Perivascular Nerve Stimulation Research Articles

Investigation of the vascular effects of short chain fatty acids in rat mesenteric resistance arteries

Fiber rich diets have been shown to prevent the onset and reduce severity of hypertension1, a condition affecting 20-30% of adults in North America. High fiber diets facilitate the production of short chain fatty acids (SCFAs) by gut bacteria of which, in humans, acetate is the most abundant, followed by propionate and butyrate. It has been proposed that these three SCFAs may have direct effects on blood vessels and so may contribute to regulation of blood pressure2. Further research is needed to determine the mechanisms underlying the vascular actions of SCFAs. We tested the hypothesis that SCFAs can induce vasodilation and limit nerve-evoked vasoconstriction in rat mesenteric vasculature. Direct effects on vascular tone were assessed by constructing cumulative concentration-response curves to SCFAs in isolated third order rat mesenteric arteries mounted in a wire myograph and pre-stimulated with phenylephrine (PE) to increase tone. The effects of SCFAs on nerve-evoked vasoconstriction were assessed by constructing frequency-responses curves to perivascular nerve stimulation in a perfused rat mesenteric bed in the presence and absence of SCFAs added to the perfusate. In both experimental approaches, pharmacological tools were used to investigate the mechanism of action of SCFAs. Propionate (3 μM - 1 mM) and acetate (0.3 μM - 1 mM) caused reductions in PE-evoked tone with maximum relaxation of 32.7 ± 7% and 31.6 ± 8.2%, respectively, and attenuated nerve-evoked vasoconstriction in mesenteric arteries at 10 mM - 60 mM propionate and 30 mM - 50 mM acetate. These effects were inhibited by blockers of nitric oxide (NO) synthase (L-NG-nitroarginine methyl ester, L-NAME, 100 μM) and endothelial Ca2+-activated K+ (KCa) channels (apamin, 50 nM; TRAM-34, 1 μM). L-NAME alone significantly inhibited relaxations elicited by acetate indicating a predominant role for NO, whereas the actions of propionate were significantly inhibited by apamin and TRAM-34, indicating a predominant role for endothelial KCa channels in this response. These data indicate that SCFAs may cause vasodilation in mesenteric resistance arteries via activation of endothelial pathways and that there may be a differential contribution of NO and KCa channels to responses of individual SCFAs. 1. Appel LJ, Moore TJ, Obarzanek E, et al. A clinical trial of the effects of dietary patterns on blood pressure. DASH Collaborative Research Group. N Engl J Med. 1997;336(16):1117-1124. doi:10.1056/NEJM199704173361601 2. Pluznick J. A novel SCFA receptor, the microbiota, and blood pressure regulation. Gut Microbes. 2014;5(2):202-207. doi:10.4161/gmic.27492

RETURN OF NERVE FUNCTION FOLLOWING RENAL DENERVATION IN THE PRESENCE OF ANGIOTENSIN-CONVERTING ENZYME INHIBITION IN SPONTANEOUSLY HYPERTENSIVE RATS

Objective: Regrowth of renal nerves and functional restoration occurs following renal denervation (RDN). There is little information regarding the impact of concomitant anti-hypertensive therapies on the extent of reinnervation following RDN. Angiotensin-II has previously been shown to promote renal vessel innervation. We hypothesised that angiotensin-converting enzyme inhibition (ACEi) prolongs the antihypertensive effects of RDN by delaying renal nerve regrowth. The aim of this study was to examine the effect of ACEi treatment on the return of nerve function at 12-weeks post-RDN in spontaneously hypertensive rats (SHR). Design and method: Male SHRs (∼12 weeks of age, n > 7/group) were implanted with a radiotelemetry probe to record mean arterial pressure (MAP). Rats were infused with enalapril (1 mg/kg/day s.c.) or vehicle for two weeks. Rats then underwent sham or surgical RDN procedures and ACEi/vehicle infusion continued for an additional 12 weeks following RDN. Vascular contraction to perivascular nerve stimulation in renal lobar arteries was assessed using wire myography to determine the extent of renal nerve function return. Results: ACEi and RDN independently caused an immediate and sustained reduction in MAP (ACEi: -12 ± 3 mmHg, RDN: -19 ± 4 mmHg, both P < 0.05) across the 12-week period compared to untreated counterparts. However, the blood pressure-lowering effect of RDN was not enhanced by concomitant ACEi. Heart mass was ∼16% less in ACEi-RDN rats compared to untreated counterparts. Return of neurovascular function was observed in RDN-treated rats, but levels were only partially restored to that of untreated rats (P < 0.001). ACEi alone attenuated the response to perivascular nerve stimulation (P < 0.05) but the effects of ACEi in combination with RDN were not additive. Conclusions: Here we show partial functional reinnervation is evident 12 weeks following RDN in the SHR. This may account for the sustained reduction in MAP following RDN. ACEi infusion alone attenuated neurovascular function, but ACEi combined with RDN did not affect the return of renal neurovascular function. These findings suggest that RDN and ACEi effectively lower blood pressure in the long term and may confer cardioprotective benefits despite the return of nerve function. However, ACE inhibition does not provide additive benefits to delaying renal reinnervation.

