Segments Of Rat Tail Artery Research Articles

Paradoxical effects of acidosis on the noradrenaline-induced and neurogenic constriction of the rat tail artery at low temperatures.

Although vasodilatation evoked by acidosis at normal body temperature is well known, the reports regarding effect of acidosis on the reactivity of the isolated arteries at low temperatures are nonexistent. This study tested the hypothesis that the inhibitory effect of acidosis on the neurogenic vasoconstriction may be increased by cooling. Using wire myography, we recorded the neurogenic contraction of the rat tail artery segments to the electrical field stimulation in the absence and in the presence of 0.03-10.0 µmol/L noradrenaline. The experiments were conducted at 37 °C or 25 °C and pH 7.4 or 6.6 which was decreased by means of CO2. Noradrenaline at concentration of 0.03-0.1 µmol/L significantly potentiated the neurogenic vasoconstriction at 25 °C, and the potentiation was not inhibited by acidosis. Contrary to our hypothesis, acidosis at a low temperature did not affect the noradrenaline-induced tone and significantly increased the neurogenic contraction of the artery segments in the absence and presence of noradrenaline. These effects of acidosis were partly dependent on the endothelium and L-type Ca2+ channels activation. The phenomenon described for the first time might be of importance for the reduction in the heat loss by virtue of decrease in the subcutaneous blood flow at low ambient temperatures.

Molecular cloning of a novel tryptophyllin peptide from the skin of the orange-legged monkey frog, Phyllomedusa hypochondrialis.

Tryptophyllins are a group of small (4-14 amino acids), heterogenous peptides, mostly from the skins of hylid frogs from the genera, Phyllomedusa and Litoria. To date, more than forty TPHs have been discovered in species from these two genera. Here, we describe the identification of a novel tryptophyllin type 3 peptide, PhT-3, from the extracts of skin of the orange-legged monkey frog, Phyllomedusa hypochondrialis, and molecular cloning of its precursor-encoding cDNA from a cDNA library constructed from the same skin sample. Full primary structural characterization was achieved using a combination of direct Edman degradation, mass spectrometry and deduction from cloned skin-derived cDNA. The open-reading frame of the precursor cDNA was found to consist of 63 amino acid residues. The mature peptide arising from this precursor contains a post-translationally modified N-terminal pyroglutamate (pGlu) residue, formed from acid-mediated cyclization of an N-terminal Gln (Q) residue, and with the structure: pGlu-Asp-Lys-Pro-Phe-Trp-Pro-Pro-Pro-Ile-Tyr-Pro-Met. Pharmacological assessment of a synthetic replicate of this peptide on phenylephrine preconstricted rat tail artery segments, revealed a reduction in relaxation induced by bradykinin. PhT-3 was also found to mediate antiproliferative effects on human prostate cancer cell lines.

Role of prostanoid IP and EP receptors in mediating vasorelaxant responses to PGI 2 analogues in rat tail artery: Evidence for G i/o modulation via EP 3 receptors

Prostanoid IP receptors coupled to Gs are thought to be the primary target for prostacyclin (PGI 2) analogues. However, these agents also activate prostanoid EP 1–4 receptor subtypes to varying degrees, which are positively (EP 2/4) or negatively (EP 3) coupled to adenylate cyclase through Gs or Gi, respectively. We investigated the role of these receptors in modulating relaxation to PGI 2 analogues cicaprost, iloprost and treprostinil in pre-contracted segments of rat tail artery. Prostanoid IP (RO1138452), EP 4 (GW627368X), EP 3 (L-798106), EP 1–3 (AH6809), and EP 1 (SC-51322) receptor antagonists were used to determine each receptor contribution. The role of G i/o was investigated using pertussis toxin (PTX), while dependence on cAMP was determined using adenylate cyclase (2′5′dideoxyadenosine, DDA) and protein kinase A (2′-O-monobutyryladenosine- 3′,5′-cyclic monophosphorothioate, Rp- isomer, Rp-2′-O-MB-cAMPS) inhibitors, and by measurement of tissue cAMP. All analogues caused relaxation which was significantly ( P < 0.01) inhibited by RO1138452; with maximum response to cicaprost, iloprost and treprostinil reduced by 51%, 66% and 37%, respectively. GW627368X had no effect when used alone, but in combination with RO1138452, caused a rightward shift of the curves for cicaprost and iloprost but not treprostinil. PTX treatment potentiated relaxation to all 3 analogues ( P < 0.01), as did L798106 and AH6809 but not SC-51322. Basal cAMP levels were higher in PTX-treated tissues and DDA- and Rp-2'-O-MB-cAMPs--sensitive responses increased to analogue concentrations < 0.1 μM. In conclusion, prostanoid EP 3 receptors via G i/o negatively modulate prostanoid IP receptor-mediated relaxation to cicaprost, iloprost and treprostinil. However, other pathways contribute to analogue-induced vasorelaxation, the nature of which remains unclear for treprostinil.

