Development of a pharmacological evidence-based anticholinergic burden scale for medications commonly used in older adults.

AIM: The present study aimed to develop a pharmacological evidence-based anticholinergic burden scale (ABS) through a direct assessment of muscarinic receptor-binding activities of 260 medications commonly used in older adults. METHODS: The muscarinic receptor-binding activities of 260 drugs were assessed by the displacement of specific [N-methyl-3 H]scopolamine methyl chloride binding in the rat brain. The maximum blood concentrations (Cmax ) of drugs after their administration to subjects were cited from their interview forms. RESULTS: In total, 96 of 260 drugs displayed concentration-dependent muscarinic receptor binding in rat brain. Based on muscarinic receptor-binding activity (IC50 ) and Cmax after the administration at clinical doses in humans, we rated ABS 3 (strong) for 33 drugs and ABS 2 (moderate) for 37 drugs. There was an approximate similarity between muscarinic receptor-binding activities (IC50 ) and Cmax of 33 drugs (ABS 3) after their administration at clinical doses in humans. Furthermore, 26 drugs were defined as ABS 1 (weak) by muscarinic receptor-binding activity. The remaining 164 drugs exhibited slight or no significant muscarinic receptor-binding activities at high concentration of 100 µM, and they were defined as ABS 0. There was a marked similarity for 28 drugs (ABS 3) between the present ABS data and their previous scoring data in the literature. CONCLUSIONS: To our knowledge, the present study developed the first comprehensive pharmacological evidence-based ABS of drugs based on muscarinic receptor-binding activity, which provides guidance as to which drugs may be discontinued to reduce anticholinergic burden. Geriatr Gerontol Int 2023; 23: 558-564.

Modulation of Vasomotor Function by Perivascular Adipose Tissue of Renal Artery Depends on Severity of Arterial Dysfunction to Nitric Oxide and Severity of Metabolic Parameters.

Perivascular adipose tissue (PVAT) enhances vascular relaxation of mesenteric arteries in SHRSP.Z-Leprfa/IzmDmcr rats (SPZF), a metabolic syndrome model. We investigated and compared the effects of PVAT on the renal artery in SPZF with those on SHR/NDmcr-cp rats (CP). Renal arteries with and without PVAT were isolated from 23-week-old SPZF and CP. The effects of PVAT on acetylcholine- and nitroprusside-induced relaxation were examined using bioassays with phenylephrine-contracted arterial rings. Acetylcholine-induced relaxations without PVAT in SPZF and CP were 0.7- and 0.5-times lower in females than in males, respectively. In the presence of PVAT, acetylcholine-induced relaxations increased 1.4- and 2-times in male and female CP, respectively, but did not differ in SPZF. Nitroprusside-induced relaxation with and without PVAT was 0.7-times lower in female than in male SPZF but did not differ in CP. Angiotensin-II type-1 receptor (AT1R)/AT1R-associated protein mRNA ratios were lower in CP than in the SPZF and negatively correlated with the difference in arterial relaxation with and without PVAT. The effects of renal artery PVAT differed between the SPZF and CP groups. Higher levels of enhanced AT1R activity in SPZF PVAT may drive these differences by impairing the vascular smooth muscle responses to nitric oxide.

Perivascular Adipose Tissue Compensation for Endothelial Dysfunction in the Superior Mesenteric Artery of Female SHRSP.Z-Leprfa/IzmDmcr Rats.

Regulation of arterial tone by perivascular adipose tissue (PVAT) differs between sexes. In male SHRSP.Z-Leprfa/IzmDmcr rats (SHRSP.ZF), PVAT exerts a compensatory relaxation effect for the loss of endothelium-mediated vasorelaxation, which occurs during the early stages of metabolic syndrome. However, this effect deteriorates by 23 weeks of age. Here, therefore, we compared the effects of PVAT in female and male SHRSP.ZF. Acetylcholine-induced relaxation in superior mesenteric artery without PVAT did not differ between 23-week-old females and males. However, the presence of PVAT enhanced relaxation in 23-week-old females, but not in males. The mRNA levels of angiotensin II type 1 receptor (AT1R) in PVAT did not differ between sexes, but AT1R-associated protein (ATRAP) and apelin levels were higher in females than in males. We observed a positive relationship between differences in artery relaxation with and without PVAT and ATRAP or apelin mRNA levels. In 30-week-old females, PVAT-enhanced relaxation disappeared, and mRNA levels of AT1R increased, while apelin levels decreased compared to 23-week-old females. These results demonstrated that in SHRSP.ZF, PVAT compensation for endothelium dysfunction extended to older ages in females than in males. Apelin and AT1R/ATRAP expression in PVAT may be predictors of favorable effects.

