Ear Artery Research Articles

Role of cytosolic phospholipase A 2 in the enhancement of α 2-adrenoceptor-mediated vasoconstriction by the thromboxane-mimetic U46619 in the porcine isolated ear artery: Comparison with vasopressin-enhanced responses

Pre-contraction with the thromboxane-mimetic U46619 enhances the subsequent α 2-adrenoceptor-mediated vasoconstriction in the porcine ear artery through an enhanced activation of ERK-MAP kinase. In this study we determined the role of cPLA 2 in this enhanced response, and determined whether vasopressin is also able to enhance α 2-adrenoceptor-mediated vasoconstriction through the same pathway. The cPLA 2 inhibitors AACOCF3 (50 μM) and MAFP (50 μM) both inhibited the U46619-enhanced α 2-adrenoceptor response, but had no effect on the direct α 2-adrenoceptor response. AACOCF3 also inhibited the enhanced ERK activation associated with the enhanced α 2-adrenoceptor-mediated vasoconstriction. Pre-contraction with arachidonic acid mimicked the effect of U46619 by enhancing the contractile response to the α 2-adrenoceptor agonist UK14304 (1 μM) and enhancing the α 2-adrenoceptor-mediated ERK activation. Pre-contraction with vasopressin also enhanced the contractile response to UK14304, but neither PD98059 (50 μM) nor AACOCF3 (50 μM) had any effect this vasopressin-enhanced response, indicating that neither the ERK pathway, nor cPLA 2 are involved in vasopressin-enhanced responses. The α 2-adrenceptor-stimulated activation of ERK was also unaffected by pre-contraction with vasopressin. On the other hand, inhibition of PKCζ inhibited the enhanced α 2-adrenoceptor contraction after pre-contraction with both U46619 and vasopressin. This study demonstrates that α 2-adrenoceptor-mediated vasoconstriction can be enhanced through two different pathways—one dependent upon the enhanced activation of ERK-MAP kinase through activation of cPLA 2, and the other through a different, ERK/cPLA 2-independent pathway.

Role of phospholipase D and diacylglycerol in activating constitutive TRPC-like cation channels in rabbit ear artery myocytes.

Previously we have described a constitutively active Ca2+-permeable non-selective cation channel in freshly dispersed rabbit ear artery myocytes that has similar properties to canonical transient receptor potential (TRPC) channel proteins. In the present study we have investigated the transduction pathways responsible for stimulating constitutive channel activity in these myocytes. Application of the pharmacological inhibitors of phosphatidylcholine-phospholipase D (PC-PLD), butan-1-ol and C2 ceramide, produced marked inhibition of constitutive channel activity in cell-attached patches and also butan-1-ol produced pronounced suppression of resting membrane conductance measured with whole-cell recording whereas the inactive isomer butan-2-ol had no effect on constitutive whole-cell or channel activity. In addition butan-1-ol had no effect on channel activity evoked by the diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Inhibitors of PC-phospholipase C (PC-PLC) and phospholipase A2 (PLA2) had no effect on constitutive channel activity. Application of a purified PC-PLD enzyme and its metabolite phosphatidic acid to inside-out patches markedly increased channel activity. The phosphatidic acid phosphohydrolase (PAP) inhibitor dl-propranolol also inhibited constitutive and phosphatidic acid-induced increases in channel activity but had no effect on OAG-evoked responses. The DAG lipase and DAG kinase inhibitors, RHC80267 and R59949 respectively, which inhibit DAG metabolism, produced transient increases in channel activity which were mimicked by relatively high concentrations (40 microm) of OAG. The protein kinase C (PKC) inhibitor chelerythrine did not prevent channel activation by OAG but blocked the secondary inhibitory response of OAG. It is proposed that endogenous DAG is involved in the activation of channel activity and that its effects on channel activity are concentration-dependent with higher concentrations of DAG also inhibiting channel activity through activation of PKC. This study indicates that constitutive cation channel activity in ear artery myocytes is mediated by DAG which is generated by PC-PLD via phosphatidic acid which represents a novel activation pathway of cation channels in vascular myocytes.