Inhibition of Src family tyrosine kinases prevents lipopolysaccharide-induced hyporeactivity in isolated rat tail arteries

Tyrosine kinases may play a role in the vascular response to sepsis. We investigated the effect of selective inhibitors of Src family tyrosine kinases (SFK) on lipopolysaccharide (LPS)-induced vascular hyporeactivity. Rat tail artery segments were mounted in an isometric wire myograph. The effect of incubation with LPS was examined on phenylephrine (PE) and high potassium (KPSS)-induced contraction, with and without the selective SFK inhibitors SU6656 or PP1. Western blotting was performed to assess SFK phosphorylation and iNOS induction. Incubation with LPS for 18 h induced marked vascular hyporeactivity to both PE ( p < 0.001) and KPSS ( P < 0.001). Incubation with SU6656 alone had no effect on contractility to PE and KPSS, and SU6656 partially prevented LPS-induced hyporeactivity to PE ( p < 0.01) and KPSS ( p < 0.001). In contrast, PP1 alone diminished contractility to PE ( p < 0.01) and KPSS ( p < 0.001), and co-incubation of LPS with PP1 completely prevented LPS-induced hyporeactivity. LPS increased tyrosine phosphorylation of SFK and this effect was inhibited by SFK inhibitors. LPS also increased levels of iNOS and this was also inhibited by SU6656 and PP1. LPS-induced hyporeactivity in vitro is mediated by activation of SFK. Selective inhibitors of SFK may have therapeutic potential in the management of septic shock.

Centrally Acting Imidazolines Stimulate Vascular Alpha 1A-Adrenergic Receptors in Rat-Tail Artery

: 1. Centrally acting imidazoline antihypertensive agents clonidine and moxonidine also act peripherally to contract blood vessels. While these agents act at both I(1)-imidazoline and alpha 2 adrenergic receptors centrally, the receptor types by which they mediate contraction require further definition. We therefore characterized the receptor subtype by which these agents mediate contraction of proximal rat-tail artery. 2. Dose-response curves were determined for phenylephrine and for several imidazoline ligands, using endothelium denuded, isolated ring segments, of tail arteries from adult male Sprague-Dawley rats. Ring segments were mounted on a force transducer with platinum wires and immersed in a tissue bath containing Krebs solution, to which drugs could be added. Signals were digitized and recorded by a computer. 3. Tail artery contractions expressed as a percent of contraction to 106 mM potassium were phenylephrine (96%), moxonidine (88%), clonidine (52%), and UK14304 (30%). Neither rilmenidine nor harmane caused contraction. Contraction of tail artery to moxonidine or clonidine could be blocked by alpha 1 antagonist urapidil or prazosin, and also by alpha 1A subtype selective antagonist WB4101. Schild plots were generated and a calculated pA2 value of 9.2 for prazosin in the presence of clonidine confirms clonidine as an agonist at alpha 1A receptors in proximal segments of rat-tail artery. 4. Our work suggests that clonidine and moxonidine are promiscuous compounds at micromolar concentrations and that harmane and rilmenidine are more selective compounds for in vivo imidazoline research.