The role of perivascular adipose tissue and endogenous hydrogen sulfide in vasoactive responses of isolated mesenteric arteries in normotensive and spontaneously hypertensive rats.

Perivascular adipose tissue (PVAT) and hydrogen sulfide (H2S) play important roles in the modulation of vasoactive responses and can interfere with the ethiopathogenesis of essential hypertension. The aim of this study was to evaluate the mutual relationship between PVAT and H2S (endogenously produced, exogenous) in vasoactive responses of isolated mesenteric arteries (MA) in adult normotensive (Wistar) and spontaneously hypertensive rats (SHR). In SHR, hypertension was associated with cardiac hypertrophy and increased contractility; however, there were no differences in the amount of retroperitoneal fat between strains. PVAT revealed the anti-contractile effect on vasoconstriction induced by exogenous noradrenaline in both strains, but surprisingly, this effect was stronger in SHR. Concurrently; PVAT exhibited a pro-contractile effect on contractions to endogenous noradrenaline released from arterial sympathetic nerves in SHR, but not in Wistar rats. We confirmed the anti-contractile effect of H2S in both, the vascular wall and PVAT of Wistar rats because the pre-treatment with propargylglycine (PPG), an inhibitor of H2S producing enzyme, significantly increased the noradrenaline-induced contraction. In SHR, H2S in the vascular wall exhibited a pro-contractile effect that was eliminated by the presence of PVAT; however, the pre-treatment with PPG did not affect noradrenaline contraction farther. Nevertheless, unlike in Wistar rats, the presence of PVAT potentiated the vasorelaxant effect of exogenously applied H2S in SHR. Our results confirmed that PVAT of MA and endogenously produced H2S could manifest as pro-contractile or as anti-contractile. In SHR, unlike in Wistar rats, the pro-contractile effect of PVAT associated with the stimulation of perivascular nerves, and the pro-contractile effect of H2S in the arterial wall could represent pathologic features. On the other hand, PVAT of SHR is endowed with compensatory vasoactive mechanisms, which include stronger anti-contractile action of an unknown factor (other than H2S) and potentiation of the vasorelaxant effect of exogenous H2S.

Rat mesenteric small artery neurogenic dilatation is predominantly mediated by β1 -adrenoceptors in vivo.

The prevailing dogma about neurogenic regulation of vascular tone consists of major vasodilatation caused by CGRP (and possibly substance P) released from sensory-motor nerves and vasoconstriction caused by noradrenaline, ATP and neuropeptode Y release from sympathetic nerves. Most studies on perivascular nerve-mediated vasodilatation are made in vitro. In the present study, we provide evidence indicating that in vivo electrical perivascular nerve stimulation in rat mesenteric small arteries causes a large β1-adrenoceptor-mediated vasodilatation, which contrasts with a smaller vasodilatation caused by endogenous CGRP that is only visible after inhibition of Y1 NPY receptors. Mesenteric arteries are densely innervated and the nerves are important regulators of vascular tone and hence blood pressure and blood flow. Perivascular sensory-motor nerves have been shown to cause vasodilatation in vitro. However, less is known about their function in vivo. Male Wistar rats (10-12 weeks old; n=72) were anaesthetized with ketamine (3mg kg-1 ) and xylazine (0.75mg kg-1 ) or pentobarbital (60mg kg-1 ). After a laparotomy, a section of second-order mesenteric artery was visualized in an organ bath after minimal removal of perivascular adipose tissue. The effects of electrical field stimulation (EFS) and drugs on artery diameter and blood flow were recorded with intravital microscopy and laser speckle imaging. EFS caused vasodilatation in arteries constricted with 1μm U46619 in the presence of 140μm suramin and 1μm prazosin. The vasodilatation was inhibited by 1μm tetrodotoxin and 5μm guanethidine, although not by the 1μm of the CGRP receptor antagonist BIBN4096bs. In the presence of 0.3μm Y1 receptor antagonist BIBP3226, BIBN4096bs partly inhibited the vasodilatation. Atenolol at a concentration 1μm inhibited the vasodilatation, whereas 0.1μm of the β2 -adrenoceptor selective antagonist ICI-118,551 had no effect. Increasing the extracellular [K+ ] to 20mm caused vasodilatation but was converted to vasoconstriction in the presence of 1μm BIBN4096bs, and constriction to 30mm potassium was potentiated by BIBN4096bs. Atenolol but not BIBN4096bs increased contraction to EFS in the absence of suramin and prazosin. In mesenteric small arteries of anaesthetized rats, EFS failed to stimulate major dilatation via sensory-motor nerves but induced sympathetic β1 -adrenoceptor-mediated dilatation.

