Harrie C.M. Boonen

Mesenteric small artery changes after vasoconstrictor infusion in young rats.

Summary We evaluated whether chronic

Increased calcium sensitivity in isolated resistance arteries from spontaneously hypertensive rats: effects of dihydropyridines

,-adrenergic stimulation, angiotensin II (All), or increased blood pressure (BP) alters resistance arterial structure and function. Structural parameters and wall tension were recorded in mesenteric small arteries (MrA) isolated from 6-week-old normotensive Wistar Kyoto rats that had been infused for 4 days with saline (WKY), 2 mg/kg/day phenylephrine (WKY + PHE), or 0.3 mg/kg/day All (WKY + ALL) and from saline-infused spontaneously hypertensive rats (SHR). During the experimental period, systolic BP (SBP) did not change in WKY but increased in WKY + PHE, WKY + All, and SHR. Relative cardiac mass did not differ between SHR and WKY, but was increased in WKY + PHE and WKY + All. Stiffness and optimal lumen diameter of MrA did not differ between WKY and SHR and were not altered in WKY + PHE or WKY + AH. Maximal contractile responses and sensitivities for vasoconstrictors and calcium in vessels of WKY + AH and SHR did not differ from those in WKY. In vessels of WKY + PHE, maximal responses to vasoconstrictors and sensitivities for norepinephrine (NE) and PHE were reduced. Relaxing responses to isoproterenol (ISO) and Na-nitroprusside did not differ between SHR and WKY and were not altered in WKY + PHE and WKY + AH. Those to acetylcholine (ACh) were reduced in WKY + PHE. Media cross-sectional area and media thickness were significantly larger in WKY + All and SHR as compared with WKY but were not altered in WKY + PHE. These data indicate that in young rats AH leads to small artery hypertrophy and that neither increased BP nor increased vasoconstriction appear to be involved therein. Chronic a,-adrenergic stimulation, on the other hand, did not modify small artery structure but resulted in nonse-lective reduction of arterial smooth muscle contractile reactivity.

Effects of a phorbol ester and staurosporine on electro- and pharmacomechanical coupling in a resistance artery.

SummaryWe evaluated whether GTP-binding regulatory proteins (G-proteins) are involved in responses of resistance arterial smooth muscle to contractile agonists. We therefore pretreated isolated sympathectomized mesenteric resistance arteries of the rat with pertussis toxin (PTX) and recorded their contractile responses to aluminium fluoride, endothelin, high potassium, phenylephrine, phorbol myristate acetate, serotonin and vasopressin. PTX reduced contractile responses to agonists with the following order of potency: phenylephrine = serotonin > vasopressin = endothelin. The toxin reduced responses to phenylephrine in both the presence and absence of extracellular Ca2+. In Ca2+-depleted vessels that were exposed to phenylephrine, PTX virtually abolished responses to Ca2+ while hardly affecting responses to Ca2+ in the presence of endothelin. Also aluminium fluoride and phorbol myristate acetate induced contractions. These were dependent on extracellular Ca2+ and inhibited by felodipine. PTX reduced responses to aluminium fluoride but not those to phorbol myristate acetate. These data indicate that PTX sensitive G-proteins are involved in both influx of Ca2+ and release of intracellular Ca2+ following α1-adrenergic and serotonergic stimulation of resistance arteries. The role of G-proteins in stimulated Ca2+ influx could involve a direct effect on calcium channels although an indirect effect through protein kinase-C can not be entirely excluded. The persistance of contractile responses to vasopressin and endothelin following PTX suggests that these agonists engage different pathways to induce contraction or have a higher efficacy in activating similar G-proteins.

Effect of Ageing on the Passive and Active Tension and Pharmacodynamic Characteristics of Rat Coronary Arteries: Age-Dependent Increase in Sensitivity to 5-HT and K+

We recorded the contractile responses to calcium in mesenteric resistance arteries of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) during depolarization or stimulation with noradrenaline. The effects of Bay-K8644 and nimodipine on these responses were evaluated. Calcium sensitivity was greater in noradrenaline-stimulated than in depolarized vessels. Nimodipine decreased and Bay-K8644 increased calcium sensitivity. Both substances were more potent in the presence of potassium than in the presence of noradrenaline. Calcium sensitivity was greater in SHR than in WKY vessels only during stimulation with noradrenaline. The rhythmic responses of SHR vessels during stimulation with noradrenaline were abolished by nimodipine. Rhythmicity could be induced in WKY vessels by Bay-K8644. Modulation of calcium sensitivity by dihydropyridines during electrochemical as well as pharmacological stimulation suggests that, in resistance arterial smooth muscle, the function of potential-operated calcium channels can be modulated by noradrenaline. This modulation could differ quantitatively between mesenteric resistance arteries of SHR and WKY.

