Rat Caudal Artery Research Articles

Dilator action of Fe2+-citrate complex on the rat caudal artery perfused in vitro

Dilator action of Fe2+-citrate complex on the rat caudal artery perfused in vitro

Effect of Ridogrel on Vascular Contractions Gaused by Vasoactive Substances Released during Platelet Activation

Ridogrel (6.3 x 10(-6) to 10(-4) M) inhibited contractions of isolated rat caudal arteries and rabbit femoral arteries caused by U-46619. The slope of an Arunlakshana-Schild plot (pA2-value: 3.4 x 10(-6) M) on the caudal artery was slightly higher than one (1.14). This effect was maximal within 20 min of incubation of the blood vessel with the compound and easily reversible. Ridogrel antagonised contractions of isolated rabbit femoral arteries caused by prostaglandin F2 alpha in the same concentration range. Ridogrel also inhibited contractions induced by aggregating rat platelets on isolated rat caudal arteries (in the presence of ketanserin 4 x 10(-7) M) and on isolated rabbit pulmonary and femoral arteries (in the absence of ketanserin). Ridogrel had no effect on Ca2(+)-induced contractions in depolarised isolated rabbit femoral arteries, and at 10(-4) M antagonised serotonin-induced contractions in this blood vessel. Its effect on serotonin-induced contractions was statistically significant but very small on isolated rat caudal arteries. These observations indicate that ridogrel is an antagonist of prostaglandin endoperoxide/thromboxane A2 and prostaglandin F2 alpha receptors on vascular smooth muscle.

P-054 - Effects of purinergic agents on endogenous norepinephrine release from adrenergic nerves of rat caudal artery

P-054 - Effects of purinergic agents on endogenous norepinephrine release from adrenergic nerves of rat caudal artery

Relaxation of rat vascular muscle by peripheral benzodiazepine modulators.

Effects of peripheral benzodiazepine receptor modulating drugs, Ro 5-4864 and PK 11195, on tension induced by K+ and the calcium agonist SDZ 202 791 (S isomer), were studied in rat caudal arteries. A significant reduction of tonic phase tension occurred with 30 nM PK 11195 or 3 microM Ro 5-4864, but decreases of the initial (first 3 min), phasic contraction were detected only at the highest concentrations of Ro 5-4864 and PK 11195. Protoporphyrin IX, the putative endogenous ligand of the peripheral benzodiazepine receptor, (at 10-100 nM) markedly increased the effectiveness of Ro 5-4864 and PK 11195 in reducing phasic contraction. Intracellular calcium localization and distribution in fura-2 loaded single vascular cells were quantitated using a high sensitivity, two-stage microchannel plate, photon-counting (PMI-VIM) camera. Peripheral benzodiazepines reduced intracellular calcium release from centrally located calcium pools, and this decrease of calcium release was potentiated by protoporphyrin IX. The decrease in intracellular calcium activity, which was more pronounced in the central regions where sarcoplasmic reticular elements are numerous, was probably the major mechanism of these vasodilator properties. Measurements of soluble guanylate cyclase activity also supported the intracellular Ca2+ release mechanism. Under conditions where protoporphyrin IX did not significantly stimulate guanylate cyclase, Ro 5-4864 alone or more effectively in combination with protoporphyrin IX stimulated cGMP production and caused relaxation. Guanylate cyclase forms a possible target for these benzodiazepine modulators, a hypothesis that merits further investigation.