Evidence that inward rectifier K+ channels mediate relaxation by the PGI2 receptor agonist cicaprost via a cyclic AMP-independent mechanism

We investigated the role of the inward rectifier potassium (KIR) channel and the cyclic AMP-dependent pathway in mediating vasorelaxation induced by the prostacyclin analogue cicaprost. Small vessel myography was used to assess responses to cicaprost in segments of rat tail artery contracted with phenylephrine. Microelectrode recordings were made from helical strips to assess effects on membrane potential. Cicaprost caused relaxation and hyperpolarisation that were significantly inhibited by Ba2+ (30-100 microM), a known blocker of KIR channels. Raising extracellular K+ from 5 to 15 mM elicited membrane hyperpolarisation and an endothelium-independent relaxation that was blocked by Ba2+ (30-100 microM), suggesting the existence of functional KIR channels on the smooth muscle. In contrast, neither glibenclamide (10 microM), a blocker of ATP-sensitive K+ channels, nor fluoxetine hydrochloride (100 microM), a blocker of G-protein-gated inward rectifier K+ channels, nor pertussis toxin (PTX; 1 microg/ml), which irreversibly inhibits Gi/Go, reduced relaxation to cicaprost. Indeed, PTX significantly potentiated responses. Relaxation to cicaprost was not mediated by NO but was partially endothelium-dependent, consistent with a similar inhibition by a combination of charybdotoxin (0.1 microM) and apamin (0.5 microM), blockers of endothelium-derived hyperpolarising factor (EDHF). However, relaxation was unaffected by adenylyl cyclase (SQ22536, dideoxyadenosine) or protein kinase A (Rp-2-O-monobutyryl-cAMP) inhibitors, consistent also with Ba2+ only weakly inhibiting relaxation to the adenylyl cyclase activator forskolin. We conclude that cicaprost relaxes rat tail artery by activating KIR channels with some involvement from EDHF. The mechanism appears to be largely independent of cyclic AMP and Gi/Go, although the latter appears to counteract relaxation through an unknown pathway and/or receptor.

Ca2+-sensitivity and cGMP-independent effects of NO in vascular smooth muscle

The effects of NO on Ca2+-sensitivity of vascular smooth muscle (VSM) myofilaments have been the focus of this study. Simultaneous measurements of [Ca2+]i and force were carried out in rat tail artery segments. NO, 10(-7) M, evoked a transient decrease in [Ca2+]i accompanied by sustained relaxation (45.3+/-6.3 vs. 69.45+/-7.2%, P<0.05, respectively) of VSM precontracted with K+ (70 mM), suggesting a decrease in Ca2+-sensitivity of VSM. This decrease in Ca2+-sensitivity was completely abolished by preincubation of VSM with ODQ (10(-6) M) (63.9+/-7.8% for [Ca2+]i vs. 20.5+/-8.4% for relaxation, P<0.05). Ca2+-presensitization of VSM myofilaments with PE (10(-6) M) decreased the efficacy of NO to relax VSM (44.25+/-6.9% vs. 69.45+/-7.2%, P<0.05), but increased its ability to lower [Ca2+]i (70.5+/-6.8% vs. 45.3+/-6.3%, P<0.05). Application of DTT (10(-3) M) together with ODQ (10(-6) M) to subtract possible cGMP-independent effects revealed the total suppression of both the relaxant responses and [Ca2+]i of VSM under high-K+ preactivation of VSM. The data indicate that NO not only relaxes VSM and lowers [Ca2+]i in K+-preactivated VSM, but also decreases Ca2+-sensitivity of VSM myofilaments and these effects are strongly cGMP-dependent. In PE-induced contractions of VSM, NO relaxed VSM of rat tail artery and lowered [Ca2+]i, but failed to reverse Ca2+-presensitized myofilaments. We suggest that alternative cGMP-independent effects of NO are primarily manifested via activation of K+-channels and inhibition of Ca2+ current rather than to affect relaxation. An importance of reduced SH-groups within VSM myoplasm for both relaxation and [Ca2+]i disposal evoked by NO is evident whatever Ca2+-mobilization pathways are involved.