Translational value of mechanical and vasomotor properties of mouse isolated mesenteric resistance-sized arteries.

We examined the role of protein kinase-C in contractile responses of small arteries of the rat by stimulating and inhibiting protein kinase-C with phorbol myristate acetate and staurosporine, respectively. The experiments were performed in isolated mesenteric resistance arteries that had been sympathectomized and mounted for recording of isometric force development. Phorbol myristate acetate (i) at concentrations lower than 3 nM increased sensitivity for the contractile effect of potassium, but not for the effect of noradrenaline or BAY-K8644, (ii) at concentrations higher than 30 nM increased the sensitivity of depolarized vessels to extracellular calcium and (iii) at concentrations higher than 30 nM induced a contractile effect that depended on the presence of extracellular calcium and that was reduced by the calcium antagonist felodipine. Neither the phorbol ester nor staurosporine affected contractile responses to caffeine in calcium-free solution. Staurosporine (10 nM) reduced the response of resistance arteries to potassium but not to noradrenaline. These results are in agreement with direct observations by others that protein kinase-C plays a role in the activation of voltage-operated calcium channels. Protein kinase-C could participate in this way in electro-mechanical coupling in resistance arterial smooth muscle and, when strongly activated, sensitize the contractile apparatus to calcium.

Early Onset Inflammation in Pre-Insulin-Resistant Diet-Induced Obese Rats Does Not Affect the Vasoreactivity of Isolated Small Mesenteric Arteries

The influence of ageing on the passive and active tension and pharmacodynamic characteristics of intramural coronary arteries from 3-month-old and 2-year-old male Wistar rats was investigated using an isometric myograph. The passive vessel wall tension measured in Ca2+-free physiological salt solution at L₀ was significantly greater in arteries from old rats (1.46 ± 0.10 Nm–1, n = 7) than in young rats (1.13 ± 0.13 Nm–1, n = 6). However, the maximal active tension at L₀ was similar. The spontaneous myogenic tone was increased by age and the vasorelaxation induced by extracellular K+ was significantly higher in coronary arteries of old rats. The sensitivity (pD2) to 5-HT was significantly higher in arteries from old (6.43 ± 0.11, n = 22) than from young rats (6.16 ± 0.08, n = 29). Ketanserin induced a concentration-dependent rightward shift of the 5-HT concentration-response curve in arteries from both young and old rats. The slopes of the regression lines of the Schild plots were not significantly different from unity and the estimated pKB values for ketanserin were similar. In conclusion, ageing is associated with changes in passive mechanical characteristics as well as changes in pharmacological properties in rat coronary small arteries.

Characterization of capsaicin induced responses in mice vas deferens: evidence of CGRP uptake.

Mice are increasingly used in vascular research for studying perturbations and responses to vasoactive agents in small artery preparations. Historically, small artery function has preferably been studied in rat isolated mesenteric resistance‐sized arteries (MRA) using the wire myograph technique. Although different mouse arteries have been studied using the wire myograph no establishment of optimal settings has yet been performed. Therefore, the purposes of this study were firstly to establish the optimal settings for wire myograph studies of mouse MRA and compare them to those of rat MRA. Second, by surveying the literature, we aimed to evaluate the overall translatability of observed pharmacological vasomotor responses of mouse MRA to those obtained in rat MRA as well as corresponding and different arteries in terms of vessel size and species origin. Our results showed that the optimal conditions for maximal active force development in mouse MRA were not significantly different to those determined in rat MRA. Furthermore, we found that the observed concentration‐dependent vasomotor responses of mouse MRA to noradrenaline, phenylephrine, angiotensin II, sarafotoxin 6c, 5‐hydroxytryptamine, carbachol, sodium nitroprusside, and retigabine were generally similar to those described in rat MRA as well as arteries of different sizes and species origin. In summary, the results of this study provide a framework for evidence‐based optimization of the isometric wire myograph setup to mouse MRA. Additionally, in terms of translational value, our study suggests that mouse MRA can be applied as a useful model for studying vascular reactivity.