Muscarinic receptor binding activity in rat tissues by vibegron and prediction of its receptor occupancy levels in the human bladder.

OBJECTIVES: To examine the effects of vibegron, a selective ß3 -adrenoceptor agonist, used to treat overactive bladder, on muscarinic receptors in the rat bladder, and to predict the occupancy levels of muscarinic receptors by vibegron in the bladders of humans orally administered a clinical dose. METHODS: Muscarinic receptors in the rat bladder and other tissues were examined by a radioligand binding assay using [N-methyl-3 H]scopolamine chloride. The occupancy levels of muscarinic receptors by vibegron in bladders of humans after its oral administration were predicted from the estimation of unbound concentrations in human plasma and urine in the literature. RESULTS: Vibegron (0.1-100 µmol/L) inhibited specific [N-methyl-3 H]scopolamine chloride binding in the bladder and other tissues of rats in a concentration-dependent manner. The 50% inhibitory concentration value of vibegron in the bladder was approximately twofold higher than that in the heart, and approximately 315- and 3.5-fold lower than those in the submaxillary gland and brain, respectively. Therefore, the binding affinity of vibegron for muscarinic receptors was higher in the heart and bladder than in the submaxillary gland and brain. By using the rat bladder receptor binding affinity, occupancy levels of muscarinic receptors in the human bladder were predicted to be 51-91% until 24 h after its oral administration at 50 mg of vibegron. CONCLUSIONS: This is the first study to suggest that vibegron binds to muscarinic receptors in the rat bladder and other tissues, with a potentially higher affinity for the M2 subtype than the M1 and M3 subtypes. These results might be clinically relevant for pharmacotherapy with vibegron for overactive bladder.

A Sodium Glucose Cotransporter 2 Inhibitor Fails to Improve Perivascular Adipose Tissue-Mediated Modulation of Vasodilation and Cardiac Function in Rats With Metabolic Syndrome.

Arterial perivascular adipose tissue (PVAT) can elicit vasodilator signals complementary to those elicited by the endothelium in SHRSP.Z-Leprfa/IzmDmcr (SHRSP.ZF) rats, an animal model of metabolic syndrome (MetS). Here, we tested whether a glucose cotransporter 2 inhibitor (SGLT2-i; tofogliflozin) increased this PVAT effect to prevent the deterioration of cardiac function in aging SHRSP.ZF rats. Tofogliflozin treatments (1 or 10 mg/kg/day) or vehicle (control) were administered for 10 weeks by oral gavage to SHRSP.ZF rats, starting at 13 weeks of age. At 23 weeks of age, glucose levels in the serum and urine (24 h after the last administration) were determined using commercial kits. Vasodilator responsiveness of PVAT-surrounded or PVAT-free superior mesenteric arteries was determined using acetylcholine with organ-bath methods. Cardiac ventricular function and coronary flow were determined using Langendorff heart preparations. Serum and urine glucose levels in SGLT2-i treatment groups did not differ from those in the controls, but the ratios of glycated to non-glycated albumin were lower than those in the controls. Tofogliflozin treatments did not alter relaxations in the presence of PVAT or affect relaxations of PVAT-free arteries. Left ventricular systolic pressures, maximum rate of pressure decline, and coronary flow in ex vivo hearts did not differ among the treatment groups. PVAT effects and cardiac dysfunction were not altered by tofogliflozin treatment in SHRSP.ZF rats with MetS. These results do not provide strong evidence to support the use of SGLT2-i as a cardiovascular protective therapy in MetS, which occurs prior to the onset of type 2 diabetes.