Diabetes abolishes the gender difference in vasopressin-mediated potentiation of sympathetic vasoconstriction

Electrical field stimulation (4 Hz, 0.2 ms pulse duration, at a supramaximal voltage of 70 V, for 1 s) of isolated rat tail artery segments produced contraction which was lower in female than in male rats, and was reduced by streptozotocin-induced diabetes in both genders. This contraction was potentiated by vasopressin (10 −12–10 −10 M) more in normoglycemic male than in normoglycemic female rats, and this effect of vasopressin was increased by the cyclooxigenase inhibitor meclofenamate (10 −5 M) in female control rats, but not in diabetic female, or control and diabetic male rats, and it was not modified by the nitric oxide synthase inhibitor N ω-nitro- l-arginine methyl ester ( l-NAME, 10 −4 M). Endothelin-1 (10 −10–3×10 −9 M) also potentiated the contraction to electrical stimulation. This potentiation was similar in all experimental groups, and it was not modified by meclofenamate or l-NAME. These results suggest that the potentiating effect of vasopressin, but not that of endothelin-1, on the sympathetic vasoconstriction, is lower in females than in males, probably by an increased release of vasodilating prostanoids, and this release may be reduced by diabetes in females.

Modification of vascular tone using iNOS under the control of a radiation-inducible promoter.

It may be therapeutically advantageous to alter tumour blood supply specifically. Nitric oxide is a potent vasodilator which is produced in many tissues by the enzyme nitric oxide synthase (NOS). We have transfected cDNA for the inducible isoform of this enzyme (iNOS), under the control of the radiation-inducible promoter WAF1. The activity of the promoter was initially assessed using green fluorescent protein (GFP) in both endothelial cells and rat tail artery segments. Induction of protein expression by 9.5- and 4.5-fold respectively, was observed after a radiation dose of 4 Gy. Artery sections were then transfected with the WAF1/iNOS construct; this gave five-fold induction of iNOS protein after a dose of 4 Gy. The transfected artery was also tested functionally for relaxation, indicative of NO production. One hour after exposure to 4 Gy there was a significant (65%) relaxation of artery segments that had been preconstricted with phenylephrine. This could be partially reversed by the NOS inhibitor nitro-L-arginine. This study demonstrates that we can regulate vascular tone using an X-ray inducible promoter.

Changes in reactivity of rat arteries subjected to dynamic stretch.

The effect of dynamic stretch on the reactivity of the rat tail and mesenteric artery segments was studied. Segments mounted on a myograph were stretched by a computer-controlled motorized micromanipulator. Dynamic stretch (1, 5 or 7 Hz) inhibited the artery constriction induced by noradrenaline (10 microM), 5-hydroxytryptamine (0.7 microM), or electrical field stimulation of intramural nerves. In contrast, dynamic stretch enhanced the tetrodotoxin-insensitive dilation induced by electrical field stimulation of noradrenaline-contracted arteries. Maximal increase of dilation evoked by electrical field stimulation (24.5 +/- 5.0% in mesenteric and 50.3 +/- 15.6% in the tail artery) was observed at a dynamic stretch-frequency of 5 Hz. An inhibitor of nitric oxide synthesis, NG-nitro-L-arginine (100 microM), abolished the difference in reactivity between static and dynamic conditions. The results indicate that dynamic stretch of the arteries activates nitric oxide synthesis/secretion, thus reducing constrictor and increasing dilator responses to the stimuli used.

Maintained vasodilatory response to cromakalim after inhibition of nitric oxide synthesis.

Activation of vascular smooth-muscle adenosine triphosphate-sensitive potassium channels (KATP channels) causes membrane hyperpolarization, reduced entry of Ca2+ through L-type voltage-gated Ca2+ channels, and subsequent smooth-muscle relaxation. Conversely, opening of endothelial KATP channels elicits hyperpolarization but may induce Ca2+ influx and stimulation of endothelium-derived nitric oxide (EDNO) because these cells appear not to possess L-type Ca2+ channels. We therefore hypothesized that EDNO contributes to KATP channel-mediated vasodilation. To test this hypothesis, we examined vasodilatory responses to the KATP channel opener cromakalim in conscious rats, perfused rat tail artery segments, and isolated perfused rat lungs in the presence or absence of the EDNO synthesis inhibitor Nomega-nitro-L-arginine (L-NNA). Additionally, we compared the effect of cromakalim with the EDNO-dependent dilator bradykinin on NO production and intracellular Ca2+ in cultured rat pulmonary artery endothelial cells. Vasodilatory profiles to cromakalim were unaffected by L-NNA in conscious rats, tail arteries, and isolated lungs. Consistent with these results, cromakalim had no apparent effect on either NO synthesis or Ca2+ levels in cultured endothelial cells. These data suggest a lack of a role for EDNO in contributing to KATP-channel-mediated vasodilation in the rat.