Lipopolysaccharides, but not Angiotensin ll, lnduces Direct Pro-lnflammatory Effects in Cultured Mouse Arteries and Human Endothelial and Vascular Smooth Muscle Cells.

Background: Obesity is an increasing burden affecting developed and emerging societies since it is associated with an increased risk of diabetes and consequent cardiovascular complications. Increasing evidence points towards a pivotal role of inflammation in the etiology of vascular dysfunction. Our study aimed to investigate signs of inflammation and their relation to vascular dysfunction in rats receiving a high fat diet. Methods: Diet-induced obese (DIO) rats were used as a model since these rats exhibit a human pre-diabetic pathology. Oral glucose and insulin tolerance tests were conducted on DIO rats and their controls prior to the development of insulin resistance. Furthermore, the plasma contents of selected cytokines [macrophage chemoattractant protein (MCP-1), interleukin-6 (IL-6), and interleukin-1 (IL-1)] and the concentration of adiponectin were measured. Using wire myography, we tested the vascular function of isolated small mesenteric arteries. Results: DIO animals had significantly (p < 0.05) increased body weight (721.2 ± 6.3 g) compared to age- and sex-matched controls (643.4 ± 14.6 g), as well as a significant increase (p < 0.01) in body fat percentage (29.7 ± 1.7% and 22.7 ± 0.97%, respectively). No significant difference in fasting plasma insulin levels could be detected between the two groups (chow-fed group 141.5 ± 15.1 pmol/l; high fat-fed group 125.9 ± 18.8 pmol/l). However, the levels of MCP-1 (89.7 ± 4.2 pg/ml vs. 60.8 ± 7.7 pg/ml) and IL-6 (61.6 ± 3.1 pg/ml vs. 41.6 ± 7.4 pg/ml) were significantly elevated in DIO animals (p < 0.05) as compared to controls. Adiponectin levels were also significantly increased (p < 0.01) in DIO rats (10.8 ± 0.7 ng/ml) versus controls (6.9 ± 0.5 ng/ml). No difference in vascular or endothelial function was evident as determined by responses to acetylcholine, sodium nitroprusside, endothelin-1, and calcitonin gene-related peptide. Conclusion: In DIO rats, which have not yet developed hyperinsulinaemia or glucose intolerance, the levels of inflammatory mediators MCP-1 and Il-6 are significantly increased without concomitant vascular dysfunction. The results show that inflammation and obesity are tightly associated, and that inflammation is manifested prior to significant insulin resistance and vascular dysfunction.

Functional network analysis of obese and lean Göttingen minipigs elucidates changes in oxidative and inflammatory networks in obese pigs.

Calcitonin gene-related peptide (CGRP) is extensively distributed in primary afferent sensory nerves, including those innervating the genitourinary tract. Capsaicin can stimulate the release of CGRP from intracellular stores of these nerves, but this phenomenon has not been investigated in-depth in isolated preparations. The present study sets out to study and characterize the capsaicin as well as CGRP-induced responses in isolated mouse vas deferens. The effects of capsaicin and CGRP family of peptides were studied on electrically-induced twitch responses in the absence or presence of transient receptor potential cation channel vanilloid subfamily member 1 (TRPV1) antagonist and CGRP receptor antagonists. Twitch responses were attenuated by capsaicin (1nM-30nM) and CGRP family of peptides. The potency order was CGRP>intermedin-long (IMDL)~[Cys(Et)(2,7)]αCGRP~adrenomedullin (AM)>[Cys(ACM)(2,7)]αCGRP>amylin (AMY). These responses were disinhibited by the CGRP receptor antagonists and TRPV1 antagonists. The addition of CGRP receptor antagonists caused a transient potentiation of the twitch response and this potentiation was blocked by pretreatment with capsaicin and enhanced by incubation with exogenous CGRP. During the second consecutive cumulative concentration-response curve with capsaicin, the first phase of concentration-response curve disappeared and this was partially restored when the mouse vas deferens was preincubated with CGRP, suggesting the uptake of exogenous CGRP by nerves. Besides showing capsaicin-induced CGRP releases this study shows that exogenous CGRP can be taken up in vas deferens and can be re-released. CGRP uptake will add another dimension in understanding the homeostasis of this neuropeptide.