined. It was found that the combination of both antagonist drugs abolished both the arterial and venous constrictor effects of noradrenaline. However, there was a greater prazosin-resistant response to noradrenaline in the venous compartment as compared with the arterial effects of noradrenaline. Yohimbine caused approximately equal reductions in the effect of noradrenaline in both arteries and veins, which was greater than that observed with prazosin. 4 These results suggest that, in the cat hindquarters, both alpha 1- and alpha 2-adrenoceptors are present in the arterial circulation, whereas there are mainly alpha 2-adrenoceptors in the venous circulation.

Comparison of the regional haemodynamic effects of the AT1-receptor antagonists, losartan and EXP 3174, in water-deprived Brattleboro rats.

1. Two separate groups (n = 8) of Brattleboro rats, chronically instrumented for the measurement of regional haemodynamics, underwent a 2-stage experimental protocol. Initially, animals had their drinking water removed and cardiovascular recordings were made every 30 min for the following 6 h. Then animals received either the AT1-receptor antagonist, losartan (3 mg kg-1, i.v., n = 8), or an initially equi-hypotensive dose of its active metabolite, EXP 3174 (1 mg kg-1, i.v., n = 8), and the resultant cardiovascular changes were monitored for a further 2 h. A third group of Brattleboro rats (n = 8) was water-deprived for 8 h to serve as a time control. 2. In all 3 groups of animals, mesenteric and hindquarters vasoconstriction (beginning approximately 30 and 120 min, respectively, after water was removed) occurred much earlier than changes in blood pressure, since increases in blood pressure were significant only after 5-6 h of water deprivation; renal perfusion was largely unchanged. The observation of a differential onset of haemodynamic changes (i.e. mesenteric > hindquarters >> renal) extends our previous findings, in which measurements were made only at the end of a 14 h period of water-deprivation. 3. When given after 6 h of water deprivation, losartan and EXP 3174 produced directionally similar, but temporally disparate, haemodynamic effects. EXP 3174 caused a depressor response associated with marked renal, mesenteric and hindquarters vasodilatations which were well maintained over 2 h. Losartan evoked similar changes to EXP 3174 in the first 5 min, but in contrast to EXP 3174, blood pressure showed some recovery and all 3 vascular conductance values returned to baseline (i.e. pre-drug)levels over the following 10-20 min. Thereafter, hypotension and renal, mesenteric and hindquarters vasodilatation again occurred, and these changes were maintained for the rest of the 2 h. However,compared with losartan, EXP 3174 caused significantly greater mesenteric and hindquarters vasodilatation,even at times when both compounds lowered blood pressure to the same extent.4. The biphasic cardiovascular response caused by losartan is consistent with the conversion of the parent compound to EXP 3174. Whether or not the enhanced vasodilator effect of EXP 3174 over losartan is related to pharmacodynamic differences (i.e., noncompetitive versus competitive antagonism,respectively), and/or to differences in the amount of EXP 3174 generated from losartan is not known at present.

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.

The influence of atropine and atenolol on the cardiac haemodynamic effects of NG-nitro-L-arginine methyl ester in conscious, Long Evans rats.