Influence of Age on Anticontractile Effect of Perivascular Adipose Tissue in Normotensive and Hypertensive Rats.

Perivascular adipose tissue (PVAT) and its vasomodulatory effects play an important role in the physiology and pathophysiology of blood vessels. Alterations in PVAT associated with reduction in its anticontractile influence are proven to contribute to vascular dysfunction in hypertension. The aim of this study was to examine whether the changes in PVAT properties could participate in progression of vascular abnormalities in developing spontaneously hypertensive rats (SHR). Normotensive Wistar-Kyoto (WKY) rats and SHR, both in 5th and in 12th week of age, were used. Systolic blood pressure was similar between WKY rats and SHR in 5th week of age; however, in 12th week, it was significantly increased in SHR comparing to WKY rats. The amount of retroperitoneal fat was higher in WKY rats in both age groups, whereas body weight was higher in WKY rats only in 12th week, when compared to age-matched SHR. From isolated superior mesenteric arteries, two ring preparations were prepared for isometric tension recording, one with PVAT intact and other with PVAT removed. In WKY rats as well as in SHR, arterial contractile responses to noradrenaline, applied cumulatively on rings, were significantly inhibited in the presence of intact PVAT. In both age groups, anticontractile effect of PVAT was higher in WKY rats than in SHR. Neurogenic contractions, induced by electrical stimulation of perivascular sympathoadrenergic nerves, were significantly attenuated in the presence of PVAT in WKY mesenteric arteries from both age groups; however, in arteries from SHR, intact PVAT had no influence on this type of contractile responses. The results suggest that in SHR impairment of anticontractile effect of PVAT precedes hypertension and might contribute to its development.

Vasoconstrictor stimulus determines the functional contribution of myoendothelial feedback to mesenteric arterial tone.

In isolated resistance arteries, endothelial modulation of vasoconstrictor responses to α1 -adrenoceptor agonists occurs via a process termed myoendothelial feedback: localized inositol trisphosphate (InsP3 )-dependent Ca2+ transients activate intermediate conductance Ca2+ -activated K+ (IKCa ) channels, hyperpolarizing the endothelial membrane potential to limit further reductions in vessel diameter. We demonstrate that IKCa channel-mediated myoendothelial feedback limits responses of isolated mesenteric arteries to noradrenaline and nerve stimulation, but not to the thromboxane A2 mimetic U46619 or to increases in intravascular pressure. In contrast, in the intact mesenteric bed, although responses to exogenous noradrenaline were limited by IKCa channel-mediated myoendothelial feedback, release of NO and activation of endothelial small conductance Ca2+ -activated K+ (SKCa ) channels in response to increases in shear stress appeared to be the primary mediators of endothelial modulation of vasoconstriction. We propose that (1) the functional contribution of myoendothelial feedback to arterial tone is determined by the nature of the vasoconstrictor stimulus, and (2) although IKCa channel-mediated myoendothelial feedback may contribute to local control of arterial diameter, in the intact vascular bed, increases in shear stress may be the major stimulus for engagement of the endothelium during vasoconstriction. Constriction of isolated resistance arteries in response to α1 -adrenoceptor agonists is limited by reciprocal engagement of inhibitory endothelial mechanisms via myoendothelial feedback. In the current model of feedback, agonist stimulation of smooth muscle cells results in localized InsP3 -dependent Ca2+ transients that activate endothelial IKCa channels. The subsequent hyperpolarization of the endothelial membrane potential then feeds back to the smooth muscle to limit further reductions in vessel diameter. We hypothesized that the functional contribution of InsP3 -IKCa channel-mediated myoendothelial feedback to limiting arterial diameter may be influenced by the nature of the vasoconstrictor stimulus. To test this hypothesis, we investigated the functional role of myoendothelial feedback in modulating responses of rat mesenteric resistance arteries to the adrenoceptor agonist noradrenaline, the thromboxane A2 mimetic U46619, increases in intravascular pressure and stimulation of perivascular sympathetic nerves. In isolated arteries, responses to noradrenaline and stimulation of sympathetic nerves, but not to U46619 and increases in intravascular pressure, were modulated by IKCa channel-dependent myoendothelial feedback. In the intact mesenteric bed perfused under conditions of constant flow, responses to exogenous noradrenaline were modulated by myoendothelial feedback, but shear stress-induced release of NO and activation of endothelial SKCa channels appeared to be the primary mediators of endothelial modulation of vasoconstriction to agonists and nerve stimulation. Thus, we propose that myoendothelial feedback may contribute to local control of diameter within arterial segments, but at the level of the intact vascular bed, increases in shear stress may be the major stimulus for engagement of the endothelium during vasoconstriction.