The efficacy of deferiprone on tissues aluminum removal and copper, zinc, manganese level in rabbits

The effect of 1,2-dimethyl-3-hydroxypyrid-4-one [deferiprone (DE)] on aluminum mobilization and elimination from tissues and serum as well as the influence on the excretion of trace elements, copper, zinc and manganese in rabbits was investigated. Sixteen New Zealand rabbits were randomly divided into three groups: control, Al-only and Al + DE. The Al-only and Al + DE animals received injections of Al 2(SO 4) 3 · 18H 2O 600 μmol Al/kg 5 days per week for 3 weeks. One week after the last Al injection the Al + DE rabbits were given deferiprone 750 μmol/kg/day intragastrically for 2 weeks. At the 42nd day the animals were sacrificed and the organs were taken and digested. Blood was taken from the ear artery three times (at the initiation of the experiment, before and after deferiprone administration). The aluminum and copper, zinc, manganese were determined by atomic absorption spectrophotometry. Our results showed that deferiprone could highly mobilize aluminum stores from tissues. At the end of experiment the aluminum contents of bone, kidney, liver and brain in Al + DE were significantly lower than that in Al-only rabbits. The copper, zinc, manganese contents were not affected by deferiprone administration.

Potential Binding Sites for Relaxin in Pregnant Rabbits

The purpose of this study was to identify tissues in the day 25 pregnant rabbit that bind relaxin. First, the clearance half-life of relaxin from the rabbit (n = 6) was determined by injecting 10 g porcine relaxin via the marginal ear vein. One-milliliter blood samples were collected via a cannula in the central ear artery. Samples were collected at 10- and 5-min pre-relaxin and at 1-min intervals post-relaxin injection, and the relaxin concentrations determined by radioimmunoassay. The clearance half-life was 4 min. Next, pregnant rabbits were infused with [125I]-relaxin. Control rabbits (n = 3) received 10 g radio-inert relaxin via the marginal ear vein in order to saturate endogenous receptors. Ten minutes later, 10 ng [125I]-relaxin was similarly injected. Treated rabbits (n = 3) received only [125I]-relaxin. After allowing sufficient time for clearance (24 min), a 1-mL blood sample was removed via the central ear vein. Rabbits were euthanized, tissues of maternal and fetal origin excised, and cpm/mg of tissue divided by cpm/mL of blood was determined. Differences in uptake of [125I]-relaxin between control and treated animals, using the Student paired t test, were found for the uterus (P < .05), uterine cervix (P < .03), and mammary gland (P < .05). These data suggest potential rabbit tissues with the LGR-7 receptor.

Transcutaneous Fluorescence Dilution Cardiac Output and Circulating Blood Volume during Hemorrhagic Hypovolemia

Cardiac output and circulating blood volume are important parameters for assessing cardiac function in the intensive care setting and during major surgeries. The authors tested in an animal model of hemorrhagic hypovolemia the feasibility of measuring these parameters simultaneously by transcutaneous fluorescence monitoring of an intravenous bolus injection of indocyanine green. Fluorescence dilution cardiac output was measured in seven anesthetized rabbits and compared to thermodilution cardiac output. The optical probe used to excite the indocyanine green fluorescence was in contact with the skin above the ear artery. Local heating enhanced blood perfusion of the measurement site. Cardiac output was measured during baseline conditions, during hemorrhagic hypovolemia, and after partial restoration of the blood volume with reinfused blood. Estimates of the circulating blood volume were simultaneously obtained from the analysis of the fluorescence dilution traces. Cardiac output measured by fluorescence dilution (thermodilution) averaged 455 +/- 16 (450 +/- 13) ml/min in baseline conditions and 323 +/- 15 (330 +/- 13) ml/min during hypovolemia. Fluorescence dilution cardiac output was linearly related to thermodilution cardiac output (slope = 1.13 +/- 0.05, ordinate = -50 +/- 19 ml/min, R = 0.92). Interanimal differences explained most of the variance between cardiac output estimates obtained with the two techniques. Circulating blood volume decreased from 204 +/- 5 ml in baseline conditions to 174 +/- 8 ml after bleeding and reflected blood volume changes in this acute bleeding-reinfusion model. The study extends the applicability of the fluorescence dilution technique for cardiac output measurement to hypovolemic conditions and demonstrates its ability to produce accurate estimates of the circulating blood volume in experimental animals.

