Raised tone reveals purinergic-mediated responses to sympathetic nerve stimulation in the rat perfused mesenteric vascular bed.

Noradrenaline and ATP are sympathetic co-transmitters. In rat isolated mesenteric small arteries, activation of sympathetic nerves can produce a vasoconstrictor response mediated by ATP. In contrast, the rat perfused mesenteric bed displays vasoconstrictor responses that are blocked solely by alpha1-adrenoceptor antagonists. This study assessed the effect of raising tone with a vasoconstrictor on purinergic and noradrenergic responses to sympathetic nerve stimulation in the rat perfused mesentery. Rat mesenteric vascular beds were perfused with physiological salt solution and responses to nerve stimulation, or P2X-receptor agonists, were determined under basal conditions and after raising tone with endothelin-1. The contribution of noradrenaline and ATP to sympathetic nerve-mediated responses was assessed using the alpha1-adrenoceptor antagonist, prazosin and the P2X-receptor desensitizing agent, alpha,beta-methyleneATP. The effect of endothelin-1 on excitatory junction potentials generated in response to nerve stimulation in isolated mesenteric arteries was also assessed. Under baseline conditions, responses to nerve stimulation were mediated solely by activation of alpha1-adrenoceptors. After raising perfusion pressure with endothelin-1 or the thromboxane mimetic 9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F2alpha (U44619), sympathetic nerve-mediated responses were larger than under basal conditions and the response was partly sensitive to P2X-receptor desensitization. Responses to exogenous P2X-receptor agonists were enhanced after treatment with endothelin-1, while endothelin-1 decreased the amplitude of excitatory junction potentials. These results indicate that ATP acts as an important, functional, sympathetic neurotransmitter in the perfused mesentery under raised tone conditions, where the perfusion pressure is closer to that found in vivo. This effect is due to a postjunctional enhancement of purinergic function.

Angiotensin-converting enzyme inhibition restores endothelial but not medial connexin expression in hypertensive rats.

OBJECTIVE AND DESIGN: Remodelling in the media and decreases in connexin (Cx) expression and size of endothelial cells occur in the caudal artery of spontaneously hypertensive rats (SHR). The objective of this study was to determine whether similar changes are found in the aorta and whether effects in both aorta and caudal artery are present in the pre-hypertensive period or can be reversed by antihypertensive treatment. METHODS AND RESULTS: In the aorta of SHR, there was no difference in endothelial cell size although Cxs 37 and 40 were decreased, compared with normotensive Wistar-Kyoto rats. Cxs 37 and 43 were also reduced in the media. These differences were not apparent in pre-hypertensive SHR. Inhibition of angiotensin-converting enzyme (ACE) in SHR decreased blood pressure and restored Cx expression in the endothelium of both aorta and caudal artery. The decreased endothelial cell size in the caudal artery or the reduced Cxs in the media of the aorta of SHR were unaffected by ACE inhibition. CONCLUSION: We conclude that cellular coupling is reduced in the endothelium of arteries of SHR, but this can be restored by inhibition of the renin-angiotensin system. Decreased cellular coupling in the media or decreased endothelial size in SHR were not reversed by this antihypertensive treatment.

Connexin37 is the major connexin expressed in the media of caudal artery.

OBJECTIVE: To determine the connexins (Cxs) involved in intercellular coupling within vascular muscle, the present study has quantified mRNA and protein expression for Cx37, Cx40, Cx43, and Cx45 in the caudal artery (CA) and thoracic aorta (ThA) of the rat. METHODS AND RESULTS: Real-time polymerase chain reaction and immunohistochemistry identified Cx37 as the most abundantly expressed Cx in the CA, with fine punctate staining observed in the media. Conversely, mRNA for Cx43 was 40-fold greater in the ThA than in the CA, with punctate staining in the endothelium and media of the ThA but confined to the endothelium in the CA. Western blotting confirmed the differences in the relative amounts of Cx43 between the 2 vessels. For both arteries, Cx45 was expressed to a lesser degree in the media but not in the endothelium, whereas Cx40 was found only in the endothelium. Cx37, Cx40, and Cx43 were expressed in the endothelium of both vessels, although the density of Cx40 plaques was significantly greater in the CA. CONCLUSIONS: The demonstration of Cx37 as the dominant Cx in the media of the CA highlights the potential heterogeneity in Cx involvement in vascular smooth muscle.