Activation of protease-activated receptor 2 is associated with blood pressure regulation and proteinuria reduction in metabolic syndrome.

Metabolic syndrome (MetS) increases the risk of kidney disease. In SHRSP.Z-Leprfa /IzmDmcr (SHRSP.ZF) rats with MetS, protease-activated receptor 2 (PAR2)-mediated vasorelaxation is preserved in the aorta at 20 weeks of age (weeks) via enhancement of nitric oxide production but impaired at 30 weeks by oxidative stress. However, impairment of PAR2-mediated vasorelaxation of renal arteries and its possible implications for kidney disease are unclear. We used organ baths to assess PAR2-mediated vasorelaxation of isolated renal arteries, colorimetric methods to measure urinary protein levels as an index of renal function, and western blot to determine expression of PAR2 and nephrin proteins in the kidneys of SHRSP.ZF rats at 10, 20, and 30 weeks. We assessed renal arteries and kidney function for effects of orally administered GB88, a pathway-dependent PAR2 antagonist, from 10 to 18 weeks, and azilsartan, an angiotensin II type 1 receptor blocker, from 13 to 23 weeks. PAR2-mediated vasorelaxation was slightly lower at 20 weeks and attenuated significantly at 30 weeks compared with those at 10 weeks. Urinary protein levels were increased at 20 and 30 weeks. Decreased protein expression of PAR2 and nephrin in the kidney were observed at 30 weeks. Administration of GB88 increased blood pressure (BP) and proteinuria. Azilsartan reduced the high BP and the impaired PAR2-mediated vasorelaxation, but did not restore the increase in urinary protein levels and decreased PAR2 and nephrin protein expression in the kidney. PAR2 activation in the kidney may be associated with maintenance of BP and urinary protein excretion in MetS.

Vasorelaxant effects of benzodiazepines, non-benzodiazepine sedative-hypnotics, and tandospirone on isolated rat arteries.

Benzodiazepines (BDZs) and non-BDZ sedative-hypnotics are effective for the management of chronic insomnia; however, they are associated with adverse effects such as headache, dizziness, and palpitations. Furthermore, long-term use of these medications is associated with decreased blood pressure (BP) or depressed baroreflex function. Therefore, here, we assessed whether BDZs and non-BDZs cause vasorelaxation directly. Vasorelaxation in response to 22 BDZs, 2 non-BDZs, and tandospirone was determined by myograph methods using isolated Wistar rat thoracic aortas. All the drugs relaxed phenylephrine-contracted rat aortas in a concentration-dependent manner. Zolpidem and tandospirone caused over 80% relaxation at a concentration of 10 µM; diazepam, estazolam, etizolam, and tofisopam caused 60-70% relaxation; whereas 18 other BDZs (alprazolam, bromazepam, brotizolam, chlordiazepoxide, clobazam, clonazepam, clorazepate, ethyl loflazepate, flunitrazepam, flurazepam, lorazepam, lormetazepam, midazolam, nimetazepam, nitrazepam, oxazepam, temazepam, and triazolam) and zaleplon caused less than 50% relaxation. The relaxation was partially but significantly inhibited to the same extent by a nitric oxide (NO) synthase antagonist and after endothelium removal. Binding assay of gamma-aminobutyric acid type A receptors was performed using [3H]flunitrazepam. No correlation was observed between vasorelaxation at a concentration of 10 µM and the binding affinities for 23 drugs. The study demonstrated that zaleplon, zolpidem, tandospirone, and many BDZs cause vasorelaxation to different extents via endothelial NO-dependent and endothelium-independent pathways. In conclusion, the direct vasodilatory effects of these drugs may be involved in the mechanisms underlying their adverse effects. Additionally, the decreased BP observed in persons who take BDZs or non-BDZs may be partly due to direct vasodilation.

Acanthopanax senticosus Root Extract Exerts Dual Action on Mouse Ileal Smooth Muscle Function, Leading to Modulation of Gastrointestinal Motility.