Electrical coupling and release of K+ from endothelial cells co‐mediate ACh‐induced smooth muscle hyperpolarization in guinea‐pig inner ear artery

The physiological basis of ACh-elicited hyperpolarization in guinea-pig in vitro cochlear spiral modiolar artery (SMA) was investigated by intracellular recording combined with dye labelling of recorded cells and immunocytochemistry. We found the following. (1) The ACh-hyperpolarization was prominent only in cells that had a low resting potential (less negative than -60 mV). ACh-hyperpolarization was reversibly blocked by 4-DAMP, charybdotoxin or BAPTA-AM, but not by N(omega)-nitro-L-arginine methyl ester, glipizide, indomethacin or 17-octadecynoic acid. (2) Ba(2)(+) (100 microm) and ouabain (1 microm) each attenuated ACh-hyperpolarization by approximately 30% in smooth muscle cells (SMCs) but had only slight or no inhibition in endothelial cells (ECs). A combination of Ba(2)(+) and 18beta-glycyrrhetinic acid near completely blocked the ACh-hyperpolarization in SMCs. (3) High K(+) (10 mm) induced a smaller hyperpolarization in ECs than in SMCs, with an amplitude ratio of 0.49 : 1. Ba(2)(+) blocked the K(+)-induced hyperpolarization by approximately 85% in both cell types, whereas ouabain inhibited K(+)-hyperpolarization differently in SMCs (19%) and ECs (35%) and increased input resistance. 18beta-Glycyrrhetinic acid blocked the high K(+)-hyperpolarization in ECs only. (4) Weak myoendothelial dye coupling was detected by confocal microscopy in cells recorded with a propidium iodide-containing electrode for longer than 30 min. A sparse plexus of choline acetyltransferase-immunoreactive (ChAT) fibres was observed around the SMA and its up-stream arteries. (5) Evoked excitatory junction potentials (EJP) were partially blocked by 4-DAMP in half of the cells tested. We conclude that ACh-induced hyperpolarization originates from ECs via activation of Ca(2)(+)-activated potassium channels, and is independent of the release of NO, cyclo-oxygenase or cytochrome P450 products. ACh-induced hyperpolarization in smooth muscle cells involves two mechanisms: (a) electrical spread of the hyperpolarization from the endothelium, and (b) activation of inward rectifier K(+) channels (K(ir)) and Na(+)-K(+) pump current by elevated interstitial K(+) released from the endothelial cells, these being responsible for about 60% and 40% of the hyperpolarization, respectively. The role ratio of K(ir) and pump current activation is at 8 : 1 or less.

Platelet Aggregation and Vasoconstriction Related to Platelet Cyclooxygenase and 12-Lipoxygenase Pathways

Many thrombotic angiopathies originate as a result of an abnormal interaction between platelets and blood vessel walls. The present study focused on the platelet activating factor (PAF)-induced platelet aggregation mechanism and was undertaken to clarify the association between platelet aggregation and vasocontractility. Vasoconstriction was examined in perfused artery segments dissected from the central ear artery of male rabbits. Autologous platelet rich plasma (PRP) was infused into the perfusion system. Vasocontractility was examined in response to PRP plus PAF or collagen. The vasocontractile response to noradrenaline (NA-R) on perfusion of PRP plus PAF was initially augmented, then gradually attenuated. The platelet aggregation in response to PAF was constant and longlasting and moderately inhibited by nordihydroguaiaretic acid, a 12-lipoxygenase (LOX) inhibitor, but was unaffected by indomethacin, a cyclooxygenase inhibitor. The attenuated responses disappeared and NA-R was returned to the initial level on pretreatment with tetrodotoxin which excluded neurogenic components from the arterial preparation. In contrast, a repetitive NA-R to PRP plus collagen gradually increased. Thus, it may be concluded that PAF-induced platelet aggregation participates in activation of the 12-LOX pathway and is accompanied by the release of vasodepressor nerve-stimulating substances from platelets.