Structure, function, and endothelium-derived hyperpolarizing factor in the caudal artery of the SHR and WKY rat.

OBJECTIVE: To quantify structural and functional characteristics of the caudal artery from spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats with particular reference to endothelium-derived hyperpolarizing factor (EDHF). METHODS AND RESULTS: Ultrastructural studies showed that the number of myoendothelial gap junctions, smooth muscle cell (SMC) layers, and medial cross-sectional area were significantly greater in SHR than WKY. Intracellular dye labeling demonstrated hyperplasia of SMCs in SHR. Analysis of nerve-mediated excitatory junction potentials recorded in SMCs at the adventitial and luminal borders demonstrated decreased radial coupling of SMCs in SHR. In both SHR and WKY, in the presence of NG-nitro-L-arginine methyl ester and indomethacin, acetylcholine-elicited EDHF was abolished by charybdotoxin and apamin, while iberiotoxin had no effect, implicating the involvement of small and intermediate, but not large, calcium-activated potassium channels. EDHF was abolished by Gap-mimetic peptides, 18beta-glycyrrhetinic acid, and endothelial removal but not affected by the NO scavengers hydroxocobalamin and carboxy-PTIO. CONCLUSIONS: Significant differences in SMC morphology and homocellular and heterocellular coupling exist between the caudal artery of SHR and WKY rats. In the caudal artery of SHR, significantly greater heterocellular coupling compensates for other structural changes in the media to maintain a functional role for EDHF.

Decreased endothelial size and connexin expression in rat caudal arteries during hypertension.

OBJECTIVES: Hypertension is accompanied by endothelial dysfunction. The present study has investigated endothelial cell morphology and connexin expression in the caudal artery of the rat during the development of hypertension. METHODS: A significant increase in systolic blood pressure was detected from 9 weeks of age in spontaneously hypertensive male rats (SHR) compared to normotensive Wistar-Kyoto (WKY) rats, reaching a maximum by 11-12 weeks of age. Immunohistochemistry was used to quantify cell size and expression of connexins (Cxs) 37, 40 and 43 in the endothelium of prehypertensive (3-week-old) and hypertensive (12-week-old) rats. RESULTS: At 12 weeks, the size of endothelial cells and the expression of all three Cxs per endothelial cell were significantly less in SHR than WKY rats. At 3 weeks, there was no significant difference in cell size nor in the expression of Cxs 37 or 43; however, expression of Cx40 was significantly lower in SHR than in WKY rats. Between 3 and 12 weeks in WKY rats, there was no change in endothelial cell size, nor in the expression of Cxs 37, 40 and 43. In SHR, both cell size and Cx expression per endothelial cell were significantly decreased during the same developmental period, with a significant decrease in the density of Cx40 plaques. CONCLUSION: The development of hypertension in the SHR is accompanied by significant decreases in endothelial cell size and expression of Cx40, which may contribute to the endothelial dysfunction present in hypertension.

Two mechanisms underlie the slow noradrenergic depolarization in the rat tail artery in vitro.

In rat tail artery, short trains of electrical stimuli evoke both ATP-mediated excitatory junction potentials (EJPs) and a slow noradrenaline (NA)-mediated depolarization (NAD). Here we have investigated the contribution of α(1)- and α(2)-adrenoceptors to the NAD. The α(1)-adrenoceptor antagonist, prazosin (0.1µM), and the α(2)-antagonist, rauwolscine (1µM), reduced the amplitude of the NAD and in combination these agents virtually abolished the NAD. The K(ATP) channel blocker, glibenclamide (10µM) abolished the α(2)-adrenoceptor-mediated component of the NAD, indicating that activation of these receptors produces closure of K(ATP) channels. The α(1)-adrenoceptor-mediated component of the NAD was increased in amplitude by glibenclamide. Changes in membrane conductance were monitored by measuring the time constant of decay of EJPs (τEJP). The τEJP was increased during α(1)-adrenoceptor-mediated depolarization, indicating a decrease in membrane conductance; i.e. closure of K(+) channels. Broad-spectrum K(+) channel blockers (tetraethylammonium, 4-aminopyridine, Ba(2+)) and the TASK-1K(+) channel blocker, anandamide (10µM), did not reduce the α(1)-adrenoceptor-mediated NAD. The α(1)-adrenoceptor-mediated NAD was unaffected by the Cl(-) channel blockers, 9-anthracene carboxylic acid (100µM) and niflumic acid (10µM) or by the non-selective cation channel blocker, SKF 96365 (10µM). These findings indicate that the NAD is produced by activation of both α(1)-and α(2)-adrenoceptors. The α(2)-adrenoceptor-mediated component is produced by closure of K(ATP) channels whereas the α(1)-adrenoceptor-mediated component is most likely mediated by closure of another type of K(+) channel.