Calmodulin loss in vascular smooth muscle following Triton X-100 or saponin skinning

The calmodulin (CaM) content of intact and chemically skinned strips of rat caudal artery was measured using a 125I-CaM radioimmunoassay. The total CaM measured following homogenization of arterial tissue with EGTA and EGTA/Triton X-100 was 2.58 mumol/kg wet tissue. Based on a smooth muscle volume of 40%, this value corresponds to a cellular CaM concentration of 6.5 microM. Approximately 97% of total CaM was soluble and approximately 3% was EGTA-nonextractable. Permeabilization of the plasmalemma with 0.15 mg/ml saponin or 0.5% Triton X-100 caused significant detergent-dependent loss of CaM. At the end of a 1 h skinning period, tissues exposed to saponin lost 30% of total CaM. By comparison, tissues skinned under the same conditions with Triton X-100 lost 50%. During a subsequent 4 h exposure to relaxing solution, total tissue CaM continued to decline. The exponential loss over the 5 h period was described by a first order model having diffusible and nondiffusible CaM components. The diffusible CaM component of saponin skinned tissue (59%) was significantly less than the diffusible component of those skinned with Triton X-100 (88%); however, the rate coefficients for CaM diffusion (0.78 h-1 and 0.91 h-1, respectively) did not statistically differ. The nondiffusible component of CaM was significantly larger in saponin treated strips (42%) than in Triton X-100 permeabilized tissue (12%). Arterial strips skinned with Triton X-100, which were subsequently exposed to relaxing solution for up to 22 h, lost significantly more CaM than those retained in Triton X-100 skinning solution for a comparable duration.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetate causes endothelium-independent increases in cyclic AMP in rat caudal artery

The mechanism of the vasorelaxant action of acetate is unknown. Because cyclic nucleotides have been linked to vasorelaxation in vascular smooth muscle, we studied the effects of acetate on tissue adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) levels in rat caudal artery. Acetate (4 mM) induced an increase in tissue cAMP levels (control: 5.1 +/- 0.67, acetate: 7.1 +/- 0.97 pmol/mg protein, P less than 0.05), with the increase noted as early as 15-30 s after acetate exposure, and peaking at 60 s. The time course of the cAMP response was compatible with the vasorelaxant effect of acetate against an arginine vasopressin (AVP, 2 X 10(-9) M) contraction. Both the increase in cAMP and the vasorelaxant effect were completely blocked by 10(-3) M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Acetate increased cAMP levels in 3-isobutyl-1-methylxanthine (IBMX)-treated tissue with an effective concentration producing 50% of the maximum response (EC50) of 1.5 mM, similar to the relaxant EC50 (without IBMX) of 2.2 mM against an AVP contraction. In other experiments, the effect of acetate on cAMP was shown to be independent of endothelium. In contrast, acetate had no effect on tissue cGMP levels, whether the endothelium was present or absent. The results suggest that acetate causes an increase in tissue cAMP levels that is not dependent on presence of a functioning endothelium. The changes in cAMP may be contributory to the vasorelaxant effect of acetate in the caudal artery.

Alpha-1 and alpha-2 adrenoceptor agonists induce vasoconstriction of the normotensive rat caudal artery in vitro by stimulation of a heterogeneous population of alpha-1 adrenoceptors.

Although alpha-2 adrenoceptor agonists rapidly induce arterial vasoconstriction in vivo, such responses have proven difficult to obtain in vitro. We have investigated the vasoconstrictor effects of various alpha-1 and alpha-2 adrenoceptor agonists in the perfused superfused caudal artery of the normotensive rat. Intrinsic activities were; methoxamine; 1, phenylephrine; 0.94, noradrenaline; 0.93, guanfacine; 0.88, clonidine; 0.47, UK 14,304 [5-bromo-6-(2-imidazoline-2-ylamino)-quinoxaline tartrate]: 0.10, azepexole; 0. Antagonism by the selective alpha-1 agent, prazosin of the vasoconstrictor responses provoked by methoxamine, guanfacine or clonidine, showed a high affinity with--log KB values in the range of 8.5 to 9.4. There were no significant differences between the KB values obtained with the three agonists. Antagonism by the selective alpha-2 antagonist, yohimbine showed a low affinity with KB values between 6.7 to 7.6 for the three agonists. The calcium entry blocker, nicardipine, antagonized responses to clonidine at nanomolar concentrations and those to phenylephrine at micromolar concentrations. We conclude that vasoconstrictor responses in this isolated tail artery preparation are primarily mediated via an alpha adrenoceptor which can be classified, on the basis of the results with specific antagonists, as being of the alpha-1 type. The results obtained with nicardipine suggest that the population of alpha adrenoceptors is not, however, homogeneous.

