Mesenteric Artery Preparations Research Articles

A molecular complex of Cav1.2/CaMKK2/CaMK1a in caveolae is responsible for vascular remodeling via excitation–transcription coupling

Elevation of intracellular Ca2+ concentration ([Ca2+]i) activates Ca2+/calmodulin-dependent kinases (CaMK) and promotes gene transcription. This signaling pathway is referred to as excitation–transcription (E-T) coupling. Although vascular myocytes can exhibit E-T coupling, the molecular mechanisms and physiological/pathological roles are unknown. Multiscale analysis spanning from single molecules to whole organisms has revealed essential steps in mouse vascular myocyte E-T coupling. Upon a depolarizing stimulus, Ca2+ influx through Cav1.2 voltage-dependent Ca2+ channels activates CaMKK2 and CaMK1a, resulting in intranuclear CREB phosphorylation. Within caveolae, the formation of a molecular complex of Cav1.2/CaMKK2/CaMK1a is promoted in vascular myocytes. Live imaging using a genetically encoded Ca2+ indicator revealed direct activation of CaMKK2 by Ca2+ influx through Cav1.2 localized to caveolae. CaMK1a is phosphorylated by CaMKK2 at caveolae and translocated to the nucleus upon membrane depolarization. In addition, sustained depolarization of a mesenteric artery preparation induced genes related to chemotaxis, leukocyte adhesion, and inflammation, and these changes were reversed by inhibitors of Cav1.2, CaMKK2, and CaMK, or disruption of caveolae. In the context of pathophysiology, when the mesenteric artery was loaded by high pressure in vivo, we observed CREB phosphorylation in myocytes, macrophage accumulation at adventitia, and an increase in thickness and cross-sectional area of the tunica media. These changes were reduced in caveolin1-knockout mice or in mice treated with the CaMKK2 inhibitor STO609. In summary, E-T coupling depends on Cav1.2/CaMKK2/CaMK1a localized to caveolae, and this complex converts [Ca2+]i changes into gene transcription. This ultimately leads to macrophage accumulation and media remodeling for adaptation to increased circumferential stretch.

Abstract 931: Compensatory Response of Perivascular Adipose Tissue to Vascular Dysfunction in Metabolic Syndrome Rats Involves Apelin

Perivascular adipose tissue (PVAT) regulates vascular homeostasis including vascular tone via release of adipokines. Using SHRSP.Z- Lepr fa /IzmDmcr (SHRSP.ZF) rats, an animal model of metabolic syndrome (MetS), we have demonstrated that mesenteric PVAT enhances vasodilation under impaired vasodilation, in response to nitric oxide. We have also shown that the compensatory effect of PVAT disappears later in the course of MetS. Apelin is an adipokine that is produced at high levels in obesity and acts as a vasorelaxing factor. In this study, we evaluated the role of apelin in the enhancing effect of mesenteric PVAT on relaxations in SHRSP.ZF rats, by examining whether apelin canenhance vasorelaxation in the mesenteric arteries of SHRSP.ZF rats without PVAT, as it does in those with PVAT. Furthermore, we investigated whether apelin expression changes with increasing age. SHRSP.ZF rats at 17, 20, 23, and 30 weeks old were used. The mesenteric artery and surrounding adipose tissue were isolated from each rat; PVAT samples were isolated from surrounding adipose tissue; and the mRNA transcript level of apelin was examined by quantitative real-time PCR assays. Mesenteric arterial ring preparations with and without PVAT were made, and after a stable contraction was obtained by adding phenylephrine, relaxation was elicited using acetylcholine in the presence or absence of apelin (100 ng/mL, 20 min). There was no significant difference in apelin mRNA levels between 17 and 20 weeks, but there was a decrease at 23 and 30 weeks of age, which was when the enhancing effect of PVAT on vasodilation in SHRSP.ZF rats was impaired. In the presence of apelin, acetylcholine-induced relaxation in arteries without PVAT was significantly increased to the level of the response in arteries with PVAT in SHRSP.ZF rats at 20 weeks. These findings suggest that apelin is involved in the enhancing effect of PVAT in MetS rat mesenteric artery, and decreases in the level is associated with deterioration of PVAT function. Further study to investigate the mechanisms underlying the decrease in apelin production with increasing age in metabolic syndrome is needed, butthe apelin level could be one of the predictors for development of cardiovascular complications in patients with MetS.