A comparative study of invasive and oscillometric methods of arterial blood pressure measurement in the anesthetized rabbit

Introduction: The aim of this project was to evaluate the reliability and accuracy of direct, using the central ear artery (CEA), and oscillometric, using limb-cuffs, methods of arterial blood pressure (AP) measurement in the anesthetized rabbit. Methods: New Zealand rabbits were anesthetized using a xylazine-ketamine-isoflurane protocol. Using the abdominal aorta (ABA) as direct “gold standard” for AP measurements, ABA pressure readings, via femoral artery catheterization, were compared with those made simultaneously from the ascending aorta after median sternotomy. Thereafter, direct CEA as well as forelimb-(FL) and hindlimb-(HL) cuff oscillometric readings were compared with those made simultaneously from ABA. Results: The blood pressure in the ABA correlated with that from ascending aorta. Furthermore, CEA correlated with the ABA readings. Nevertheless, at high pressures, their divergence from “true” pressure tended to increase. Oscillometric readings at the FL site correlated well with “true” pressure while those at the HL site did not. Their divergence tended to increase at high pressures when using the FL site, while it varied when using the HL site. The accuracy of measurements was moderate for the FL site while poor for the HL site. Discussion: Our results suggest that the CEA can be readily used with high reliability and accuracy for direct AP measurements in the anesthetized rabbit. On the other hand, the FL-cuff oscillometric method should only be used for the evaluation of AP at low and normal pressure ranges.

Inhibitory regulation of constitutive transient receptor potential-like cation channels in rabbit ear artery myocytes.

In the present study we have investigated an inhibitory pathway regulating a constitutively active Ca(2+)-permeable non-selective cation conductance (I(cat)) in rabbit ear artery smooth muscle cells. Constitutive single channel activity of I(cat) was recorded in cell-attached and inside-out patches with similar unitary conductance values. In inside-out patches with relatively high constitutive activity the G-protein activator GTPgammaS inhibited channel activity which was reversed by the protein kinase C (PKC) inhibitor chelerythrine indicating a G-protein pathway inhibits channel activity via PKC. Spontaneous channel activity was also suppressed by the G-protein inhibitor GDPbetaS suggesting a G-protein is also involved in initiation of constitutive channel activity. Bath application of antibodies to G(alphaq)/G(alpha11) enhanced channel activity whereas anti-G(alpha1-3)/G(alphao) antibodies decreased basal channel activity which suggests that G(alphaq)/G(alpha11) and G(alphaiota)/G(alphao) proteins initiate, respectively, the inhibitory and excitatory cascades. The phospholipase C (PLC) inhibitor U73122 increased spontaneous activity which implies a role for PLC in the inhibitory pathway. Bath application of the diacylycerol (DAG) analogue 1-oeoyl-2-acetyl-sn-glycerol (OAG) decreased the probability of channel opening (NP(o)) and this was reversed by chelerythrine. Application of the PKC activator phorbol 12, 13-dibutyrate (PDBu) and chelerythrine, respectively, decreased and increased NP(o). These data indicate that spontaneously active cation channels are inhibited by a tonic inhibitory pathway involving G(alphaq)/G(alpha11)-mediated stimulation of PLC to generate DAG which activates PKC to inhibit channel opening. There were some patches with relatively low NP(o) and it was evident that the inhibitory pathway was particularly marked in these cases. Moreover in the latter patches GTPgammaS and OAG caused marked increases in NP(o). Together with inhibitory effects of GDPbetaS and anti-G(alpha1-3)/G(alphao) antibodies the results suggest that there is constitutive G(alphai)/G(alphao) protein activity leading to channel opening via a DAG-mediated but PKC-independent mechanism. Finally, with whole-cell recording it is shown that noradrenaline increases I(cat) and the noradrenaline-evoked response is markedly potentiated by PKC inhibition. This latter observation shows that PKC also limits agonist-evoked I(cat) in these arterial myocytes.