1. In the present study, the extent to which baroreflexes contribute to the cardiac effects of NG-nitro-L-arginine methyl ester (L-NAME) was assessed in conscious, Long Evans rats chronically instrumented with thoracic electromagnetic flow probes for the measurement of cardiac haemodynamics. 2. L-NAME (10 mg kg-1, i.v.) was administered in the absence (n = 6) and in the presence (n = 7) of atropine (1 mg kg-1) and atenolol (1 mg kg-1). 3. L-NAME caused a marked increase in mean arterial pressure and marked reductions in total peripheral conductance, cardiac output, heart rate, stroke volume, peak thoracic flow and the maximum rate of rise of aortic flow. 4. Administration of atropine, after the maximal bradycardic effect of L-NAME was established, restored the heart rate to resting levels. Concurrently, there was a reduction in stroke volume, such that cardiac output, although transiently elevated, did not show a sustained increase. No other variables were significantly affected by atropine. Additional administration of atenolol had no effect other than to cause a slight bradycardia, such that in the presence of atropine and atenolol, heart rate was not different from that in animals receiving atropine and atenolol before L-NAME. 5. In the presence of atropine and atenolol, L-NAME had similar pressor, vasoconstrictor and cardiac haemodynamic effects to those in untreated animals, although the bradycardia was significantly attenuated. However, there was still a significant reduction in heart rate following L-NAME in the presence of atropine and atenolol.6. These results indicate that the major component of the bradycardia following L-NAME is indirect and mediated through an increase in vagal efferent activity. However, the substantial reduction in cardiac function caused by L-NAME is not dependent on the autonomic control of the heart but rather, may depend on the increase in afterload and/or a direct effect of L-NAME on the heart and/or its vasculature.

Inhibition of the haemodynamic effects of angiotensin II in conscious rats by AT2-receptor antagonists given after the AT1-receptor antagonist, EXP 3174.

1. Conscious, Long Evans rats (n = 10), chronically instrumented for the measurement of regional haemodynamics, were studied on 3 consecutive experimental days to assess responses to angiotensin II (AII) (125 pmol kg-1, i.v.) and noradrenaline (1 nmol kg-1, i.v.) in the absence and presence of the AT2-receptor antagonist, PD 123319 (10 mg kg-1, i.v.) (day 1), the AT1-receptor antagonist, EXP 3174 (1 mg kg-1, i.v.) (day 2), and PD 123319 (10 mg kg-1, i.v.) given 24 h after EXP 3174 (day 3). 2. In naive rats (day 1), PD 123319 did not antagonize the haemodynamic effects of AII or noradrenaline. EXP 3174 (day 2) caused a marked, prolonged blockade of the haemodynamic effects of AII but not those of noradrenaline. Twenty four h after administration of EXP 3174 (day 3) there was still significant attenuation of the haemodynamic effects of AII. However, administration of PD 123319 at this time caused a further inhibition (lasting 1 h) of the effects of AII but not those of noradrenaline. 3. An identical 3 day protocol was used in a separate group of rats (n = 6) in which the AT2-receptor antagonist, PD 123177, was given instead of PD 123319, and the results were essentially the same, i.e., PD 123177 significantly attenuated the haemodynamic effects of AII but only when given 24 h after EXP 3174.4. In a separate group of rats (n = 4), a low dose of EXP 3174 (60 pg kg-' i.v.) was given to naive rats in order to simulate the degree of inhibition of the effects of All seen after administration of AT2-receptor antagonists in animals pretreated with EXP 3174. This low dose of EXP 3174 did not produce a sustained inhibition of the effects of All and the time course of recovery of All responses was similar to that seen with PD 123319 or PD 123177 given after the high dose of EXP 3174.5. The apparent inhibition of the effects of AII by the AT2-receptor antagonists, PD 123319 and PD 123177, when these were administered 24 h after the AT,-receptor antagonist, EXP 3174, may have been due to the functional activation of AT2-receptors and/or loss of AT2-receptor antagonist selectivity,and/or the displacement of nonspecifically bound EXP 3174 by AT2-receptor antagonists. While the latter explanation seems the most likely, these results raise the possibility that nonpeptide, All-receptor antagonists that act at both AT,- and AT2-receptors may have therapeutic advantages over selective AT,-receptor antagonists.

The effect of clonidine on venous haemodynamics in cats and dogs.