Nerve-evoked constriction of rat tail veins is potentiated and venous diameter is reduced after chronic spinal cord transection.

Despite reduced sympathetic activity below the level of a spinal cord injury (SCI), venoconstriction during autonomic dysreflexia increases venous return to the heart. Here, contractions of isometrically mounted tail veins from rats with spinal transection at T4 performed 8 - 10 weeks earlier are compared with those from sham-operated rats. After SCI, lumen diameter was reduced by ∼30% and the contractions evoked by electrical stimulation of the perivascular axons were larger than control. This augmentation of neurovascular transmission was not associated with enhanced sensitivity to α-adrenoceptor agonists or to adenosine-5'-triphosphate (ATP) although contractions to depolarization with K(+) were larger after SCI. The percentage reduction in nerve-evoked contraction after SCI produced by the α(1)-adrenoceptor antagonist prazosin (10 nM) was unchanged but that by the α(2)-adrenoceptor antagonist rauwolscine (0.1 µM) was reduced. The relative contribution of P2-purinoceptors to nerve-evoked contractions after α-adrenoceptor blockade, revealed by adding suramin (0.1 mM), was unchanged. The greater depolarization-induced contraction and the reduced contribution of α(2)-adrenoceptors to nerve-evoked contraction suggest that changes in the venous smooth muscle underlie the potentiation of neurovascular transmission after SCI. Furthermore, the smaller lumen diameter after SCI will increase the pressure that the veins exert on the luminal contents when they are neurally activated.

Sympathetic vasoconstriction is potentiated in arteries caudal but not rostral to a spinal cord transection in rats.

Sympathetic nerve-mediated contractions of mesenteric and tail arteries controlled by preganglionic neurones decentralized by a spinal cord injury (SCI) are potentiated, and likely contribute to autonomic dysreflexia. However, reactivity to the α(1)-adrenoceptor agonist phenylephrine has been reported to be enhanced in vascular beds controlled by preganglionic neurones lying both rostral and caudal to an SCI in vivo. Here responses of isometrically-mounted median and saphenous arteries isolated from rats 2 and 8 weeks after transection of the T4 spinal cord have been compared with those from sham-operated rats. After SCI, contractions of median arteries to perivascular nerve stimulation, to α-adrenoceptor agonists (phenylephrine and clonidine), to the P2X-purinoceptor agonist α,ß-methylene ATP, and to 60 mM K(+) were unchanged. Blockade of nerve-evoked contractions by α-adrenoceptor antagonists (prazosin and idazoxan) was not affected by SCI in either the median or saphenous arteries. In contrast, at 2 and 8 weeks after SCI, nerve-evoked contractions of saphenous arteries were potentiated. Saphenous arteries were less sensitive to phenylephrine 8 weeks after SCI, and their contractions to 60 mM K(+) were reduced. However, the sensitivity of saphenous arteries to clonidine was unchanged by SCI. Eight weeks after SCI, the reactivity of saphenous arteries to α,ß-methylene ATP was unchanged, but the P2-antagonist suramin produced more blockade of nerve-evoked contractions. These findings demonstrate that neurovascular transmission is enhanced in arteries located caudal, but not rostral, to a spinal transection. In the saphenous artery, the most likely explanation seems to be an increase in neurotransmitter release, as may occur in other inactive sympathetic pathways caudal to the lesion.

ATP is the predominant sympathetic neurotransmitter in rat mesenteric arteries at high pressure.