Differences in the vascular selectivity and tolerance between the NO donor/β-blocker PF9404C and nitroglycerin

The vascular selectivity of PF9404C ((2′ S),(2 S)-3-isopropylamine,1-[4-(2,3-dinitroxy)propoxymethyl]-phenoxy-2′-propanol), a new β-blocker with nitric oxide (NO)-donor and vasodilator properties, was studied in different rabbit arteries and veins. Phenylephrine (10 −6 M) or 35 mM K + were used to pre-contract the arteries and veins prior to study the relaxant effects of PF9404C and nitroglycerin. The potency of both drugs to depress the phenylephrine-induced contraction was greater than that shown in the blockade of the K +-evoked contraction in most of the vessels studied, with the exception of the central ear artery. PF9404C exhibited about three-fold higher potency than nitroglycerin to relax the majority of the vessels studied, especially when they were contracted with K +, and showed a certain selectivity of action for the renal artery. PF9404C produced autotolerance but this effect was about 20-fold less pronounced than that observed with nitroglycerin. Cross-tolerance in those preparations pre-exposed to PF9404C that were relaxed later on with nitroglycerin was much greater than autotolerance. The tolerance for nitroglycerin was practically abolished in the presence of N-acetylcysteine. However, this was not the case for PF9404C. These results indicate that, although sharing the property of being NO donors, PF9404C and nitroglycerin show a different profile in causing vasodilation; furthermore, the tolerance to this effect is lesser for PF9404C and seems to be mediated by a mechanism different to that of nitrates. This makes PF9404C a nice pharmacological tool to further develop novel NO-donor compounds with a lesser degree of vascular tolerance than those now available.

Effects of gene delivery on collateral development in chronic hypoperfusion: Diverse effects of angiopoietin-1 versus vascular endothelial growth factor

The aim of this research was to test the effects of vascular endothelial growth factor (VEGF)/angiopoietin-1 (Ang-1) on adult hypoperfused tissues. Angiopoietin-1 and VEGF act separately and synergistically in vascular development during embryogenesis. However, little is known regarding their relative roles in collateral development after chronic arterial obstruction and tissue ischemia in the adult. Central and caudal ear arteries of 32 rabbits were ligated to induce ischemia. At two months, when flow was about 65% of pre-ligation values, we injected intradermally 10(9) plaque-forming unit adenovirus with the following transgenes: Ang-1, VEGF, or a combination of both. Ear perfusion was followed up for four weeks, and vessel leakage was assessed by Evens Blue test. Before injection, flow was 65% of baseline, and endogenous VEGF levels in ischemic tissue were increased. Adenovirus-encoding VEGF gene (Ad.VEGF) at one week caused a visible inflammatory response associated with a 24% flow increase (p = 0.018). Adenovirus-encoding Ang-1 gene (Ad.Ang-1) increased flow 22% (p = 0.004) with no visible inflammation; Ad.VEGF caused three times as much vessel leakage as Ad.Ang-1 (142.5 +/- 38 vs. 49.5 +/- 9.8 ng Evens Blue/mg tissue; p < 0.001). However, at four weeks, compared with baseline, VEGF decreased flow 18% (p = 0.004), whereas Ang-1 increased tissue perfusion 26% (p < 0.001). This effect was abolished when Ad.Ang-1 was injected with soluble VEGF receptor [Ad.Flt(1-3)-Fc], which blocks VEGF-dependent signaling. Exogenous Ang-1 did not increase perfusion in a normally perfused ear, in which endogenous VEGF is not expressed. Exogenous Ang-1 enhances perfusion in hypoperfused tissues only in the presence of increased levels of endogenous VEGF. Overexpression of VEGF, however, after causing an inflammatory response, does not improve collateral blood flow.