Diarrhea is often caused by changes in lifestyle, stress, or side effects of drugs. Acanthopanax senticosus root extract (ASRE) has long been used as a functional food remedy with anti-fatigue, neuroprotective, and immunomodulatory activities. However, it is unclear whether ASRE has beneficial effects on gastrointestinal (GI) motility. Therefore, we first investigated whether ASRE directly affects contractile functions of the isolated mouse ileum, and then assessed its effects on GI transit of a charcoal meal in normal mice and a carbachol (CCh)-induced diarrhea mouse model. ASRE caused contraction of the isolated mouse ileum and the maximum contraction was approximately half of that induced by acetylcholine (ACh) administration. In the presence of atropine, this ASRE-induced contraction disappeared, while relaxation responses were observed. However, ASRE reduced potassium chloride- and ACh-induced contractions, and the inhibitory effect was not counteracted by a ß-blocker. Administration of a nitric oxide synthase inhibitor or potassium channel blockers did not affect the ASRE-induced relaxation. Oral administration of ASRE for 1 and 4 d reduced the increased GI transit in CCh-treated but did not affect the GI transit of normal mice. These results indicate that ASRE exhibited dual effects of contraction via muscarinic receptors and direct relaxation on mouse ileal function, and its relaxant effect could be useful in treating diarrhea symptoms, resulting in an increase in the parasympathetic nerve activities.

Direct in vitro and in vivo demonstration of muscarinic receptor binding by the novel radioligand, [3H]5-hydroxymethyltolterodine, in the bladder and other tissues of rats.

In vitro and in vivo binding sites of [3H]-labeled 5-hydroxymethyltolterodine (5-HMT), a new radioligand for labeling muscarinic receptors in rat tissues were characterized. Specific [3H]5-HMT binding in rat tissues was saturable and of high affinity in each tissue. The dissociation constant (Kd) was significantly lower in bladder and heart than in submaxillary gland. Significant levels of in vivo specific [3H]5-HMT binding by intravenous injection of the radioligand were detected in tissues, except for cerebral cortex. Thus, [3H]5-HMT was shown to specifically label muscarinic receptors in rat tissues, suggesting a useful radioligand for labeling muscarinic receptors with high affinity.

Effects of Anticholinergic Drugs Used for the Therapy of Overactive Bladder on P-Glycoprotein Activity.

We evaluated the effects of anticholinergic drugs principally used for the therapy of overactive bladder (OAB) on the activity of P-glycoprotein, an efflux transport protein, in Caco-2 cells. The time-dependent changes in the fluorescence of residual rhodamine 123, a P-glycoprotein activity marker, in the apical region of Caco-2 cells were measured in the presence of anticholinergic drugs using time-lapse confocal laser scanning microscopy. The effect of anticholinergic drugs on human P-glycoprotein ATPase activity was also measured. The fluorescence of residual rhodamine 123 in untreated Caco-2 cells decreased over time. The gradual decrease in the fluorescence was significantly inhibited by treatment with cyclosporine A, darifenacin, and trospium. In contrast, oxybutynin, N-desethyl-oxybutynin (DEOB), propiverine, and its active metabolites (M-1, M-2), imidafenacin, solifenacin, or tolterodine had little effect on the efflux of rhodamine 123. P-Glycoprotein ATPase activity was increased by darifenacin. Darifenacin and trospium reduced the rhodamine 123 transfer across the apical cell membrane. These data suggest that darifenacin and trospium interact with P-glycoprotein. Additionally, darifenacin influenced P-glycoprotein ATPase activity. These results suggest that darifenacin may be a substrate of P-glycoprotein. This study is the first paper to test simultaneously the effects of 10 anticholinergic drugs used currently for the therapy of OAB, on the P-glycoprotein.

trans-2-Pentenal, an Active Compound in Cigarette Smoke, Identified via Its Ability to Form Adducts with Glutathione.