Vasorelaxant activity and acute toxicity of the ethanolic extract of Zanthoxylum rhoifolium Lam leaves

The study evaluated the vasorelaxant effect induced by the ethanolic extract of the leaves of Zanthoxylum rhoifolium Lam (EEtOH-Zr/leaves). Wistar rats were treated with the leaf extract containing a single dose of 2,000 mg / kg, v.o. After 14 days, the animals were anesthetized for blood collection and subsequent analysis of the biochemical parameters; they were then euthanized (sodium pentobarbital-100 mg/kg, i.p.) for the removal and morphological analysis of the heart, lung, liver and kidney. The vasorelaxation activity the and vascular reactivity of EEtOH-Zr/leaves were evaluated on artery mesenteric rings isolated from rats. The extract showed no signs of toxicity and no significant difference in the values of the biochemical parameters between the control group and the group of treated animals. In the evaluation of pharmacological activity in the smooth muscle, the EEtOH-Zr/leaves caused vasorelaxant effect on the tonic contraction induced by phenylephrine in mesenteric artery preparations in the presence (pD2=2.17±0.05 µg/mL; Emax=99.8±5.2%) and absence (pD2=2.14±0.05 µg/mL; Emax=95.3±6.4%) of the vascular endothelium. Oral administration of EEtOH-Zr/leaves reduced the contraction induced by the cumulative addition of PHE. It is concluded that the EEtOH-Zr/leaves promote vasorelaxation and reduce vascular reactivity of adrenergic alpha-1 agonist in the mesenteric artery. The results did not show toxic effects of the extract.

The Effect of Umbellularia californica Essential Oil on Blood Vessel Diameter in Frogs

Plant essential oils contain many chemicals that are physiologically active in vertebrates. Terpenoids, which represent the largest category of these compounds, have been shown to lower blood pressure by reducing cardiac output and causing vasodilation. Most studies on the vasoactivity of terpenoids have been done on aortic rings and mesenteric artery preparations, and little is known about their effects on microvasculature. We applied Umbellularia californica essential oil, which contains several different terpenoids, to the cutaneous microvasculature of frogs and measured changes in blood vessel diameter. The U. californica oil caused a sustained, reversible vasoconstriction of these blood vessels, whereas no significant change in diameter was caused by medical grade sesame oil, which served as our control. The vasoconstriction we observed was therefore induced by compounds in the U. californica oil, many of which have previously been shown to cause vasodilation in arteries. Our results suggest that the vasoactive effects of terpenoids may differ across blood vessel types, and are more variable than previously thought. KEYWORDS: Cardiovascular; Terpenoid; Vasoactive; Arteriole; Secondary Metabolite; Vasoconstriction; Essential Oil; Microvasculature

Effects of Resveratrol on Contractions of the Rat Tail Artery

In 2000, Naderali et. al. reported in Clinical Science (98:537-43) that resveratrol's direct vasorelaxant effect in vitro (on either adrenergic receptor-induced or high potassium-induced vascular smooth muscle contractions) was significantly greater in ring segments cut from mesenteric resistance arteries than from the main uterine conductance artery of female guinea pigs. Since then this important difference has not been confirmed in any other resistance versus conductance arterial tissues, and isolated from the same vasculature perfusing the same organ rather than different vessels perfusing different organs. The most distal and most proximal portions of the notably long ventral tail artery of the laboratory rat are commonly known for their unique structural and functional similarities to other resistance and conductance arteries (respectively) found throughout the rest of the body. Accordingly, we tested for relaxant effects of resveratrol in vitro in adrenergically pre-contracted rings cut from short segments of the distal and proximal portions of the tail artery from male rats. Its half-maximally effective concentration values were essentially identical (25±3 versus 27±3 micromolar) in distal versus proximal segments; results which do not confirm a greater relaxant effect of resveratrol in resistance versus conductance arterial tissues. Therefore, what Naderali et. al. previously observed may have been due to some other property that differed between their mesenteric and uterine arterial preparations. Support: MWU Master's Program.

Vasorelaxation induced by methyl cinnamate, the major constituent of the essential oil of Ocimum micranthum, in rat isolated aorta.