Most studies of neurovascular transmission in isolated small mesenteric arteries have used either isometric recording techniques or measured vasoconstriction in vessels with no distending pressure. Here we have used pressure myography to assess the contribution of noradrenaline and ATP to sympathetic neurotransmission in rat second-order mesenteric arteries. In arteries pressurized to 30 or 90 mmHg, activation of sympathetic axons with trains of electrical stimuli (50 pulses, 0.5-10 Hz) evoked frequency-dependent vasoconstrictions that increased in amplitude at higher pressure. In the presence of the P2-receptor antagonist suramin (0.1 mM), the amplitude of vasoconstrictions to trains at 2 and 10 Hz did not differ at 30 and 90 mmHg. In contrast, in the presence of the alpha(1)-adrenoceptor antagonist prazosin (0.1 microm) vasoconstrictions at 90 mmHg were larger than those at 30 mmHg. At both pressures, the combination of prazosin and suramin virtually abolished constrictions. The purinergic component of vasoconstriction (prazosin-resistant) was almost abolished by the L-type Ca(2+) channel antagonist nifedipine (1 microm). Increasing pressure from 30 to 90 mmHg decreased the resting membrane potential and increased the amplitude of purinergic excitatory junction potentials. These findings indicate that the contribution of ATP to neurovascular transmission increases when the pressure is raised from 30 to 90 mmHg, which is similar to the pressure second-order mesenteric arteries experience in vivo, and that Ca(2+) influx through L-type Ca(2+) channels is largely responsible for purinergic activation of the vascular smooth muscle.

Vascular gap junctions and implications for hypertension.

Four connexin (Cx) molecules, namely Cx37, Cx40, Cx43 and Cx45, are expressed in the gap junctions that exist within and between the cellular layers of arteries. Endothelial cells are well coupled by large gap junctions expressing Cx37, Cx40 and, to a lesser extent, Cx43, whose expression may be more subject to regulation by physical factors. Smooth muscle cells are more heterogeneously coupled by gap junctions that are small and rare. The identity of the Cx expressed in the media may vary among different arteries. Myoendothelial gap junctions are small and more common in resistance arteries with fewer layers of smooth muscle cells. Given the small size of these gap junctions and the rapid turnover rate of Cxs, homocellular coupling in the media and heterocellular coupling between the cell layers may be subject to more dynamic control than coupling in the endothelium. Vascular gap junctions have been implicated in a number of vasomotor responses that may regulate vascular tone and blood pressure. These include the mechanism of action of the vasodilator, endothelium-derived hyperpolarizing factor (EDHF), the myogenic constriction to intramural pressure increase, the spontaneous or agonist-induced vasomotion of arteries and arterioles and the spreading vasodilation and constriction observed in microcirculatory networks. Few data are available on Cx expression in the media of resistance arteries during hypertension. Changes in the expression of Cx43 described in the media of the aorta of hypertensive rats vary with the hypertensive model studied and are likely to represent adaptations to structural changes in the vascular wall. In contrast, in the endothelium of the caudal and mesenteric arteries of spontaneously hypertensive rats, expression of Cxs is significantly decreased compared with arteries from normotensive rats and this decrease is reversed by inhibitors of the renin-angiotensin system. During hypertension, the activity of EDHF is decreased in the mesenteric artery, but this occurs much later than the initial increase in blood pressure and the decrease in endothelial Cxs, suggesting that changes in EDHF may not be causally related to hypertension or to the changes in endothelial Cxs. Upregulation of the myogenic response and the incidence of vasomotion has been reported in hypertension. Little is currently known of the effects of hypertension on spreading vasomotor responses. Deletion of specific Cxs in genetically modified mice is complicated by neonatal lethality or coordinate regulation and compensatory changes in the remaining Cxs. Nevertheless, mice in which Cx40 has been deleted are hypertensive and spreading vasodilatory responses are significantly impaired. Determination of a role for specific Cxs in the control of blood pressure must await the development of animals in which Cx expression can be modulated in a more complex temporal and tissue-specific manner.

Developmental changes in myoendothelial gap junction mediated vasodilator activity in the rat saphenous artery.