α,ß-Unsaturated carbonyl compounds readily form adducts with SH or NH2 residues, which are nucleophilic agents, by Michael addition. Glutathione (GSH) is a tripeptide that contains an SH residue and functions as an antioxidant. We demonstrated previously that acrolein (ACR), crotonaldehyde (CA), and methyl vinyl ketone (MVK) are present in nicotine- and tar-removed cigarette smoke extract (CSE) and reacted with GSH in B16-BL6 mouse melanoma cells to form GSH-ACR, GSH-CA, and GSH-MVK adducts, suggesting a possible mechanism for CSE-induced cytotoxicity. In this study, we searched for novel α,ß-unsaturated carbonyl compounds other than ACR, CA, and MVK. We selected candidate compounds in CSE based on accurate mass values generated using LC/MS analysis of products formed between CSE and GSH, and identified these using GC/MS analysis and library screening. As a result, we isolated trans-2-methyl-2-butenal, 2-methyl-2-cyclopenten-1-one, 3-methyl-2-cyclopenten-1-one, and furfural, which were poorly reactive with GSH and only very weakly inhibited growth of Colon-26 mouse carcinoma cells and BALB/3T3 clone A31 mouse normal cells. We also isolated 2-cyclopenten-1-one, trans-2-pentenal, 3-methyl-2-butenal and ethyl vinyl ketone, which were highly reactive with GSH and significantly inhibited the growth of both cell lines. Our data suggest that the reactivity of compounds in CSE with GSH may be positively correlated with the effect on inhibiting cell growth. Notably, trans-2-pentenal showed marked inhibition of carcinoma cells growth, whereas this compound exhibited little inhibitory effect on normal cells. trans-2-Pentenal may be a potent candidate or seed for antitumor agents.

Angiotensin II Type 1 Receptor Antagonist Azilsartan Restores Vascular Reactivity Through a Perivascular Adipose Tissue-Independent Mechanism in Rats with Metabolic Syndrome.

PURPOSE: Perivascular adipose tissues (PVAT) are involved in the regulation of vascular tone. In mesenteric arteries, the compensatory vasodilatory effects of PVAT appear when vascular relaxation is impaired and disappear at around 23 weeks of age in SHRSP.Z-Leprfa/IzmDmcr (SHRSP.ZF) rats with metabolic syndrome (MetS). The renin-angiotensin system is involved in the development of endothelium and vascular dysfunction. Therefore, we investigated whether azilsartan, a potent angiotensin II type 1 (AT1) receptor antagonist, can protect against the deterioration of the PVAT compensatory vasodilator function that occurs with aging in MetS. METHODS: Two age groups of SHRSP.ZF rats (13 and 20 weeks of age) were administered azilsartan or vehicle through oral gavage once daily for 10 weeks. The vasodilation response of the isolated superior-mesenteric arteries upon addition of endothelium-dependent and -independent agonists was determined in the presence or absence of PVAT using organ bath methods. RESULTS: In vivo treatment with azilsartan improved the acetylcholine-induced vasodilation in mesenteric arteries with and without PVAT at both time-points. The mRNA levels of AT1 receptor and AT1 receptor-associated protein were unchanged in PVAT upon azilsartan treatment. Furthermore, in vitro treatment with azilsartan (0.1 and 0.3 µM for 30 min) did not affect the compensatory effect of PVAT on vasodilation in response to acetylcholine in SHRSP.ZF rat mesenteric arteries. CONCLUSIONS: Our results provide evidence supporting the use of azilsartan for the long-term protection against vascular dysfunctions in MetS. Azilsartan did not improve the dysfunction of PVAT-mediated modulation of vascular tone during MetS. The protective effect of azilsartan is mediated by restoring the endothelium- and vascular smooth muscle-mediated mechanisms.

Acanthopanax senticosus Induces Vasorelaxation via Endothelial Nitric Oxide-Dependent and -Independent Pathways.