The aim of the present study was to investigate the vascular effects of the E-isomer of methyl cinnamate (E-MC) in rat isolated aortic rings and the putative mechanisms underlying these effects. At 1-3000μmol/L, E-MC concentration-dependently relaxed endothelium-intact aortic preparations that had been precontracted with phenylephrine (PHE; 1μmol/L), with an IC50 value (geometric mean) of 877.6μmol/L (95% confidence interval (CI) 784.1-982.2μmol/L). These vasorelaxant effects of E-MC remained unchanged after removal of the vascular endothelium (IC50 725.5μmol/L; 95% CI 546.4-963.6μmol/L) and pretreatment with 100μmol/L N(G) -nitro-l-arginine methyl ester (IC50 749.0μmol/L; 95% CI 557.8-1005.7μmol/L) or 10μmol/L 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (IC50 837.2μmol/L; 95% CI 511.4-1370.5μmol/L). Over the concentration range 1-3000μmol/L, E-MC relaxed K(+) -induced contractions in mesenteric artery preparations (IC50 314.5μmol/L; 95% CI 141.9-697.0μmol/L) with greater potency than in aortic preparations (IC50 1144.7μmol/L; 95% CI 823.2-1591.9μmol/L). In the presence of a saturating contractile concentration of K(+) (150mmol/L) in Ca(2+) -containing medium combined with 3μmol/L PHE, 1000μmol/L E-MC only partially reversed the contractile response. In contrast, under similar conditions, E-MC nearly fully relaxed PHE-induced contractions in aortic rings in a Ba(2+) -containing medium. In preparations that were maintained under Ca(2+) -free conditions, 600 and 1000μmol/L E-MC significantly reduced the contractions induced by exogenous Ca(2+) or Ba(2+) in KCl-precontracted preparations, but not in PHE-precontracted preparations (in the presence of 1μmol/L verapamil). In addition, E-MC (1-3000μmol/L) concentration-dependently relaxed the contractions induced by 2mmol/L sodium orthovanadate. Based on these observations, E-MC-induced endothelium-independent vasorelaxant effects appear to be preferentially mediated by inhibition of plasmalemmal Ca(2+) influx through voltage-dependent Ca(2+) channels. However, the involvement of a myogenic mechanism in the effects of E-MC is also possible.

Sympathetic nerve stimulation induces local endothelial Ca2+ signals to oppose vasoconstriction of mouse mesenteric arteries

It is generally accepted that the endothelium regulates vascular tone independent of the activity of the sympathetic nervous system. Here, we tested the hypothesis that the activation of sympathetic nerves engages the endothelium to oppose vasoconstriction. Local inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) signals ("pulsars") in or near endothelial projections to vascular smooth muscle (VSM) were measured in an en face mouse mesenteric artery preparation. Electrical field stimulation of sympathetic nerves induced an increase in endothelial cell (EC) Ca(2+) pulsars, recruiting new pulsar sites without affecting activity at existing sites. This increase in Ca(2+) pulsars was blocked by bath application of the α-adrenergic receptor antagonist prazosin or by TTX but was unaffected by directly picospritzing the α-adrenergic receptor agonist phenylephrine onto the vascular endothelium, indicating that nerve-derived norepinephrine acted through α-adrenergic receptors on smooth muscle cells. Moreover, EC Ca(2+) signaling was not blocked by inhibitors of purinergic receptors, ryanodine receptors, or voltage-dependent Ca(2+) channels, suggesting a role for IP(3), rather than Ca(2+), in VSM-to-endothelium communication. Block of intermediate-conductance Ca(2+)-sensitive K(+) channels, which have been shown to colocalize with IP(3) receptors in endothelial projections to VSM, enhanced nerve-evoked constriction. Collectively, our results support the concept of a transcellular negative feedback module whereby sympathetic nerve stimulation elevates EC Ca(2+) signals to oppose vasoconstriction.

Relaxant and contractile responses of detrusor muscle strips obtained from bladder outlet-obstructed rats treated with doxazosin enantiomers

(-)Doxazosin, one of (±)doxazosin enantiomers, was speculated to have a pharmacological enantioselectivity between the cardiovascular system and the urinary system by comparison with (+)doxazosin. Therefore, to evaluate the potential benefits of (-)doxazosin in the treatment of benign prostate hyperplasia, we compared the effects of the 3 agents, using rat mesenteric artery preparations and obstructed bladder strips. Concentration-response curves for carbachol (contractile response) and isoprenaline (relaxant response) in detrusor muscle strips of the bladder outlet obstruction (BOO) rats were shifted to the left, with significant increases in the Emax values, and significant decreases in the EC50 values by comparison with the sham-operated rats (P< 0.05, n = 10). The enhanced responses in detrusor muscle strips of the BOO rats treated with (±)doxazosin and its enantiomers at 3mg·(kg body mass)(-1)·day(-1) for 2weeks returned to normal levels, and the 3 agents inhibited the enhanced responses to carbachol and isoprenaline to the same extent. On the other hand, the 3 agents uncompetitively inhibited the vasoconstrictive response curves for NA in the rat isolated mesenteric artery, and the pKB value of (-)doxazosin at vascular α1-adrenoceptors was significantly smaller (P< 0.05, n = 6) than that of (+)doxazosin or (±)doxazosin. In conclusion, although (-)doxazosin inhibits vascular functional α1-adrenoceptors more weakly than (+)doxazosin, both agents equally ameliorate the enhanced responses in detrusor muscle of BOO rats, suggesting that the chiral carbon atom in the molecular structure of doxazosin does not affect its beneficial effects in the bladder smooth muscle of BOO rats.