Norepinephrine stimulates arachidonic acid release from vascular smooth muscle via activation of cPLA2.

The mechanism of agonist-activated arachidonate release was studied in segments of rat tail artery. Tail artery segments were prelabeled with [3H]arachidonate and then stimulated with norepinephrine (NE), and the radioactivity of the extracellular medium was determined. NE stimulated arachidonate release from the tissue without increasing arachidonic acid levels within cellular cytosol or crude membranes. About 90% of the extracellular radioactivity was shown to be unmetabolized arachidonate by TLC. Arachidonic acid release was not inhibited by the removal of the endothelium from the artery. NE exerted a half-maximal effect at a concentration of 0.2 microM. NE-stimulated arachidonate release was not inhibited by blockers of phospholipase C (U-73122), diacylglycerol lipase (RHC-80267), secretory phospholipase A2 (manoalide), calcium-insensitive phospholipase A2 (HELSS), or beta-adrenergic receptors (propranolol). NE-stimulated arachidonic acid release was inhibited by blockers of cytosolic phospholipase A2 (cPLA2) (AACOCF3), alpha 1-adrenergic receptors (prazosin), and specific G proteins (pertussis toxin). This indicated that NE stimulated arachidonate release from vascular smooth muscle via activation of alpha-adrenergic receptors, either Gi or Go, and cPLA2. NE-activated arachidonic acid release from vascular smooth muscle may play a role in force generation by the tissue. Perhaps arachidonic acid extends the effect of NE on one specific smooth muscle cell to its nearby neighbor cells.

Neuropeptide Y Y1 receptor blockade does not alter adrenergic nerve responses of the rat tail artery.

Using the selective neuropeptide Y Y1 receptor antagonist, BIBP3226 [(N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-D-argininamide], the role of endogenous neuropeptide Y in mediating vasoconstrictor responses to adrenergic nerve stimulation was investigated by recording isometric force from isolated rat tail artery segments. BIBP3226 had no effect on contractile responses to adrenergic nerve stimulation (10 pulses; 0.5-2 Hz), but it completely blocked the enhancement of contraction produced by exogenous neuropeptide Y. When frequency and train length of the transmural nerve stimulation were increased (100 pulses; 1-16 Hz), contractile responses were still unaffected by BIBP3226. A peptidase inhibitor mixture known to increase responses to exogenous neuropeptide Y was added; however, BIBP3226 still did not influence contractile responses to adrenergic nerve stimulation. Thus, contractile responses to adrenergic nerve stimulation in the rat tail artery do not appear to involve the release and postjunctional action of endogenous neuropeptide Y; however, exogenous neuropeptide Y does potentiate these responses by acting on Y1 receptors.

Measurement of changes in sarcoplasmic reticulum [Ca 2+] in rat tail artery with targeted apoaequorin delivered by an adenoviral vector