Characterization of prejunctional purinoceptors on adrenergic nerves of the rat caudal artery.

The effects of a number of purinoceptor agents on the release of endogenous noradrenaline from the electrically stimulated rat caudal artery were determined. Noradrenaline was quantified by high performance liquid chromatography-electrochemical detection techniques. Both P1-receptor and P2-receptor agonists reduced the release of noradrenaline; the relative order of potency being 2-chloroadenosine greater than beta, gamma methylene ATP greater than ATP greater than or equal to adenosine. The adenosine uptake inhibitor S-p-nitro-benzyl-6-thioguanosine potentiated the effects of adenosine but not those of the adenine nucleotides. This suggests that the nucleotides do not need to be converted to adenosine to produce a prejunctional inhibition of the release of noradrenaline. The P1-receptor antagonist 8-(p-sulfophenyl) theophylline reduced the inhibitory effects of both P1- and P2-receptor agonists as did the photolysis of tissues with an intense light source. The findings indicate that prejunctional purinoceptors that mediate an inhibition of the release of noradrenaline from the adrenergic nerves of the caudal artery may not be adequately defined as either P1- or P2-receptors and thus appear to represent a unique receptor. We suggest referring to these receptors as P3-purinoceptors.

Influence of pH on isometric force development and relaxation in skinned vascular smooth muscle.

The effects of pH (from pH values 6.50-7.10) on isometric tension development and relaxation were investigated in Triton X-100 "skinned" rat caudal artery. Helically cut skinned strips contracted in 21 microM Ca2+ were studied with respect to maximal isometric tension (Po) and rate of contraction (T0.5 C), and following relaxation in 18 nm Ca2+, the rate of relaxation (T0.5 R). Acidic pH (pH 6.50) decreased Po to 87% of isometric force obtained at pH 6.90, and increased the rate of contraction as shown by a decrease of T0.5 C to 80%. In contrast, T0.5 R increased 4.5-fold, indicating that with a change of only 0.40 pH units, relaxation rates were dramatically decreased. pCa-tension curves at pH values 6.50, 6.70, 6.90 and 7.10 indicated no significant shift in half maximal activation (pCa50) between pH 6.50 and 6.70, but a significant (P less than 0.01) shift in pCa50 between pH 6.70 [( Ca2+] = 0.46 microM) and pH 7.10 [( Ca2+] = 0.87 microM). Compared to contractions at pH 6.90, myosin light chain (LC20) phosphorylation at pH 6.50 was significantly greater at 30 and 60 s into contraction but not significantly different at 3-10 min. At both pH 6.50 and 6.90, dephosphorylation was rapid and substantially preceded relaxation; LC20 dephosphorylation and relaxation occurred more rapidly at pH 6.90 than at 6.50. At pH 6.50 and 6.90, relax solutions made with increased Ca2+ buffering capacity showed no effect in enhancing T0.5 R, suggesting the difference between relaxation rates was not due to Ca2+ diffusion limitations from the skinned strip.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological actions of the calcium antagonist propyl-methylenedioxyindene in skinned vascular smooth muscle.