Cardiovascular and vasoconstrictive actions of skate bradykinin in the little skate, Leucoraja erinacea (Elasmobranchii)

The vasoconstrictive and cardiovascular actions of a recently identified bradykinin (BK)-related peptide (Gly-Ile-Thr-Ser-Trp-Leu-Pro-Phe) from the little skate, Leucoraja erinacea were examined in the unanesthetised little skate. Intra-arterial administration of a skate BK (0.1–1 nmol kg −1) produced a hypertensive response with a rise in blood pressure reaching a maximum elevation of 28.7 ± 4.8% over baseline ( P < 0.05, n = 8) that was sustained for at least 12 min following administration of a 1 and 0.3 nmol kg −1 dose of skate BK. Further, in vivo administration of 1 nmol kg −1 skate BK induced a significant delayed increase in stroke volume (reaching a maximum of 54.4 ± 14.7% above baseline) without significant effect on either cardiac output or heart rate. In vitro, skate BK constricted the 1st branchial, mesenteric (EC 50 2.7 × 10 −9 M) and coeliac (EC 50 3.1 × 10 −9 M) arterial preparations of the skate. In contrast, skate [Arg 9]BK, the mammalian B 1 receptor agonist des-[Arg 9]BK, and the mammalian B 2 receptor antagonist HOE-140 failed to induce vasoconstriction in these isolated arterial preparations. The vasoconstrictor actions of skate BK in the isolated mesenteric, coeliac and branchial arterial preparations were significantly inhibited when co-administrated with esculetin and phentolamine. Indomethacin also inhibited the vasoconstrictor actions of skate BK in the isolated branchial artery. We conclude that, as in mammals and teleost fish, multiple pathways involving at least the alpha adrenergic and leukotriene synthesis pathway are involved in mediating the vasoconstrictive actions of BK in vascular smooth muscle of the little skate.

Activation of sphingosine kinase by muscarinic receptors enhances NO-mediated and attenuates EDHF-mediated vasorelaxation

Local formation of the sphingomyelin metabolite sphingosine-1-phosphate (S1P) within the vascular wall has been shown to modulate vascular reactivity. In this study we investigated whether sphingosine kinase, the enzyme responsible for S1P synthesis, plays a role in muscarinic receptor-mediated NO production and vascular relaxation in different blood vessel types. For this purpose, sphingosine kinase translocation and sphingolipid-dependent NO-production after muscarinic receptor stimulation were assessed in an endothelial cell line. Furthermore, we used the sphingosine kinase inhibitor N,N-dimethylsphingosine (DMS) to investigate the role of sphingosine kinase in the relaxant responses to the muscarinic agonist methacholine (MCh) in isolated rat aorta and mesenteric arteries. Activation of M(3)-receptors in an endothelial cell line induced a fast translocation of YFP-tagged sphingosine kinase-1 from the cytosol to the plasma membrane. Concomitant NO-production in this cell line was partially inhibited by DMS. Accordingly, in rat aorta the relaxant responses to MCh were attenuated in the presence of DMS, while the responses to the NO-donor sodium nitroprusside were unaltered. In contrast, DMS enhanced the relaxant responses to MCh in mesenteric artery preparations. This effect could also be observed in the presence of NO synthase and cyclooxygenase inhibitors, indicating that sphingosine kinase inhibition specifically enhanced endothelium-derived hyperpolarizing factor-mediated (i.e. non-NO and non-prostacyclin-dependent) relaxation. We conclude that sphingosine kinase differentially regulates vascular tone in different vessel types, enhancing NO-dependent vasorelaxation but counteracting EDHF-dependent vasorelaxation. This observation enhances our understanding of the complex mechanisms by which sphingolipids regulate vascular homeostasis. Moreover, a disturbed regulation of sphingolipid metabolism in the vascular wall may therefore play a role in the aetiology/pathology of disease states characterized by endothelial dysfunction.