Although Acanthopanax senticosus root extract (ASRE), a functional food used in Japan, improves peripheral blood circulation and exerts vasorelaxant effects in rats under healthy conditions, the underlying mechanisms currently remain unclear. Therefore, we investigated the mechanisms responsible for ASRE-induced relaxation in isolated thoracic aortas using organ bath techniques and examined whether ASRE affects systemic and peripheral circulation using a photoplethysmographic tail-cuff system and noncontact laser tissue blood flow meter in Wistar rats. Similar to acetylcholine (ACh), ASRE induced dose-dependent relaxation in aortas pre-contracted with phenylephrine; however, in contrast to ACh, ASRE-induced relaxation was partially inhibited by treatments with antagonists of nitric oxide (NO) synthase and soluble guanylyl cyclase as well as by endothelium removal. Contractile responses to phenylephrine or potassium chloride were observed in the presence of ASRE. The oral administration of ASRE (900 mg/kg/d for 1 wk) decreased systolic blood pressure in rats 3 h after the treatment and did not affect heart rate, tail blood flow, mass, or velocity; this decreasing effect was not observed on day 2. A 1-wk treatment with ASRE did not affect vasorelaxation in response to ASRE. These results demonstrate that ASRE induces vasorelaxation via endothelial NO production and an NO-independent pathway in rats. Based on these findings, positive impacts of ASRE on blood pressure and peripheral blood circulation cannot be expected under healthy conditions as the systemic effects of ASRE are temporary. Instead, caution is needed to prevent the occurrence of side effects (i.e., orthostatic dizziness) at the beginning of ASRE dosing.

Effect of Royal Jelly on Mouse Isolated Ileum and Gastrointestinal Motility.

Royal jelly (RJ) is widely used as a cosmetic or dietary supplement to relieve various health disorders, such as dry skin, fatigue, and menopause. RJ has been recommended to improve constipation on a commercial basis. However, the detailed mechanisms by which RJ influences intestinal motility and whether RJ improves constipation remain unclear. Therefore, we investigated the effects of RJ on the motility of mouse ileum both in vitro and in vivo. Using myograph methods, RJ dose-dependently induced contractions of isolated ileal segments, which were inhibited by treatment with atropine. Eserine sulfate, a cholinesterase inhibitor, enhanced the RJ-induced contractions, whereas RJ treated with acetylcholinesterase did not result in ileum contraction. RJ-induced contractions were not affected by NG-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor, although nicotine-induced contractions were significantly enhanced. In contrast, in a gastrointestinal (GI) transit model, single oral administration of 300 mg/kg RJ did not affect GI transit in both normal mice and the loperamide-induced constipation model mice. These results demonstrate that acetylcholine in RJ directly acted on the muscarinic receptors of the mouse intestinal smooth muscle, causing it to contract in vitro. In contrast, single oral administration of RJ did not improve constipation. This study is the first to evaluate the effects of RJ on the motility of mouse ileum in in vitro and in vivo experiments for the validation of application of RJ as a gentle laxative.

Royal jelly increases peripheral circulation by inducing vasorelaxation through nitric oxide production under healthy conditions.

AIMS: Royal jelly (RJ) has a variety of reported biological activities, including vasorelaxation and blood pressure-lowering effects. Although functional foods are positively used for health, the effects of RJ on the cardiovascular system in healthy individuals have not been well studied. Therefore, we investigated the mechanisms underlying the vasorelaxation effects of RJ in healthy control rats to evaluate whether the peripheral circulation was increased. MAIN METHODS: We used fresh RJ to examine the vasorelaxation effects and related mechanisms in Wistar rats using organ bath techniques. Furthermore, we measured changes in tail blood circulation, systolic blood pressure (sBP), and heart rate (HR) after the oral administration of RJ to control rats and nitro-l-arginine methyl ester (l-NAME)-treated rats (0.5 mg/ml dissolved in distilled drinking water for 1 week). Concentrations of acetylcholine (ACh) in the RJ were measured using a commercial kit. KEY FINDINGS: RJ caused vasorelaxation of isolated rat aortas and superior mesenteric arteries, and this effect was inhibited by atropine (10-5 M, 15 min) or L-NAME (10-4 M, 20 min) and endothelium-denuded arterial ring preparations. Oral RJ increased tail blood flow and mass in control rats 1 h after treatment without affecting velocity, sBP, or HR. These effects were not observed in L-NAME-treated rats. RJ contained approximately 1000 µg/g of ACh. SIGNIFICANCE: The present study demonstrated that RJ is composed of muscarinic receptor agonist(s), likely ACh, and induces vasorelaxation through nitric oxide (NO) production from the vascular endothelium of healthy rats, leading to increased tail blood circulation. Thus, fresh RJ may improve peripheral circulation in healthy individuals.