Experiments were undertaken to investigate a possible action of clonidine on venous haemodynamics, using cats and dogs with autoperfused hindquarters. This provided an arterial input to the region independent of cardiac output. Venous capacitance in the hindquarters was indicated by blood flow in the abdominal vena cava, as measured by an electromagnetic flow probe around this vessel. It was found that in both cats and dogs, clonidine given intravenously (5 micrograms kg-1 i.v.) or into the cisterna magna (1 microgram kg-1 i.c.m.) reduced mean arterial pressure and heart rate, but did not decrease hindquarter perfusion pressure. With the exception of dogs given i.v. clonidine, this drug caused a decrease in inferior vena cava (IVC) blood flow and this closely paralleled the hypotensive effect. However, when clonidine was given i.v. to dogs, the decrease in IVC blood flow preceded the hypotension. At the doses used, the reduction in IVC blood flow was larger following i.v. than i.c.m. clonidine in both species, and in all cases, it remained below control levels for at least 30 min, in spite of the constant arterial input to the region. In cats, dose-response curves were constructed to noradrenaline and adrenaline given into the perfusion circuit (i.a.) before and after clonidine. Following i.v. and i.c.m. clonidine, there was a selective depression of the venoconstrictor actions of the catecholamines, but no change in the arterial pressor action. Radiographic and latex studies in the dog and cat respectively were performed in order to visualize collateral blood flow that could account for the persistent decreases in IVC blood flow. In both species, intraspinal collateral flows were demonstrated which returned blood to the heart after ligation of the inferior vena cava below the renal veins. However, it was not possible to demonstrate radiographically any changes produced by clonidine in the collateral flow because of technical difficulties. These results suggest that clonidine causes a selective reduction in sympathetic tone to the veins that is mediated at least partly by a central action, as well as an expansion of the collateral venous routes. This, together with the selective impairment of venoconstrictor responses to both noradrenaline and adrenaline, may account for the decrease in cardiac output that is most often reported following clonidine.

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.

Preprogalanin mRNA is increased in vagal motor neurons following axotomy.

Expression of preprogalanin and tyrosine hydroxylase mRNA was examined in the rat dorsal vagal complex following nodose ganglionectomy and cervical vagotomy, using in situ hybridization of specific 35S-labelled oligonucleotides. Seven days after unilateral cervical vagotomy (and nodose ganglionectomy), neurons in the ipsilateral dorsal motor nucleus of the vagus and nucleus ambiguus expressed 6- to 10-fold increased levels of preprogalanin mRNA. In contrast, tyrosine hydroxylase mRNA was no longer expressed by cells of the dorsal motor nucleus of the vagus after the lesion. These results demonstrate that changes in the expression of the galanin and tyrosine hydroxylase genes occur in vagal motor neurons following lesion of their axons. More generally, these results, and those from other laboratories, demonstrate that specific alterations of neuropeptide and neurotransmitter production, are part of the reactive process activated by nerve injury.

Expression of c-fos and NGFI-A messenger RNA in the medulla oblongata of the anaesthetized rat following stimulation of vagal and cardiovascular afferents.

Messenger RNA encoding the immediate early genes (IEGs) c-fos and NGFI-A was localized by in situ hybridization of specific 35S-labelled oligonucleotides to detect activated neurones in the medulla oblongata following unilateral electrical stimulation of the vagus (nX) and aortic depressor nerve (ADN), and following mechanical stimulation of the left carotid sinus (CS). In electrically stimulated rats, c-fos and NGFI-A mRNA was strongly expressed in the nucleus tractus solitarius (NTS) (predominantly ipsilaterally), area postrema (AP) and in a dorsal subregion of the paratrigeminal nucleus (PTN). Lower levels of c-fos and NGFI-A mRNA were seen in the ipsilateral NTS and PTN following mechanical stimulation of the left CS. In general these data correlate with the topography of innervation by the different nerve afferents, although the expression in the PTN (and in some cases the AP) would not be predicted on the basis of neuronal innervation patterns reported for the rat. Expression of these IEGs also occurred in the rostral and caudal ventrolateral medulla and inferior olive of both stimulated and sham-operated rats; presumably due to effects of the anaesthesia and surgical procedures. In conclusion the localization of the expression of c-fos and NGFI-A mRNAs represents a useful neuroanatomical technique for detecting the cell bodies of neurones that are activated by cardiovascular nerve afferents and should allow the further characterization of the neurochemical identity of these neurones.