Social stress-induced blood pressure increase in borderline hypertensive rats is associated with endothelial dysfunction in the resistant arteries

ObjectivesSeveral studies have observed that altered endothelial function is involved in the development of stress-induced hypertension. The aim of this study was to investigate the effects of chronic social stress (crowding) on endothelium-dependent relaxation (EDR) of the superior mesenteric artery (SMA) and of small resistant mesenteric arteries (MA) as well as on neurogenic contractions of SMA in adult borderline hypertensive rats (BHR).MethodsTwelve-week-old BHR (offspring of spontaneously hypertensive dams and Wistar-Kyoto sires) males were exposed to crowding (living space: 200 cm2/rat) for eight weeks. Control BHR were kept in the groups of four rats per cage (living space: 480 cm2/rat). Systolic blood pressure (sBP) was determined by the tail-cuff method.Vascular function was investigated in the isolated arteries at isometric conditions.EDR was assessed using acetylcholine test.ResultsCrowding significantly increased sBP of BHR to the hypertensive values. Results showed that stress did not affect total acetylcholine-induced relaxation and its nitric oxide (NO)-dependent and NO-independent components in the SMA. In the resistant MA, stress reduced total acetylcholine-induced relaxation by reducing NO-independent component, without the alterations of its NO-dependent component.However, stress failed to affect significantly neurogenic contractions of SMA elicited by electrical stimulation of perivascular sympathetic nerves and vasoconstriction induced by exogenous noradrenaline in SMA.ConclusionIn conclusion, chronic social stress can accelerate the development of hypertension in BHR, which seems to be associated with NO-independent endothelial dysfunction in small resistant arteries.Supported by the grants VEGA No. 2/0190/17 and APVV-16-0263.

PS 13-30 ROUX-EN-Y GASTRIC BYPASS SURGERY AMELIORATES HIGH BLOOD PRESSURE THROUGH ANTAGONIZING OVERDRIVE OF THE SYMPATHETIC NERVE

Objective: Roux-en-Y Gastric Bypass (RYGB) Surgery can effectively prevent cardiometabolic dysfunction, however its mechanism is little known. We investigated the hypothesis that the favorable effects on hypertension (HTN) and cardiovascular (CV) disturbances are mediated by antagonizing overdrive of the sympathetic nervous system (SNS). Design and Method: Hypertensive patients with metabolic disturbances underwent laparoscopic RYGB surgery, and spontaneously hypertensive rats (SHRs) underwent RYGB or sham surgery. Blood pressure (BP), heart rate (HR), endothelium-dependent flow-mediated dilation, and anthropometric as well as laboratory parameters were measured at baseline and during follow-up. Changes of BP and HR in response to cold stress, renal sympathetic nervous activity (RSNA), vasoconstriction induced by electrical field stimulation, microinjection of nucleus of the solitary tract (NTS), and CV function and structure were examined in SHRs with or without surgery. Results: Compared with baseline, BP and HR were significantly reduced in both hypertensive patients with type 2 diabetes and rats. Impaired endothelial-dependent vasodilatation and metabolic disturbances in hypertensive patients were also ameliorated after surgery. CV disturbances were reversed by surgery in SHRs. Under acute cold exposure, the variations in BP and HR were smaller in surgically treated SHRs, compared to sham SHRs. RSNA and vasoconstriction induced by perivascular nerve stimulation as well as NTS-mediated changes of BP were decreased in surgically treated SHRs, compared to sham SHR. Weight loss did not affect BP and RSNA in sham SHRs. Conclusions: RYGB surgery ameliorates HTN in both hypertensive patients and rats by inhibiting overdrive of the SNS. Therefore, targeting gastrointestine could be a novel strategy to treat HTN with metabolic disturbances.

Agmatine Modulation of Noradrenergic Neurotransmission in Isolated Rat Blood Vessels.

Agmatine, a vasoactive metabolite of L-arginine, is widely distributed in mammalian tissues including blood vessels. Agmatine binding to imidazoline and α₂-adrenoceptors induces a variety of physiological and pharmacological effects. We investigated the effect of agmatine on contractile responses of the rat pulmonary artery and portal vein induced by electrical stimulation of perivascular nerves and by exogenous adrenergic substances. Experiments were performed on isolated segments of rat main pulmonary artery and its extralobular branches, and portal vein suspended in organ bath containing modified Krebs bicarbonate solution and connected to a force-displacement transducer for isometric tension recording. Electrical field stimulation (EFS) produced tetrodotoxin-sensitive contractile responses of pulmonary artery and portal vein. Besides the well known vasorelaxant actions, we found that agmatine also produced a concentration-dependent inhibition of neurogenic contractions induced by EFS in pulmonary arteries; however, the agmatine treatment did not influence the responses to exogenous noradrenaline. The inhibitory effect on EFS-induced contractions was not abolished by the α₂-adrenoceptor antagonist rauwolscine. In portal vein, in contrast, agmatine increased spontaneous mechanical contractions and enhanced the contractions induced by EFS. The results suggest that agmatine can significantly influence vascular function of pulmonary arteries and portal veins by modulating sympathetically mediated vascular contractions by pre- and postsynaptic mechanisms.