Invasion-inhibiting Effects of Gaseous Components in Cigarette Smoke on Mouse Rectal Carcinoma Colon-26 Cells.

We investigated the anti-metastatic action of nicotine- and tar-removed cigarette smoke extract (CSE) on highly metastatic mouse Colon-26 cells using syngeneic BALB/c mice. Colon-26 cells were injected into the spleen of mice, cells were grown in the spleen as the primary lesion, and some metastasized from the spleen to liver and established a metastatic lesion. CSE (10, 30, and 100%) was intraperitoneally administered daily to the mice for 14 days after tumor inoculation. As a result, the relative spleen weights of CSE-administered mice did not differ significantly from those of the control mice. However, the relative liver weights of CSE 30%-administered mice significantly decreased compared to control mice. In order to identify the active component in CSE, we examined the action of methyl vinyl ketone (MVK) on the invasiveness of Colon-26 cells. MVK significantly reduced the invasiveness of cells. MVK may be a candidate active component of CSE.

Perivascular Adipose Tissue-Enhanced Vasodilation in Metabolic Syndrome Rats by Apelin and N-Acetyl⁻l-Cysteine-Sensitive Factor(s).

Perivascular adipose tissue (PVAT) can regulate vascular tone. In mesenteric arteries of SHRSP.Z-Leprfa/IzmDmcr rats (SHRSP.ZF) with metabolic syndrome, vascular dysfunction is compensated by PVAT-dependent mechanisms that disappear with increasing age. In this study, we investigated the mechanisms of the age-related changes and responsible factor(s) involved in the enhancing effects of mesenteric arterial PVAT in SHRSP.ZF. Acetylcholine- and sodium nitroprusside-induced relaxations of isolated arteries were greater with PVAT than without PVAT at 17 and 20 weeks of age (wks), and as expected, this enhancement by the presence of PVAT disappeared at 23 wks. PVAT mRNA levels of angiotensin II type 1 (AT1) receptor-associated protein was less and AT1 receptor was unchanged at 23 wks when compared to 20 wks. At 20 wks, the enhanced acetylcholine-induced relaxation by the presence of PVAT was inhibited by N-acetyl-l-cysteine (NAC). Acetylcholine-induced relaxation of arteries without PVAT was increased in the presence of exogenously added apelin. PVAT mRNA level of apelin was higher in SHRSP.ZF than in control Wistar-Kyoto rats, and the level was decreased with aging. These results suggest that AT1 receptor activation in PVAT, and changes in the regulation of apelin and a NAC-sensitive factor are related to the age-dependent deterioration of the vasodilation enhancing effects of mesenteric arterial PVAT in SHRSP.ZF.

Progression of Time-Dependent Changes to the Mechanisms of Vasodilation by Protease-Activated Receptor 2 in Metabolic Syndrome.

Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor activated by serine proteases released from tissues or by synthetic peptide ligands administered pharmacologically. Its wide expression in the cardiovascular system, particularly within the endothelium, vasodilation activity, and link to increased expression of inflammatory cytokines positions PAR2 as a potentially important regulator of vascular pathology under conditions of tissue inflammation, and injury; and thus, a pharmaceutical target for new therapeutics. Obesity is considered a chronic low-grade systemic inflammatory condition as inflammatory cytokines released from adipocytes are closely related to development of metabolic syndrome and related disorders. Our work over the past five-years has focused on the changes in vasomotor functions of PAR2 in metabolic syndrome, using an animal model known as the SHRSP.Z-Leprfa/IzmDmcr rats (SHRSP.ZF). In young SHRSP.ZF that had already developed impaired responses to nitric oxide, we reported that PAR2-induced endothelium-dependent vasodilation is preserved. However, this PAR2 vasodilation decreased with increasing age and further chronic exposure to the conditions of metabolism disorder. These findings raise the possibility that PAR2 regulates tissue perfusion and can protect organs from injury, which is an increasing clinical concern at later stages of metabolic syndrome. Here we present our studies on the time-dependent changes in vasoreactivity to PAR2 in metabolic syndrome and the underlying mechanisms. Furthermore, we discuss the implications of these age-related changes in PAR2 for the cardiovascular system in metabolic syndrome.