The physiologic relevance of Ca 2+ release from the sarcoplasmic reticulum in arterial smooth muscle contraction is controversial. Therefore, we sought to measure changes in sarcoplasmic reticulum free [Ca 2+] (i.e. [Ca 2+] sr) in the intact rat tail artery. We exploited a novel technique to measure [Ca 2+] sr with genetically targeted apoaequorin acting as a pseudo-luciferase rather than as classic aequorin. Intact rat tail arteries were infected with a replication deficient adenoviral vector (RAdER) containing the apoaequorin gene targeted to the sarcoplasmic reticulum. Addition of apoaequorin's substrate, coelenterazine, to the perfusate increased light production in a [Ca 2+] dependent manner, consistent with apoaequorin action on coelenterazine. Within the limits of the photon counting system, imaging of infected rat tail artery segments revealed light production from the whole thickness of the vascular wall. Phenylephrine stimulation decreased apoaequorin generated light and induced a contraction. Washout of phenylephrine relaxed the tissues and increased light indicating refilling of the sarcoplasmic reticulum with Ca 2+. Incubation in 10 μM cyclopiazonic acid, a SERCA inhibitor, did not alter apoaequorin generated light or induce a contraction. In the presence of cyclopiazonic acid, phenylephrine contractions were enhanced and apoaequorin generated light decreased further than that observed in the absence of cyclopiazonic acid. Cyclopiazonic acid also prevented the increase in apoaequorin generated light upon washout of phenylephrine, consistent with its inhibition of sarcoplasmic reticulum refilling. These results suggest that light production from targeted apoaequorin, delivered by a replication deficient adenovirus, is a valid measure of changes in [Ca 2+] sr in the intact arterial wall. There appeared to be a correlation between Ca 2+ release and contraction in these lightly loaded arteries.

Vasodilator responses of rat isolated tail artery enhanced by oxygen‐dependent, photochemical release of nitric oxide from iron‐sulphur‐nitrosyls

1. The vasodilator properties and photochemical decomposition of two synthetic iron-sulphur-nitrosyl clusters (cluster A: [Fe4S4(NO)4], tetranitrosyl-tetra-mu 3-sulphido-tetrahedro-tetrairon; and B:[Fe4S3 (NO)7]-1, heptanitrosyl-tri-mu 3-thioxotetraferrate(-1)) have been investigated. Experiments were carried out on isolated, internally-perfused segments of rat tail artery. 2. Bolus injections (10 microliters) of A or B ( > 0.25 mM) delivered into the internal perfusate generated sustained (or S-type) vasodilator responses, characterized by a persistent plateau of reduced tone due to NO released from clusters which enter and become trapped within endothelial cells. Clusters were therefore irradiated with visible laser light (lambda = 457.9 or 514.5 nm) either (a) in solution, while passing through a glass tube en route to the artery; or (b) when retained within the endothelium, by illuminating the artery directly during the plateau of an S-type response. Irradiation produced an additional vasodilator response, the magnitude of which depended upon wavelength and laser beam energy. 3. The nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (100 microM), had no effect on light-induced vasodilator responses. However, they were (a) blocked entirely by adding oxyhaemoglobin (5 microM) to the internal perfusate; and (b) greatly enhanced by the enzyme superoxide dismutase (150 u ml-1). 4. Photolysis of cluster B was measured by absorption spectroscopy and by detecting NO released with an electrochemical sensor. The photochemical reaction was found to be oxygen-dependent. The half-time for inactivation of cluster-derived NO was measured by interposing different lengths of tubing (i.e. time delays) between the photolysis tube and NO sensor. The steady-state probe current decayed exponentially with increasing delay time, with a t 1/2 of 21 s. The amplitudes of vasodilator responses of the tail artery also decreased exponentially by increasing the time delay (t 1/2 = 58 s). Superoxide dismutase (150 u ml-1) prevented this from happening, showing that "inactivation' of cluster-derived NO was caused by reaction with superoxide anions formed during photolysis. 5. We conclude that potentiation of vasodilator responses to iron-sulphur-nitrosyl clusters by visible light is due to an oxygen-dependent photochemical reaction which accelerates the release of ligated nitrosyl groups as free NO. Based on our measurements, we estimate that ca 100 pM NO is sufficient to produce a just-detectable additional vasodilatation and that the ED50 dose is ca 3.7 nM.

Effect of NG-nitro-L-arginine methylester (L-NAME) on functional and biochemical alpha 1-adrenoceptor-mediated responses in rat blood vessels.