Impaired Purinergic Neurotransmission to Mesenteric Arteries in Deoxycorticosterone Acetate-Salt Hypertensive Rats

Sympathetic nerves release norepinephrine and ATP onto mesenteric arteries. In deoxycorticosterone acetate (DOCA)-salt hypertensive rats, there is increased arterial sympathetic neurotransmission attributable, in part, to impaired prejunctional regulation of norepinephrine release. Prejunctional regulation purinergic transmission in hypertension is less well understood. We hypothesized that alpha(2)-adrenergic receptor dysfunction alters purinergic neurotransmission to arteries in DOCA-salt hypertensive rats. Mesenteric artery preparations were maintained in vitro, and intracellular electrophysiological methods were used to record excitatory junction potentials (EJPs) from smooth muscle cells. EJP amplitude was reduced in smooth muscle cells from DOCA-salt (4+/-1 mV) compared with control arteries (9+/-1 mV; P<0.05). When using short trains of stimulation (0.5 Hz; 5 pulses), the alpha(2)adrenergic receptor antagonist yohimbine (1 micromol/L) potentiated EJPs in control more than in DOCA-salt arteries (180+/-35% versus 86+/-7%; P<0.05). Norepinephrine (0.1 to 3.0 micromol/L), the alpha(2)adrenergic receptor agonist UK 14304 (0.001 to 0.100 micromol/L), the A(1) adenosine receptor agonist cyclopentyladensosine (0.3 to 100.0 micromol/L), and the N-type calcium channel blocker omega-conotoxin GVIA (0.0003 to 0.1000 micromol/L) decreased EJP amplitude equally well in control and DOCA-salt arteries. Trains of stimuli (10 Hz) depleted ATP stores more completely, and the latency to EJP recovery was longer in DOCA-salt compared with control arteries. These data indicate that there is reduced purinergic input to mesenteric arteries of DOCA-salt rats because of decreased ATP bioavailability in sympathetic nerves. These data highlight the potential importance of impaired purinergic regulation of arterial tone as a target for drug treatment of hypertension.

Direct Modulation of P2X1 Receptor-Channels by the Lipid Phosphatidylinositol 4,5-Bisphosphate

The P2X(1) receptor-channels activated by extracellular ATP contribute to the neurogenic component of smooth muscle contraction in vascular beds and genitourinary tracts of rodents and humans. In the present study, we investigated the interactions of plasma membrane phosphoinositides with P2X(1) ATP receptors and their physiological consequences. In an isolated rat mesenteric artery preparation, we observed a strong inhibition of P2X(1)-mediated constrictive responses by depletion of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] with the phosphatidylinositol 4-kinase inhibitor wortmannin. Using the Xenopus laevis oocyte expression system, we provided electrophysiological evidence that lowering PI(4,5)P(2) levels with wortmannin significantly decreases P2X(1) current amplitude and recovery. Previously reported modulation of recovery of desensitized P2X(1) currents by phospholipase C-coupled 5-hydroxytryptamine(2A) metabotropic receptors was also found to be wortmannin-sensitive. Treatment with wortmannin alters the kinetics of P2X(1) activation and inactivation without changing its sensitivity to ATP. The functional impact of wortmannin on P2X(1) currents could be reversed by addition of intracellular PI(4,5)P(2), but not phosphatidylinositol 3,4,5-trisphosphate, and direct application of PI(4,5)P(2) to excised inside-out macropatches rescued P2X(1) currents from rundown. We showed that the proximal region of the intracellular C terminus of P2X(1) subunit directly binds to PI(4,5)P(2) and other anionic phospholipids, and we identified the basic residue Lys(364) as a critical determinant for phospholipid binding and sensitivity to wortmannin. Overall, these results indicate that PI(4,5)P(2) plays a key role in the expression of full native and heterologous P2X(1) function by regulating the amplitude, recovery, and kinetics of ionotropic ATP responses through direct receptor-lipid interactions.