Depressed perivascular sensory innervation of mouse mesenteric arteries with advanced age

The dilatory role for sensory innervation of mesenteric arteries (MAs) is impaired in Old (∼24months) versus Young (∼4months) mice. We investigated the nature of this impairment in isolated pressurized MAs. With perivascular sensory nerve stimulation, dilatation and inhibition of sympathetic vasoconstriction observed in Young MAs were lost in Old MAs along with impaired dilatation to calcitonin gene-related peptide (CGRP). Inhibiting NO and prostaglandin synthesis increased CGRP EC50 in Young and Old MAs. Endothelial denudation attenuated dilatation to CGRP in Old MAs yet enhanced dilatation to CGRP in Young MAs while abolishing all dilatations to ACh. In Old MAs, sensory nerve density was reduced and RAMP1 (CGRP receptor component) associated with nuclear regions of endothelial cells in a manner not seen in Young MAs or in smooth muscle cells of either age. With advanced age, loss of dilatory signalling mediated through perivascular sensory nerves may compromise perfusion of visceral organs. Vascular dysfunction and sympathetic nerve activity increase with advancing age. In the gut, blood flow is governed by perivascular sensory and sympathetic nerves but little is known of how their functional role is affected by advanced age. We tested the hypothesis that functional sensory innervation of mesenteric arteries (MAs) is impaired for Old (24months) versus Young (4months) C57BL/6 male mice. In cannulated pressurized MAs preconstricted 50% with noradrenaline and treated with guanethidine (to inhibit sympathetic neurotransmission), perivascular nerve stimulation (PNS) evoked dilatation in Young but not Old MAs while dilatations to ACh were not different between age groups. In Young MAs, capsaicin (to inhibit sensory neurotransmission) blocked dilatation and increased constriction during PNS. With no difference in efficacy, the EC50 of CGRP as a vasodilator was ∼6-fold greater in Old versus Young MAs. Inhibiting nitric oxide (l-NAME) and prostaglandin (indomethacin) synthesis increased CGRP EC50 in both age groups. Endothelial denudation reduced the efficacy of dilatation to CGRP by ∼30% in Old MAs yet increased this efficacy ∼15% in Young MAs while all dilatations to ACh were abolished. Immunolabelling revealed reduced density of sensory (CGRP) but not sympathetic (tyrosine hydroxylase) innervation for Old versus Young MAs. Whereas the distribution of CGRP receptor proteins was similar in SMCs, RAMP1 associated with nuclear regions of endothelial cells of Old but not Young MAs. With advanced age, the loss of sensory nerve function and diminished effectiveness of CGRP as a vasodilator is multifaceted and may adversely affect splanchnic perfusion.

Gastrointestinal intervention ameliorates high blood pressure through antagonizing overdrive of the sympathetic nerve in hypertensive patients and rats.

BackgroundWe investigated the hypothesis that the favorable effects of gastrointestinal (GI) intervention on hypertension (HTN) and cardiovascular (CV) disturbances are mediated by antagonizing overdrive of the sympathetic nervous system (SNS).Methods and ResultsHypertensive patients with metabolic disturbances underwent laparoscopic Roux‐en‐Y gastric bypass surgery, and spontaneously hypertensive rats (SHRs) underwent RYGB or sham surgery. Blood pressure (BP), heart rate (HR), endothelium‐dependent flow‐mediated dilation, and anthropometric as well as laboratory parameters were measured at baseline and during follow‐up. Changes of BP and HR in response to cold stress, renal sympathetic nervous activity (RSNA), vasoconstriction induced by electrical field stimulation, microinjection of nucleus of the solitary tract (NTS), and CV function and structure were examined in SHRs with or without surgery. Compared with baseline, BP and HR were significantly reduced in both hypertensive patients with type 2 diabetes and rats. Impaired endothelial‐dependent vasodilatation and metabolic disturbances in hypertensive patients were also ameliorated after surgery. CV disturbances were reversed by surgery in SHRs. Under acute cold exposure, the variations in BP and HR were smaller in surgically treated SHRs, compared to sham SHRs. RSNA and vasoconstriction induced by perivascular nerve stimulation as well as NTS‐mediated changes of BP were decreased in surgically treated SHRs, compared to sham SHR. Weight loss did not affect BP and RSNA in sham SHRs.ConclusionsGI intervention ameliorates HTN in both hypertensive patients and rats by inhibiting overdrive of the SNS. Therefore, targeting gastrointestine could be a novel strategy to treat HTN with metabolic disturbances.