A role for myoendothelial gap junctions (MEGJs) has been proposed in the action of the vasodilator endothelium-derived hyperpolarizing factor (EDHF). EDHF activity varies in disease and during ageing, but little is known of the role of EDHF during development when, in many organ systems, gap junctions are up-regulated. The aims of the present study were therefore to determine whether an up-regulation of heterocellular gap junctional coupling occurs during arterial development and whether this change is reflected functionally through an increased action of EDHF. Results demonstrated that in the saphenous artery of juvenile WKY rats, MEGJs were abundant and application of acetylcholine (ACh) evoked EDHF-mediated hyperpolarization and relaxation in the presence of N(omega)-nitro-l-arginine methyl ester (L-NAME) and indomethacin to inhibit nitric oxide and prostaglandins, respectively. Responses were blocked by a combination of charybdotoxin plus apamin, or 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) plus apamin, or by blockade of gap junctions with the connexin (Cx)-mimetic peptides, (43)Gap26, (40)Gap27 and (37,43)Gap27. On the other hand, we found no evidence for the involvement of the putative chemical mediators of EDHF, eicosanoids, L-NAME-insensitive nitric oxide, hydrogen peroxide or potassium ions, since 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE), hydroxocobalamin, catalase or barium and ouabain were without effect. In contrast, in the adult saphenous artery, MEGJs were rare, EDHF-mediated relaxation was absent and hyperpolarizations were small and unstable. The present study demonstrates that MEGJs and EDHF are up-regulated during arterial development. Furthermore, the data show for the first time that this developmentally regulated EDHF is dependent on direct electrotonic coupling via MEGJs.

Attenuation of conducted vasodilatation in rat mesenteric arteries during hypertension: role of inwardly rectifying potassium channels.

The present study was designed to elucidate whether the conduction of vasomotor responses mediated by endothelium-derived hyperpolarizing factor (EDHF) in rat mesenteric arteries is altered during hypertension. Iontophoresed acetylcholine (ACh; 500 ms) caused EDHF-mediated hyperpolarization and vasodilatation at the local site and these responses spread through the endothelium to remote sites in 12-week-old Wistar-Kyoto rats (WKY). Conducted responses were significantly attenuated in age-matched spontaneously hypertensive rats (SHR) although the rate of decay with distance did not change. Inhibition of inwardly rectifying potassium (Kir) channels (30 microM barium) eliminated the difference between WKY and SHR by attenuating conducted responses in WKY but not SHR. At the local site, barium (30 microM) significantly reduced the duration but not the amplitude of ACh-induced hyperpolarization in WKY only. Barium had no effect when the iontophoretic stimulus was reduced to 350 ms. After blockade of EDHF in SHR, ACh elicited a depolarization which our indirect data suggest spreads along the vessel in the endothelium. Messenger RNA expression of Kir2.0 genes did not differ between the strains nor did the amplitude of K(+)-induced hyperpolarization, which was abolished by disruption of the endothelium. Immunohistochemistry revealed a decrease in connexin (Cx)37 but not Cx40 or Cx43 protein in endothelial cells of SHR compared to WKY. Results suggest that conduction of EDHF-mediated responses in WKY, but not in SHR, is facilitated by activation of Kir channels at the site of ACh application and not by differences in endothelial connexin expression. Lack of Kir channel involvement in hypertension may result from reduction in the duration of the hyperpolarization due to the development of ACh-mediated depolarization, rather than to any difference in Kir subunit expression or function.

Myoendothelial gap junctions may provide the pathway for EDHF in mouse mesenteric artery.

Endothelium-dependent hyperpolarization of vascular smooth muscle provides a major pathway for relaxation in resistance arteries. This can occur due to direct electrical coupling via myoendothelial gap junctions (MEGJs) and/or the release of factors (EDHF). Here we provide evidence for the existence of functional MEGJs in the same, defined branches of BALB/C mouse mesenteric arteries which show robust EDHF-mediated smooth muscle relaxation. Cyclopiazonic acid (CPA, 10 microM) was used to stimulate EDHF in arteries mounted under isometric conditions and constricted with phenylephrine. Simultaneous measurement of smooth muscle membrane potential and tension demonstrated that CPA caused a hyperpolarization of around 10 mV, reversing the depolarization to phenylephrine by 94% and the associated constriction by 66%. The relaxation to CPA was endothelium dependent, associated with the opening of Ca2+-activated K channels, and only in part due to the release of nitric oxide (NO). In the presence of the NO synthase inhibitor, L-NAME (100 microM), the relaxation to CPA could be almost completely inhibited with the putative gap junction uncoupler, carbenoxolone (100 microM). Inhibition of the synthesis of prostaglandins or metabolites of arachidonic acid had no effect under the same conditions, and small rises in exogenous K+ failed to evoke consistent or marked smooth muscle relaxation, arguing against a role for these molecules and ions as EDHF. Serial section electron microscopy revealed a high incidence of MEGJs, which was correlated with heterocellular dye coupling. Taken together, these functional and morphological data from a defined mouse resistance artery suggest that the EDHF response in this vessel may be explained by extensive heterocellular coupling through MEGJs, enabling spread of hyperpolarizing current.