The impact of subchronic cadmium poisoning on the vascular effect of nitric oxide in rats

The aim of this study was to evaluate the impact of poisoning with cadmium in hypertensive doses (50 or 200 ppm in drinking water for three months) on the basal and stimulated release NO effect in the isolated and perfused rat mesenteric bed. Mesenteric artery preparation preconstricted by norepinephrine (0.5 microg/mL) was used to determine changes in its vascular resistance induced by e-NOS synthase blocker, N-omega-nitro-L-arginine (L-NOARG) injected in increasing doses from 1.0 to 200.0 microg or acetylcholine (ACh) administered in doses from 0.05 x 10(-10) to 5.0 x 10(-10) mol before and during L-NOARG infusion (1.0 microg/mL). Vascular reactivity was measured as an increase or decrease in perfusion pressure in the constant flow system. Rats poisoned with 50 or 200 ppm of cadmium demonstrated a significant decrease (P <0.05) in vascular response to L-NOARG used in doses of 50 or 100 microg. The dose-response curve obtained for L-NOARG was shifted to the right and ED50 value was greater in the group of rats given cadmium in a dose of 200 ppm than in the controls (70.3 +/- 10.7 versus 25.7 +/- 4.8 microg, P <0.01). These rats reacted with lower expressed vasodilatation to ACh in doses to 0.2 x 10(-10) mol. In all poisoned rats, L-NOARG enhanced the effect of ACh used in doses from 0.05 to 0.5 x 10(-10) mol, whereas in the control group this effect was only achieved at 0.1 x 10(-10) mol. The serum nitric oxide concentration was decreased (P <0.05) in both groups of cadmium-treated rats. These results suggest that cadmium in hypertensive doses modifies the vascular effect of NO in basal conditions and after stimulation by ACh.

Vasorelaxation induced by common edible tropical plant extracts in isolated rat aorta and mesenteric vascular bed

In this study, the vasodilatory actions of nine edible tropical plant extracts were investigated. Ipomoea batatas (sweet potato leaf), Piper betle (betel leaf), Anacardium occidentale (cashew leaf), Gynandropsis gynandra (maman leaf), Carica papaya (papaya leaf), and Mentha arvensis (mint leaf) extracts exhibited more than 50% relaxing effect on aortic ring preparations, while Piper betle and Cymbopogon citratus (lemongrass stalk) showed comparable vasorelaxation on isolated perfused mesenteric artery preparation. The vascular effect on the aortic ring preparations were mainly endothelium-dependent, and mediated by nitric oxide (NO) as supported by the inhibition of action in the presence of N ω-nitro- l-arginine (NOLA), an nitric oxide synthase (NOS) inhibitor, or by the removal of endothelium. In contrast, vasodilatory actions in resistance vessels (perfused mesenteric vascular beds) appear to involve several biochemical mediators, including NO, prostanoids, and endothelium-dependent hyperpolarizing factors (EDHFs). Total phenolic contents and antioxidant capacities varied among different extracts and found to be independent of vascular relaxation effects. This study demonstrates that many edible plants common in Asian diets to possess potential health benefits, affording protection at the vascular endothelium level.

Morphine potentiates dextromethorphan-induced vasodilation in rat superior mesenteric artery

The combined action of morphine and dextromethorphan on the superior mesenteric artery was investigated in this study. The artery was cut into rings, placed in a muscle bath and mounted to a force transducer for recording tension. Rings preconstricted with 1 μM phenylephrine produced a dose-dependent relaxation to graded doses of dextromethorphan but showed no response to morphine. An equimolar combination of morphine and dextromethorphan exhibited a marked synergism quantitated by a factor of 3.7 (1.8–7.7, 95% CI). Naloxone, which had no effect on the dextromethorphan dose–response relation, abolished the synergism. Removal of the endothelium produced a slight attenuation of the morphine–dextromethorphan synergism, but the magnitude of this attenuation was the same when dextromethorphan alone was examined in the denuded preparation. In contrast to the marked synergism seen in the mesenteric artery preparation, similar experiments on the carotid artery and the aorta produced only additive interactions.

α-lactorphin and β-lactorphin improve arterial function in spontaneously hypertensive rats