1. The modulation by NG-nitro-L-arginine methylester (L-NAME) of alpha 1-adrenoceptor-mediated contraction was investigated on isolated segments of rat tail artery and aorta. The influence of L-NAME on inositol phosphates accumulation by alpha 1-adrenoceptor agonists was also investigated to elucidate the intracellular mechanism responsible for this modulation. 2. In aorta but not in tail artery L-NAME (30 microM) enhanced the sensitivity (3.3 times) and the maximum contraction (Emax) induced by the full agonist, phenylephrine. 3. St-587, a partial alpha 1-adrenoceptor agonist, behaved as a weak agonist in the aorta (22.2% of phenylephrine Emax). However, when the same agonist was studied in tail artery rings a maximum contraction that was 78.4% of the phenylephrine induced Emax was reached. 4. L-NAME increased (3.3 times) the Emax for St-587 contraction in the aorta but not in the tail artery. Sensitivity to St-587 was slightly but significantly (P < 0.001) enhanced (1.9 times) by L-NAME in tail artery segments. 5. Contractile responses to phenylephrine after partial alkylation with phenoxybenzamine were analyzed by the nested hyperbolic null method. To elicit 50% of Emax for contraction only 1.1% of the receptors in the tail artery and 21% of the receptors in the aorta need to be occupied. These results indicate a higher receptor reserve for the tail artery than the aorta. 6. In the tail artery but not in the aorta, St-587 activates phosphoinositide turnover. The presence of L-NAME was without effect on inositol phosphates accumulation induced by this partial alpha 1-adrenoceptor agonist. 7. The maximum contraction induced by phenylephrine, after partial alpha-adrenoceptor alkylation, was enhanced by L-NAME in tail artery rings. However, the NO synthase inhibitor was unable to modify the phenylephrine-induced accumulation of inositol phosphates in the presence of phenoxybenzamine. 8. These results indicate that the differences in St-587-induced contraction and the modulation by L-NAME of alpha 1-adrenoceptor-mediated contraction observed between the tail artery and aorta are associated with differences in receptor reserve. In addition, our biochemical studies indicate that the potentiating effect of L-NAME is independent of intracellular calcium release via phosphatidylinositol turnover.

Cocaine promotes an apparent direct vasoconstrictor effect of neuropeptide Y in the rat tail artery

Neuropeptide Y is a powerful vasoconstrictor in vivo; however, in vitro it shows weak constrictor effects. This discrepancy may have led to conflicting reports concerning the contractile effects of neuropeptide Y on isolated blood vessels. Using isolated rat tail and femoral artery segments neuropeptide Y (0.1–100 nM) did not induce any contractile response. However, if the catecholamine neuronal uptake blocker cocaine was added to the tissue bath, neuropeptide Y induced a contraction which could be fully blocked by prazosin (1000 nM). Furthermore, an age-dependent increase in the contraction to neuropeptide Y plus cocaine was observed. In conclusion, in the rat tail artery an apparent direct vasoconstrictor effect of neuropeptide Y occurs only in the presence of cocaine. Since this contraction can be fully blocked by prazosin, spontaneously released norepinephrine is an important component of the contraction. The discrepancy between in vivo and in vitro effects of neuropeptide Y may be explained in part by the presence of circulating vasoconstrictors.

Neuropeptide y potentiates norepinephrine-stimulated inositol phosphate production in the rat tail artery

We have recently demonstrated the ability of neuropeptide Y to augment norepinephrine-sti,ulated contractions of rat tail artery. In [ 3H]myo- inositol labeled segments of rat tail artery, addition of a concentration of neuropeptide Y (10 −8 M) known to potentiate contractile responses produced no effect on the basal accumulation of [ 3H]inositol phosphates. Addition of norepinephrine (10 −7 M), however, resulted in the rapid accumulation of [ 3H]inositol 1,4,5-trisphosphate to 325 ± 29 % of basal levels within 5 min. This effect of norepinephrine on [ 3H]inositol 1,4,5-trisphosphate accumulation was mirrored by significant increases (274 ± 25 % of control) in total [ 3H]inositol phosphates. When neuropeptide Y and norepinephrine were added together, the norepinephrine-stimulated increase in [ 3H]inositol 1,4,5-trisphosphate was significantly augmented (467 ± 35 % of control) as was the increase measured in total [ 3H]inositol phosphates (366 ± 21 % of control). We suggest that the ability of neuropeptide Y to augment norepinephrine-induced contraction of the rat tail artery is due, at least in part, to the ability of neuropeptide Y to increase norepinephrine-stimulated phospholipase C activity in vascular smooth muscle and may represent the mechanism of action of this co-neurotransmitter in vivo.