Cholinergic innervation of human mesenteric lymphatic vessels

The cholinergic neurotransmission within the human mesenteric lymphatic vessels has been poorly studied. Therefore, our aim is to analyse the cholinergic nerve fibres of lymphatic vessels using the traditional enzymatic techniques of staining, plus the biochemical modifications of acetylcholinesterase (AChE) activity. Specimens obtained from human mesenteric lymphatic vessels were subjected to the following experimental procedures: 1) drawing, cutting and staining of tissues; 2) staining of total nerve fibres; 3) enzymatic staining of cholinergic nerve fibres; 4) homogenisation of tissues; 5) biochemical amount of proteins; 6) biochemical amount of AChE activity; 6) quantitative analysis of images; 7) statistical analysis of data. The mesenteric lymphatic vessels show many AChE positive nerve fibres around their wall with an almost plexiform distribution. The incubation time was performed at 1 h (partial activity) and 6 h (total activity). Moreover, biochemical dosage of the same enzymatic activity confirms the results obtained with morphological methods. The homogenates of the studied tissues contain strong AChE activity. In our study, the lymphatic vessels appeared to contain few cholinergic nerve fibres. Therefore, it is expected that perivascular nerve stimulation stimulates cholinergic nerves innervating the mesenteric arteries to release the neurotransmitter AChE, which activates muscarinic or nicotinic receptors to modulate adrenergic neurotransmission. These results strongly suggest, that perivascular cholinergic nerves have little or no effect on the adrenergic nerve function in mesenteric arteries. The cholinergic nerves innervating mesenteric arteries do not mediate direct vascular responses.

Abstract 538: Prolonged Functional Sympathetic Efferent Denervation Following Ablation of the Renal Nerves in SHR

Renal denervation (RDN) has been shown to be an effective and safe treatment for human resistant hypertension and is considered one of the most promising advances in the field of hypertension. However, the mechanisms mediating the sustained decrease in arterial pressure in response to RDN in humans are unknown. This issue is particularly perplexing, given reports that the depressor effect of RDN in animal models is short lived. The current dogma in the literature is that overtime the efferent sympathetic nerves regrow and so the longevity of the depressor effect of RDN in humans cannot be ascribed to the absence of efferent renal sympathetic nerve activity. We hypothesize that the renal sympathetic nerves regenerate following RDN, and arrive at the target tissues, but the functional response is not fully restored and this contributes to the long-term depressor effect of RDN. Our aim was to examine mean arterial pressure (MAP) and vascular function in spontaneously hypertensive rats (SHR) following RDN. In 10 week old male SHR radiotelemetry probes were implanted to measure MAP. At 12 weeks of age, following 3 days basal MAP recording, sham (n=5) or bilateral RDN (n=5) surgery was performed. MAP was recorded until 24 weeks of age (12 weeks post-RDN). Renal lobar arteries were mounted on a myograph and membrane potential and tension responses to perivascular nerve stimulation assessed. Perivascular nerve stimulation with single pulses at increasing voltage evoked excitatory junction potentials (Ejps) of increasing amplitude in SHR-sham. Ejp amplitude was markedly reduced in arteries from SHR-RDN (P<0.01). Repetitive stimulation (1-8Hz, 5s) evoked slow depolarisation and contraction. At 5Hz 5s, slow depolarisation was 12±2 mV in SHR-shams and 7±2 mV in SHR-RDN (P<0.01), and contractions were smaller than SHR-sham (10 ± 3% vs 22 ± 2% HiK; P<0.01). In conclusion, smaller Ejps suggest that close contacting neuroeffector junctions are not reformed and blunted vasoconstrictor responses indicate neuro-vascular function has only partially returned 12 weeks post-RDN (Fig. 2). Our finding of continued ‘functional’ denervation is compatible with our data that RDN causes a sustained decrease in arterial pressure in SHR.

PAR‐2 RECEPTORS MODULATE NERVE‐EVOKED CONTRACTION OF RESISTANCE ARTERIES

The sympathetic nervous system regulates arterial diameter and thus blood flow and blood pressure. Protease‐activated receptors (PARs) are a novel class of G‐protein coupled receptors activated by protealytic cleavage of the extracellular domain to reveal a tethered ligand. Four sub‐types of PARs have been identified, of which PAR‐1 and PAR‐2 are expressed in the vasculature. We have investigated whether activation of PAR‐2 can modulate nerve‐mediated vasoconstriction in rat isolated mesenteric resistance arteries denuded of endothelium. Stimulation of peri‐vascular nerves elicited frequency‐dependent increases in tone which were abolished by the α1‐adrenoceptor antagonist prazosin. Activation of PAR‐2 by either trypsin, the tethered ligand mimetic peptide FLIGRL or AC264613, a synthetic PAR‐2 ligand, significantly attenuated nerve‐mediated responses. Increases in tone to bath applied noradrenaline were attenuated but to a significantly lesser extent. The actions of trypsin were prevented by the trypsin inhibitor aprotinin. Thrombin, which activates PAR‐1 receptors, did not attenuate nerve‐mediated or noradrenaline‐evoked increases in tone. These data indicate that PAR‐2 receptors can modulate nerve‐mediated vasoconstriction of resistance arteries and so may be novel regulators of sympathetic vascular tone.