α-lactorphin (Tyr-Gly-Leu-Phe) lowers blood pressure in conscious adult SHR. This tetrapeptide is originally released from milk protein α-lactalbumin by enzymatic hydrolysis. In order to evaluate the antihypertensive mechanisms of α-lactorphin, the effects of the tetrapeptide on vascular function were investigated in (30−35 weeks old) spontaneously hypertensive rats (SHR) with established hypertension and age-matched normotensive Wistar-Kyoto (WKY) rats in vitro. In addition, we studied the vascular effects of another structurally related tetrapeptide, β-lactorphin (Tyr-Leu-Leu-Phe), which originates from milk protein β-lactoglobulin. Endothelium-dependent relaxation to acetylcholine (ACh) was reduced in mesenteric arterial preparations of SHR as compared to those of WKY. In SHR, the ACh-induced relaxation was augmented by α-lactorphin or β-lactorphin. The role of nitric oxide (NO) is suggested, since this improvement was abolished by the NO synthase (NOS) inhibitor N G-nitro-L-arginine methyl ester (L-NAME). Simultaneous potassium channel inhibitor tetraethylammonium (TEA) elicited no additional effect on the ACh-induced relaxation. The cyclooxygenase inhibitor diclofenac did not attenuate the augmented ACh relaxation induced by α-lactorphin or β-lactorphin, suggesting that endothelial vasodilatory prostanoids were not involved in the effect of the tetrapeptides. Endothelium-independent relaxation to the NO donor sodium nitroprusside (SNP) was augmented in mesenteric arterial preparations of SHR by simultaneous β-lactorphin. The tetrapeptides did not alter vascular responses in mesenteric arteries from WKY. In conclusion, both α-lactorphin and β-lactorphin improved vascular relaxation in adult SHR in vitro. The beneficial effect of α-lactorphin was directed towards endothelial function, whereas β-lactorphin also enhanced endothelium-independent relaxation.

Hypotension, Autonomic Failure, and Cardiac Hypertrophy in Transgenic Mice Overexpressing the α1B-Adrenergic Receptor

alpha(1)-Adrenergic receptors (alpha(1A), alpha(1B), and alpha(1D)) are regulators of systemic arterial blood pressure and blood flow. Whereas vasoconstrictory action of the alpha(1A) and alpha(1D) subtypes is thought to be mainly responsible for this activity, the role of the alpha(1B)-adrenergic receptor (alpha(1B)AR) in this process is controversial. We have generated transgenic mice that overexpress either wild type or constitutively active alpha(1B)ARs. Transgenic expression was under the control of the isogenic promoter, thus assuring appropriate developmental and tissue-specific expression. Cardiovascular phenotypes displayed by transgenic mice included myocardial hypertrophy and hypotension. Indicative of cardiac hypertrophy, transgenic mice displayed an increased heart to body weight ratio, which was confirmed by the echocardiographic finding of an increased thickness of the interventricular septum and posterior wall. Functional deficits included an increased isovolumetric relaxation time, a decreased heart rate, and cardiac output. Transgenic mice were hypotensive and exhibited a decreased pressor response. Vasoconstrictory regulation by alpha(1B)AR was absent as shown by the lack of phenylephrine-induced contractile differences between ex vivo mesenteric artery preparations. Plasma epinephrine, norepinephrine, and cortisol levels were also reduced in transgenic mice, suggesting a loss of sympathetic nerve activity. Reduced catecholamine levels together with basal hypotension, bradycardia, reproductive problems, and weight loss suggest autonomic failure, a phenotype that is consistent with the multiple system atrophy-like neurodegeneration that has been reported previously in these mice. These results also suggest that this receptor subtype is not involved in the classic vasoconstrictory action of alpha(1)ARs that is important in systemic regulation of blood pressure.

Anti-allergic activity of the Sophorae radix water extract in experimental animals.

The water extract of Sophorae radix was tested for its preventive effects against cardiovascular anaphylaxis elicited in experimental animals. In actively sensitized pithed rats, the extract partially protected the animals from death resulting from antigen challenge. Of the cardiovascular changes observed during the anaphylaxis in the pithed animals, the initial pressor response was significantly attenuated by the extract. The extract also improved anaphylactic cardiac dysfunction in passively sensitized isolated guinea hearts: improvement was noted in the contractility, arrhythmic duration and lactate dehydrogenase elevation. The perfusion pressure change to antigen challenge was not altered by the extract in passively sensitized isolated mesenteric artery preparations. The extract however significantly inhibited homologous passive cutaneous anaphylaxis responses in rats. When examined in isolated guinea pig tracheal muscle, the extract did not influence at reasonable concentrations on the contraction elicited by three major anaphylactic mediators, histamine, leukotriene D4 and serotonin. These results suggest that the water extract of Sophorae radix possesses anti-anaphylactic effect in cardiovascular system, primarily acting on the heart not the peripheral resistance vessels.

The influence of chronic inhibition of nitric oxide synthesis on contractile and relaxant properties of rat carotid and mesenteric arteries.