Temporal artery temperature measurements in healthy infants, children, and adolescents

Temporal artery temperature measurements in healthy infants, children, and adolescents

Hypotensive Effects of Eugenosedin-A with Serotonin, Alpha- and Beta-Adrenoceptor Antagonistic Activities in Spontaneously Hypertensive and Normotensive Rats

Eugenosedin-A is a newly synthesized compound with special serotonergic, α- and β₁-adrenergic blocking actions. Intravenous injection of eugenosedin-A significantly caused dose-dependent decreases in the mean arterial blood pressure and heart rate in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). The effects of eugenosedin-A-decreased blood pressure and heart rate in SHR were more potent than in WKY. In in vitro experiments, eugenosedin-A competitively antagonized the serotonin-, norepinephrine- and clonidine-induced vasocontraction in a concentration-dependent manner in isolated thoracic aorta of WKY and SHR. We also observed that eugenosedin-A competitively antagonized the isoproterenol-induced positive inotropic effects in a concentration-dependent manner in the isolated left atrium of WKY and SHR. These findings clearly suggested that eugenosedin-A possesses α₁/α₂, β₁ and 5-HT_2A receptor-blocking activities. The order of pA₂ values in isolated tissues of WKY was 5-HT_2A > α₁/α₂ > β₁. However, the order of pA₂ values in isolated tissues of SHR was α₁/α₂ >5-HT_2A > β₁. Similarly, we found that the in vitro functional activity of eugenosedin-A is quite different between WKY and SHR. On the other hand, in the isolated rabbit ear artery sensitized with 16 mmol/l K⁺, eugenosedin-A antagonized 5-nonyloxytryptamine- and serotonin-induced vasocontractions, indicating that it also blocked 5-HT_1B and 5-HT_2A receptors. In radioligand binding experiments, eugenosedin-A had significant binding affinities on α₁/α₂, β₁, 5-HT_1B and 5-HT_2A receptors. Finally, we suggest that the hypotensive effects of eugenosedin-A can be attributed to its multiple actions on the blockade of 5-HT_1B, 5-HT_2A, α and β₁ receptors in both WKY and SHR strains.

Inertial cavitation dose produced ex vivo in rabbit ear arteries with optison

Ultrasound-induced inertial cavitation (IC) effects were studied ex vivo in rabbit ear arteries with the addition of ultrasound contrast agents (UCAs). Ears were removed from New Zealand white rabbits immediately after being euthanized under a protocol approved by the University of Washington IACUC. The auricular arteries were perfused with varying concentration of UCA (Optison) in saline and exposed to 1.155-MHz pulsed high-intensity focused ultrasound (HIFU) with constant PRF (10 Hz), pulse length (20 cycles), and total treatment time (20 s). Experiments were performed for variable peak negative acoustic pressure (P−) (from 0.19 to 3.31 Mpa) and Optison volume concentration (0% [saline only], 0.1%, 0.2%, 0.5%, and 1%). Cavitation activity was quantified by IC Dose (cumulated root-mean-squared [rms] broadband noise amplitude in a particular band in the frequency domain). The results showed that (1) IC activity was induced much more easily with the addition of Optison, even at low volume concentration, such as 0.1%. (2) IC dose increased significantly with the increasing acoustic pressure and Optison concentration. (3) Higher concentrations of Optison decreased the IC threshold. [Work supported by NIH 8RO1 EB00350-2.]

Identification of alpha-1L adrenoceptor in rabbit ear artery.

The alpha-1L adrenoceptor (AR) was identified in rabbit ear artery by both functional and ligand binding studies. In functional studies using arterial rings, the contractile response to NS-49 [(R)-(-)-3'-(2-amino-1-hydroxyethyl)-4'-fluorometh-anesulfonanilide hydrochloride] (alpha-1A and alpha-1L AR-selective agonist) was competitively antagonized with low affinities by prazosin, RS-17053 [N-[2-(2-cyclopropylmethoxyphenoxy) ethyl]-5-chloro-alpha,alpha-dimethyl-1H-indole-3-ethamine hydrochloride], and 5-methylurapidil but with high affinities by tamsulosin and KMD-3213 [(-)-1-(3-hydroxypropyl)-5-[(2R)-2-([2-[(2,2,2-trifluoroethoxy)phenoxy]ethyl]amino)propyl]-2,3-dihydro-1H-indole-7-carboxamide]. In contrast, the response to noradrenaline (nonselective alpha-1 AR agonist) was inhibited noncompetitively by these antagonists (except 5-methylurapidil) with Schild slopes different from unity. These results suggest that the response to NS-49 was mediated predominantly via alpha-1L ARs, whereas the response to noradrenaline was produced through two distinct alpha-1 AR subtypes (presumably alpha-1B and alpha-1L ARs). In binding studies with intact segments of rabbit ear artery, [3H]KMD-3213 bound with high affinity (pKD=9.7) to alpha-1 ARs, which were subdivided by prazosin, RS-17053, and 5-methylurapidil into two subtypes (alpha-1A and alpha-1L ARs). In contrast, [3H]prazosin binding sites in ear artery segments (pKD = 9.8) were identified as alpha-1A and alpha-1B ARs. In conventional binding studies using isolated rabbit ear artery microsomal membranes, [3H]KMD-3213 binding sites were identified as alpha-1A ARs with high affinities for prazosin, RS-17053, and 5-methylurapidil. Our study indicates that an alpha-1L AR having a unique pharmacological profile coexists with alpha-1A and alpha-1B ARs in rabbit ear artery and can be identified either functionally or by binding studies using intact tissues but not microsomal membrane preparations.