DIFFERENTIAL ACTIVATION OF ADRENOCEPTOR SUBTYPES BY NORADRENALINE APPLIED FROM THE INTIMAL OR ADVENTITIAL SURFACES OF RAT ISOLATED TAIL ARTERY

1. The vasoconstrictor effects of noradrenaline applied to the intimal and adventitial surfaces of perfused segments of rat tail artery in the presence and absence of endothelium were studied. 2. Noradrenaline was about six times more potent as a vasoconstrictor when applied to the intimal than to the adventitial surface. Cocaine (25 mumol/L) enhanced responses to adventitial noradrenaline to a greater extent than those to intimal noradrenaline. A high concentration of propranolol (1 mumol/L) had a similar effect. 3. The vasoconstriction elicited by adventitial noradrenaline declined from a peak whereas that to intimal noradrenaline remained steady. A low concentration of propranolol (0.1 mumol/L) abolished the decline in the response to adventitial noradrenaline. 4. The alpha 1- and alpha 2-adrenoceptor antagonists prazosin (1 nmol/L) and idazoxan (100 nmol/L) significantly reduced responses to intimal and adventitial noradrenaline in the presence or absence of endothelium. 5. Removal of endothelium enhanced responses to intimal but not adventitial noradrenaline. Idazoxan produced a significantly greater reduction of responses to noradrenaline in the absence than in the presence of endothelium, and was more effective against intimal than adventitial noradrenaline. Similar effects were produced by the nitric oxide synthase inhibitor L-NAME (30 mumol/L). 6. It was concluded that noradrenaline acts on both alpha 1- and alpha 2-adrenoceptors to produce vasoconstriction: the alpha 1-adrenoceptors appear to be uniformly distributed, whereas alpha 2-adrenoceptors are located nearer the intima. Intimal noradrenaline also acts on endothelial alpha 2-adrenoceptors to release EDRF which counteracts the vasoconstrictor action of noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)

Norepinephrine stimulates the direct breakdown of phosphatidyl inositol in rat tail artery.

When segments of rat tail artery were labeled with [3H]inositol and then stimulated with norepinephrine (NE), the inositol phosphates produced were primarily IP and IP2, together with a small but significant amount of Ins(1,4,5)P3 and a very small amount of Ins(1,3,4,5)P4. It has been unclear in many studies whether or not the relatively large levels of IP and IP2 produced in [3H]inositol-labeled tissue represent indirect products of phosphatidyl inositol(4,5)bis phosphate breakdown (through Ins(1,4,5)P3) or direct products of phosphatidyl inositol 4 monophosphate and phosphatidyl inositol breakdown. In order to answer this question tail artery segments were prelabeled with [3H]inositol and then permeabilized with beta escin and stimulated with norepinephrine and GTP gamma S, so that increases in IP, IP2, and Ins(1,4,5)P3 were still observed. If these permeable segments were stimulated with agonist in the presence of compounds known to inhibit Ins(1,4,5)P3 5-phosphatase, such as glucose 6P, (2,3)diphosphoglycerate, or Ins(1,4,5)P3, the levels of labeled Ins(1,4,5)P3 and labeled IP2 were increased, while the level of stimulated labeled IP was unchanged. This indicated that some of the IP2 and IP formed in these cells was produced from PIP2 but that some of these compounds might be formed from PIP or PI. When the isomers of inositol monophosphate, Ins 1P and Ins 4P, were separated by HPLC, it was shown that after prelabeled tail artery was stimulated by norepinephrine for periods of 1-2 min, the predominant isomer formed was Ins 4P, indicating either PIP2 or PIP as the source. However, after 5-20 min stimulation, both Ins 1P and Ins 4P were formed in equal amounts, suggesting that during sustained stimulation of smooth muscle PI itself was broken down directly. Therefore it appears that within 1-2 min of norepinephrine addition to vascular smooth muscle the bulk of the IP and IP2 produced are derived from PIP2 via IP3, while after 20 min of norepinephrine treatment much of the IP comes directly from PI. This suggests that the regulation of PLC in this tissue is more complicated than has been previously believed.