Balloon denudation of the rat carotid artery leads to an immediate decrease in beta-adrenoceptor-medi-ated vasodilator response. However, this arterial function becomes significantly enhanced during subsequent formation of neointima with the endothelial cell lining still being absent. It was therefore hypothesized that chronic suppression of endothelium-dependent nitric oxide (NO) synthesis may eventually upregulate the beta-adrenoceptor system on vascular smooth muscle. To investigate this hypothesis, male Wistar rats were treated chronically with the L-arginine analogue NG-nitro-L-arginine methyl ester (L-NAME) to inhibit the synthesis of NO (i.e. 15 mg/kg per day or 0.06 mmol/kg per day for 6 weeks p.o.). Prior to the experiments the mean arterial blood pressure (MAP) was significantly elevated in the L-NAME-treated rats (i.e. 128.4+/-3.4 mmHg vs. 100.0+/-2.9 mmHg, L-NAME vs. control, n=4, P<0.05). The functional properties of the isolated vessel preparations were established by isometric force measurement in a myograph setup, in the absence of L-NAME. The maximal contractile responses to high potassium chloride solution (100 mM), to the alpha1-adrenoceptor agonist phenylephrine, and to the thromboxane A2-agonist U46619, were not influenced by chronic L-NAME-treatment in the carotid and mesenteric artery preparations. The vasodilator responses to the cholinergic agonist methacholine were significantly impaired in the carotid arteries of L-NAME-treated animals: 30.9+/-7.9% vs. 64.6+/-2.0%, P<0.05, L-NAME vs. control, n=10. However, these responses appeared not to be modulated in the mesenteric artery preparations after chronic L-NAME treatment. Separate experiments showed that these responses could be blocked both in the rat carotid and mesenteric artery with L-NAME (10 mM) in vitro. Addition of the NO synthase substrate L-arginine could partially but significantly reverse this blockade. Chronic inhibition of NO synthesis caused significant deterioration of beta-adrenoceptor-mediated vasodilator responses. For carotid arteries: Emax=36.1+/-9.4% vs. 65.9+/-6.0%, P<0.05, L-NAME vs. control, n=5; and pD2=6.7+/-0.2 and 7.4+/-0.1, respectively, P<0.05, n=5. For mesenteric arteries: pD2=7.7+/-0.0 and 8.0+/-0.0, respectively, P<0.05, n=5. From these data, it may be concluded that chronic L-NAME treatment results in a stable impairment of the endothelium-dependent NO system in the rat carotid but not mesenteric arteries. The stated hypothesis fails as the beta-adrenoceptor-induced vasorelaxation of carotid and mesenteric arteries became significantly impaired, rather than enhanced. Taken together, the beta-adrenoceptor function in the rat carotid artery is apparently more dependent on endothelial NO synthesis than that in the rat mesenteric artery.

Hypotensive mechanism of [Leu13]motilin in dogs in vivo and in vitro

The effects of [Leu13]motilin were examined in vivo after its intravenous administration into anesthetized dogs and in vitro with isolated preparations of canine mesenteric artery. [Leu13]Motilin (0.1-10 nmol·kg-1, i.v.) induced both strong and clustered phasic contractions in the gastric antrum and duodenum. At doses of over 1 nmol·kg-1, [Leu13]motilin also produced transient decreases in arterial blood pressure, left ventricular pressure, maximum rate of rise of left ventricular pressure, and total peripheral resistance, and an increase in aortic blood flow and heart rate. A selective motilin antagonist, GM-109 (Phe-cyclo[Lys-Tyr(3-tBu)-betaAla]betatrifluoroacetate), completely abolished the gastric antrum and duodenal motor responses induced by [Leu13]motilin. In contrast, hypotension induced by [Leu13]motilin (1 nmol·kg-1) was unchanged in the presence of GM-109. In isolated mesenteric artery preparations precontracted with U-46619 (10-7 M), [Leu13]motilin (10-8-10-5 M) induced an endothelium-dependent relaxation, and this was inhibited by a pretreatment with Nomega-nitro-L-arginine, a competitive inhibitor of NO synthase (10-4 M). A high dose (10-4 M) of GM-109 slightly decreased [Leu13]motilin-induced relaxation, and shifted the concentration-response curve of [Leu13]motilin to the right. However, the pA2 value (4.09) of GM-109 for [Leu13]motilin in the present study was conspicuously lower than that previously demonstrated in the rabbit duodenum (7.37). These results suggest that [Leu13]motilin induces hypotension via the endothelial NO-dependent relaxation mechanism and not through the receptor type that causes upper gastrointestinal contractions.Key words: motilin, gastrointestinal motility, hypotension, hemodynamics, anesthetized dog, mesenteric artery, endothelium, nitric oxide.