Sympathetic hyperinnervation in obesity is associated with oxidative stress and inflammatory cell NGF synthesis

Obesity is associated with an increased density of the sympathetic nerve plexus over arteries that control blood pressure. Here we examine the role of oxidative stress and inflammatory cell infiltration to changes in perivascular nerve innervation over small mesenteric arteries from C57BL/6 and NOX2−/− mice fed a normal or high fat diet. Changes in smooth muscle membrane potential were measured in response to perivascular nerve stimulation. Innervation density, nerve growth factor (NGF) and inflammatory cell type were examined by immunohistochemistry. Oxidative stress was measured using dihydroethidium fluorescence. Denser nerve plexuses over arteries from obese C57BL/6 mice were associated with larger excitatory junction potentials (EJPs; P<0.05). Oxidative stress and numbers of inflammatory cells positive for NGF were also increased (P<0.05). By contrast, the perivascular nerve plexus in obese NOX2−/− mice was less dense (P<0.05) and EJP amplitude was decreased (P<0.05). Oxidative stress and numbers of NGF positive inflammatory cells were also decreased (P<0.05). We demonstrate that obesity causes an increase in the density of the sympathetic nerve plexus via NOX2dependent and independent processes. Hyperinnervation occurs due to increased oxidative stress and infiltration of NGF producing inflammatory cells. This study was supported by the NHMRC to REH (466009).

Effect of Wine Polyphenol Resveratrol on the Contractions Elicited Electrically or by Norepinephrine in the Rat Portal Vein

We investigated the effects of resveratrol on rat portal vein (RPV) contractility without endothelium. Contractions were produced by electrical field stimulation of perivascular nerves (EFS), norepinephrine (NE), adenosine 5'-triphosphate (ATP), high K(+) solution and by calcium chloride (CaCl2 ) in Ca(2+) -free and high K(+) , Ca(2+) -free solution. The EFS-evoked contractions were more sensitive to resveratrol and to NS1619-selective openers of big calcium-sensitive (BKCa ) channels, than NE-evoked contractions. Effects of resveratrol on the ATP-evoked contractions were weak. Blockers of BKCa channels partly inhibited the effect of resveratrol only in EFS-contracted preparations. Western blotting showed that RPV expressed KCa 1.1 protein. Inhibitors of ATP- and voltage-sensitive K(+) channels did not modify the effects of resveratrol. None of the antagonists of K(+) channels affected the resveratrol inhibition of NE-evoked contractions and effect of high concentrations of resveratrol on the EFS-evoked contractions. Resveratrol more potently inhibited CaCl2 than potassium chloride contractions of RPV. Thus, BKCa channels partly mediate the inhibitory effect of resveratrol on the neurogenic contractions of RPV. The smooth muscle Ca(2+) channels and/or Ca(2+) mobilizing through cells might be involved in the effects of resveratrol on the contractility of RPV. Our results are important for better understanding the impact of resveratrol on the portal circulation.

Do cholinergic nerves innervating rat mesenteric arteries regulate vascular tone?

Vascular blood vessels have various types of cholinergic acetylcholine receptors (AChR), but the source of ACh has not been confirmed. Perivascular adrenergic nerves and nonadrenergic calcitonin gene-related peptide (CGRP)-containing (CGRPergic) nerves innervate rat mesenteric arteries and regulate vascular tone. However, function of cholinergic innervation remains unknown. The present study investigated cholinergic innervation by examining effects of cholinesterase inhibitor (neostigmine), a muscarinic AChR antagonist (atropine), and a nicotinic AChR antagonist (hexamethonium) on adrenergic nerve-mediated vasoconstriction and CGRPergic nerve-mediated vasodilation in rat mesenteric vascular beds without endothelium. In preparations treated with capsaicin (CGRP depletor) or in the presence of N(ω)-nitro-l-arginine methyl ester (nonselective nitric oxide synthase inhibitor), perivascular nerve stimulation (PNS; 2-12 Hz) evoked a frequency-dependent vasoconstriction. In the same preparations, exogenous norepinephrine induced a concentration-dependent vasoconstriction. Atropine, hexamethonium, and neostigmine had no effect on vasoconstrictor responses to PNS and norepinephrine injections. In denuded preparations, these cholinergic agents did not affect the PNS (12 Hz)-evoked release of norepinephrine in perfusate. In preconstricted preparations without endothelium in the presence of guanethidine (adrenergic neuron blocker), PNS (1-4 Hz) induced a frequency-dependent vasodilation, which was not affected by atropine, hexamethonium, and neostigmine. In denuded preparations treated with capsaicin and guanethidine, PNS did not induce vascular responses, and atropine, neostigmine, and physostigmine had no effect on PNS. Immunohistochemistry study showed choline acetyltransferase-immunopositive fibers, which were resistant to capsaicin and 6-hydroxydopamine (adrenergic toxin). These results suggest that rat mesenteric arteries have cholinergic innervation, which is different from adrenergic and capsaicin-sensitive nerves and not associated with vascular tone regulation.