Aalpha,beta-methylene ATP enhances P2Y4 contraction of rabbit basilar artery.

Interactions between different selective P2 receptor agonists have been used as tools to identify different P2 receptor subtypes. In the present study, we examined the P2 receptor subtypes and the mechanisms of potentiation of UTP contraction (P2Y contraction) by alpha,beta-methylene ATP [(2-carboxypiperazin-4-yl)propyl-1-phosphanoic acid (CPP), a P2X agonist] using isometric tension in the denuded rabbit basilar artery. We made the following observations: 1). a predominant P2X receptor contraction was observed in the rabbit ear artery by the rank order of CPP >> 2-methylthioATP > ATP > UTP; 2). functional P2Y receptors were observed in the rabbit basilar artery by the rank order of UTP >> ATP = CPP = 2-methylthioATP; 3). CPP potentiated UTP-, ATP-, and ATPgammaS-induced contractions, possibly by activation of P2Y4 receptors because ATPgammaS does not activate P2Y6 receptors; and 4). ectonucleotidase did not play a predominant role in the potentiative effect of CPP because Evans blue, Ca(2+)-free medium, or divalent cation Ni(2+) did not affect the effect of CPP. Evans blue potentiated the contraction by UTP but not by ATP or ATPgammaS. We conclude that CPP enhanced P2Y4-mediated contraction in the rabbit basilar artery, and the influence by ectonucleotidases on CPP-potentiation remains unclear.

Dopaminergic vasodilation in the choroidal circulation by d1/d5 receptor activation.

To test whether exogenous dopamine can cause choroidal vasodilation and to identify the mediating receptors in anesthetized rabbits. Mean arterial pressure (MAP), intraocular pressure (IOP), and orbital venous pressure (OVP) were measured by direct cannulation of the central ear artery, the vitreous, and the orbital venous sinus, respectively. Laser Doppler flowmetry was used to measure choroidal blood flow (ChorBF) while MAP was manipulated mechanically with occluders on the aorta and vena cava, thus changing perfusion pressure (PP) over a wide range. In the first group of animals (n = 11), pressure-flow (PF) relationships were performed at control and in response to 40 micro g/kg per minute intravenous (IV) dopamine (D40) and D40+SCH-23390 (0.5 mg/kg, bolus injection IV). In the second group of animals (n = 6), PF relationships were recorded at control and during infusion of SKF-38393 (80 micro g/kg per minute). D40 lowered IOP and caused an upward shift in the choroidal PF relationship, which was blocked by the D1/D5 antagonist SCH-23390 suggesting the involvement of the dopamine D1/D5 receptors. Stimulation of the D1/D5 receptors by infusion of the selective agonist SKF-38393 also lowered IOP and caused an upward shift in the PF relationship. Dopamine and SKF-38393 tended to decrease OVP, but the effect was not significant. Dopamine can cause choroidal vasodilation in anesthetized rabbits. Because SCH-23390 was able to block the response and SKF-38393 caused a similar vasodilation, we conclude that the vasodilation is caused by a D1/D5-receptor-mediated mechanism.