Time-Dependent Differences in the Influence of Perivascular Adipose Tissue on Vasomotor Functions in Metabolic Syndrome.

BACKGROUND: Metabolic syndrome (MetS) facilitates the development of cardiovascular disease due to atherosclerosis, which is accelerated by defects of the vascular endothelium. Vascular dysfunction in response to nitric oxide (NO) occurs in the mesenteric arteries of an animal model of MetS, SHRSP.Z-Leprfa/IzmDmcr (SHRSP.ZF) rats. Vascular responses to vasodilators are affected by perivascular adipose tissue (PVAT) that surrounds the outsides of arteries. In this study, we assessed the role of PVAT in vascular dysfunction observed in SHRSP.ZF. METHODS: To determine the effects of PVAT on vasodilators in SHRSP.ZF and control Wistar-Kyoto (WKY) rats, we used organ bath bioassay techniques to assay acetylcholine and nitroprusside-induced relaxations of isolated mesenteric arterial ring preparations with PVAT intact or removed. RESULTS: A PVAT-mediated enhancement of relaxations induced by acetylcholine and nitroprusside occurred in SHRSP.ZF at 20 weeks of age, but not at 10 and 30 weeks, and did not occur in WKY. Furthermore, the enhancing effects of PVAT from SHRSP.ZF at 20 weeks could not be substituted by replacement with PVAT from either WKY or 30-week-old SHRSP.ZF, was inhibited by NO synthase inhibitor, and abolished by removal of the arteries' endothelium. Cyclic guanosine monophosphate (cGMP) accumulation elicited by nitroprusside was higher in SHRSP.ZF arteries with PVAT than arteries without PVAT at 20 weeks, but the enhancement of cGMP accumulation did not occur at 30 weeks. CONCLUSIONS: PVAT may regulate arterial tone by releasing diffusible vasorelaxing factor(s), which, through endothelium-derived NO production, compensates for impaired vasodilations at early stages of MetS.

Age-related changes to vascular protease-activated receptor 2 in metabolic syndrome: a relationship between oxidative stress, receptor expression, and endothelium-dependent vasodilation.

Protease-activated receptor 2 (PAR2) is expressed in vascular endothelium. Nitric oxide (NO) - cyclic GMP-mediated vasodilation in response to 2-furoyl-LIGRLO-amide (2fLIGRLO), a PAR2-activating peptide, is impaired in aortas from aged SHRSP.Z-Leprfa/IzmDmcr (SHRSP.ZF) rats with metabolic syndrome. Here we investigated mechanisms linking PAR2's vascular effects to phenotypic characteristics of male SHRSP.ZF rats at 10, 20, and 30 weeks of age. We found vasodilation responses to either 2fLIGRLO or enzyme-mediated PAR2 activation by trypsin were sustained until 20 weeks and lessened at 30 weeks. PAR2 protein and mRNA levels were lower in aortas at 30 weeks than at 10 and 20 weeks. PAR2-mediated responses positively correlated with PAR2 protein and mRNA levels. Decreased cGMP accumulation in the presence of 2fLIGRLO paralleled the decreased relaxations elicited by nitroprusside and the cGMP analog 8-pCPT-cGMP, and the less soluble guanylyl cyclase protein at 30 weeks. 2fLIGRLO-induced relaxation was negatively correlated with serum thiobarbituric acid reactive substances, an index of oxidative stress, which increased with age. Forward stepwise data regression supported a model of age-related decreases in PAR2 function resulting from decreased PAR2 mRNA and increased oxidative stress. We conclude that decreased responsiveness of aortic smooth muscle to NO and downregulation of receptor expression impair PAR2 functions at later stages of metabolic syndrome in SHRSP.ZF rats.