Dysbalance in sympathetic neurotransmitter release and action in cirrhotic rats: Impact of exogenous neuropeptide Y

Splanchnic vasodilation is an essential disturbance in portal hypertension. Increased systemic sympathetic nerve activity is well known, but potential corresponding vascular desensitization is incompletely characterized. Release of splanchnic sympathetic neurotransmitters noradrenaline (NA) and co-transmitter neuropeptide Y (NPY) remains to be elucidated. Finally, the effects of exogenous NPY on these mechanisms are unexplored. Portal vein ligated cirrhotic, and control rats were used for in vitro perfusion of mesenteric arteries. Depletion of vascular pressure response was induced by repetitive electric sympathetic perivascular nerve stimulation (PNS) and performed in the absence and presence of exogenous NPY. Additionally, PNS-induced release of NA and NPY was measured. Mesenteric PNS-induced pressure response was lower in portal hypertension. Depletion of the pressure response to PNS, representing the degree of desensitization, was enhanced in portal hypertension. NA release was elevated, whereas NPY release was attenuated in cirrhosis. Administration of exogenous NPY led to marked recovery from desensitization and vasoconstrictive improvement in cirrhotic rats, being associated with more pronounced decrease of NA release. Pronounced depletion of splanchnic arterial pressure-response to repetitive sympathetic nerve stimulation in cirrhosis is partly attributable to altered NA release as well as to deficient NPY release. External NPY restores vascular contractility and attenuates pathologically elevated NA release in the portal hypertensive mesenteric vasculature, revealing post-, and prejunctional effects at the vascular smooth muscle motor endplate; therefore outlining encouraging therapeutic strategies.

50 INFLUENCE OF EARLY LIFE VITAMIN D INSUFFICIENCY ON ARTERIAL PRESSURE AND VASCULAR AND RENAL FUNCTION

Background: Many women of reproductive age are vitamin D (VitD) deficient and the implications for the cardiovascular health of their offspring are unknown. The aim of this study was to examine the effects of in utero and early life VitD insufficiency in rats on cardiovascular and renal function in the offspring. Methods: Female Sprague-Dawley rats were fed either a VitD-deplete or –replete diet before, and throughout pregnancy and lactation. Offspring were maintained on the same diet as their mothers until 3 months of age, after which time, all rats were fed VitD replete diet. Conscious arterial pressure was measured at 2 and 12 months of age. Vascular function was assessed using myography and real-time PCR was used to examine renal gene expression of elements of the renin-angiotensin system (RAS). Kidney function was measured by clearance methods. Results: At 3 days postnatal renal mRNA expression of renin was reduced in VitD deficient males but not females. At 2 months VitD deficient offspring had elevated mean arterial pressure (MAP) and heart rate (HR) compared with controls. Mesenteric arteries exhibited endothelial vasodilator dysfunction. Following long term VitD repletion, renal RAS gene expression, MAP, HR and endothelial function had normalised. However, renal arteries had enhanced responsiveness to angiotensin II and perivascular nerve stimulation, and renal glomerular filtration rate was reduced by 40% (P<0.002). Conclusions: Early life VitD insufficiency causes transient increases in MAP and changes in renal RAS and endothelial function, but causes permanent changes in renal artery constriction and kidney function.

Neurogenic and Endothelial Nitric Oxide Regulates Blood Circulation in Lingual and Other Oral Tissues

Blood flow in oral tissues, including the tongue, salivary glands, gingiva, dental pulp, and lip, plays an important role in modulating the complex oral functions involved in food intake. Oral tissue circulation is regulated by nitric oxide (NO) synthesized by neuronal NO synthase mainly present in parasympathetic vasodilator neurons and also by endothelial NO sythase. Electrical stimulation of parasympathetic nerves causes vasodilatation and blood flow increase in the tongue, submandibular gland, and lip in various mammals in vitro and in vivo. Lingual arteries isolated from Japanese monkeys respond to perivascular nerve stimulation by electrical pulses and nicotine with relaxations that are mediated via neurogenic NO. There is evidence supporting the hypothesis that the superior salivatory nucleus delivers central information through the geniculate ganglion and greater petrosal nerve to the pterygopalatine ganglion, which sends off impulses through nitrergic nerves to oral tissues. Endothelial NO also plays an important role in improving oral blood circulation not only in resting conditions but also under conditions activated by chemical and physical stimuli in the tongue, submandibular and parotid glands, dental pulp/gingiva, and cheek pouch. Maintenance of health in oral circulation by minimizing factors responsible for impairment of endothelial and neurogenic NO bioavailability would be important for the prophylaxis of life-style related diseases.