Comparative evaluation of absorbable hemostats: advantages of fibrin-based sheets

Bioactive hemostats and wound dressings consist of either inherently active materials or act as delivery vehicles which contain such materials. Fibrin is a natural hemostat and scaffold, guiding the direction of wound contraction and closure. In order to improve the ease of application of liquid fibrin glue, we have made a freeze-dried form of polymerized fibrin that supports hemostasis and wound healing. The bleeding from the middle ear artery of rabbits was found to be arrested instantaneously on application of fibrin sheets, even when the animal was heparinized systemically. As the fibrin sheet was found to be fragile, gelatin was incorporated to the sheet and thus the mechanical stability was improved without compromising the hemostatic effect. The efficacy of the fabricated fibrin and fibrin–gelatin sheets to seal traumatized rat liver was compared with commercially available hemostats, Abgel (cross-linked gelatin) and Surgicel (cross-linked cellulose). Tissue compatibility of all the hemostats was studied by analyzing the liver tissue 15 days after application. While the hemostatic effect was best with fibrin and fibrin–gelatin sheets, both Surgicel and Abgel were not capable of arresting the bleeding quickly. Gross analysis of tissue on the 15th day of application, visibly, Abgel was not only degraded but resulted in severe adhesions of internal organs and histologically capsule formation around the implant was evident. Though Surgicel was also seen as cream soft material on the site of application that joined two pieces of liver, there was no adhesion of other internal organs and histologically, immune reaction and foreign-body-type giant cells were present in large amounts. Fibrin was not found grossly on application site whereas fibrin–gelatin was seen as a small white spot. Granulation tissue formation and cell migration into the fibrin-based sheets were evident, and therefore, fibrin-based sheets are not only efficient hemostats but showed optimum degradation and wound healing.

Inhibition of rostral medullary raphé neurons prevents cold-induced activity in sympathetic nerves to rat tail and rabbit ear arteries

Sympathetically-mediated vasoconstriction of cutaneous vessels is critical for thermoregulation in the cold. We determined whether cold-induced sympathetic discharge depends on activity of neurons in the rostral medullary raphé. In urethane-anesthetized rats and rabbits, cooling the trunk skin by a water jacket reproducibly increased cutaneous sympathetic discharge recorded in the tail (rats) and the ear pinna (rabbits). When neurons in the rostral medullary raphé were inhibited by microinjection of glycine (30–100 nmol in 60–200 nl in rats), or muscimol (1 nmol in 100 nl in rabbits), cutaneous sympathetic activity was silenced and no longer responded to cooling (7±3 and 5±2% of pre-injection increase in rats and rabbits, respectively, P<0.01). Our data demonstrate that activity of rostral medullary raphé neurons is important for the CNS mediation of cold-induced increases in sympathetic cutaneous vasomotor nerves.

Feline immunodeficiency virus and retrovirus-mediated adventitial ex vivo gene transfer to rabbit carotid artery using autologous vascular smooth muscle cells

We have developed an ex vivo gene transfer technique to rabbit arterial wall using autologous smooth muscle cells (SMCs). SMCs were harvested from rabbit ear artery, transduced in vitro with vesicular stomatitis virus G-glycoprotein pseudotyped retrovirus or feline immunodeficiency virus (FIV) and returned to the adventitial surface of the carotid artery using a periadventitial silicone collar or collagen sheet placed around the artery. β-galactosidase (lacZ) and human apolipoprotein E3 (apoE3) cDNAs were used as transgenes. After retrovirus-mediated gene transfer of lacZ the selected cells implanted with high efficiency and expressed lacZ marker gene at a very high level 7 and 14 days after the operation. The level of lacZ expression decreased thereafter but was still detectable 12 weeks after the gene transfer, and was exclusively localized to the site of cell implantation inside the collar. Utilizing FIV vector expressing apoE3, low levels of apoE were measured from serum collected from a low-density lipoprotein receptor deficient Watanabe heritable hyperlipidemic rabbits 1 month after the gene transfer. The physiological effect of apoE expression was detected as transiently elevated serum cholesterol levels. The results indicate that the model can be used for high efficiency local gene transfer in arteries, e.g. during vascular surgery. The model is also valuable for studying expression, stability and safety of new gene transfer vectors and their expression products in vivo.