Previous studies provided strong evidence that propyl-methylenedioxyindene (pr-MDI) interfered with calcium at an intracellular site. To further characterize the mechanism of action of pr-MDI, its pharmacological actions on chemically skinned vascular smooth muscle were examined. Rat caudal artery strips were chemically skinned with saponin (0.15 mg/mL for 1 h). The efficiency of the skinning was evidenced by a loss of contractile response to 74 mM K+. The intactness of the regulatory and contractile proteins was ascertained by the ability of the skinned tissue to contract in response to Ca2+ (free Ca2+ concentration of 10(-4) or 10(-6)M). Caffeine (25 mM) induced contraction was used as an index of the functional integrity of the sarcoplasmic reticulum in the skinned preparations. Contraction of the skinned artery with a free Ca2+ concentration of 10(-6)M was significantly obtunded by 1 X 10(-4)M trifluoperazine (a calmodulin antagonist) but not by 1 X 10(-4)M pr-MDI. Contraction of the skinned artery evoked by 25 mM caffeine in the absence of extracellular calcium was significantly obtunded by 1 X 10(-4)M pr-MDI but not by 1 X 10(-6)M nifedipine (a calcium channel blocker). The results indicate that pr-MDI acts intracellular to block calcium mobilization from the sarcoplasmic reticulum without directly interfering with the regulatory and contractile proteins.

Mechanism of Inhibition of Rat Caudal Artery Contraction by Amiloride

Amiloride (N-amidino-3,5-diamino-6-chloropyarzine carboxamide), a potassium sparing diuretic, decreases blood pressure and inhibits vascular smooth muscle contractility in rats. The purpose of these experiments was to investigate the mechanisms by which amiloride inhibits contraction of rat caudal artery. Caudal artery rings were mounted in a muscle chamber that was continuously flushed with circulated bathing medium and the contractions were recorded through a force transducer. The results demonstrate that amiloride competitively inhibited the norepinephrine-stimulated contraction of arterial rings (pA2 = 5.2). This suggests that amiloride and norepinephrine (NE) interact at the same site. The concentration required to produce half-maximum inhibition (IC50) of contraction produced by 10(-6) M norepinephrine was 48 +/- 6 microM. Amiloride (100 microM) shifted the concentration-response curves of norepinephrine and methoxamine to the right without significantly affecting the maximum contraction produced by these agonists. The EC50 value and the threshold concentration of both norepinephrine and methoxamine were increased by about 10-fold in the presence of 100 microM amiloride. Amiloride-induced inhibition of norepinephrine-stimulated contraction was reversible following removal of the drug. Amiloride-induced inhibition of vascular smooth muscle contraction was not blocked by ouabain or monensin. Amiloride at concentrations up to 100 microM did not inhibit K+ stimulated contractions. From these results we conclude that amiloride reversibly inhibits vascular smooth muscle contraction by competing with alpha-adrenoceptor agonists for binding to alpha 1-adrenoceptors.

In vitro studies with ICI 169,369, a chemically novel 5-HT antagonist

ICI 169,369 is a chemically novel 5-HT antagonist that has higher affinity for the 5-HT 2 binding sites in rat cortex than it has for 5-HT 1 sites (K i 1.79 × 10 −8 and 1.58 × 10 −6 M, respectively). In isolated tissue preparations ICI 169,369 was shown to be a competitive antagonist of 5-HT on the rabbit aorta, pig coronary artery and rat caudal artery. In the latter preparation it had a similar pA 2 value to ketanserin (pA 2 8.18 ± 0.5 and 8.42 ± 0.06, respectively). Unlike ketanserin, which was inactive, ICI 169,369 was a non-surmountable antagonist at the rat stomach fundus 5-HT ‘D’ receptor, recently reclassified as 5-HT IC. It was inactive (> 10 −6 M) at the 5-HT 3 receptors found in the isolated perfused rabbit heart and the myenteric plexus of the guinea-pig ileum. At receptors other than those for 5-HT (α 1, α 2, β 1, β 2, H 1, H 2 and muscarinic), ICI 169,369 was inactive at concentrations of either 10 −6 or 10 −5 M. Thus the profile of ICI 169,369 should make it useful in the analysis of the role of 5-HT in physiological and pathological states.

Contractile response of spontaneously hypertensive rat caudal artery to phorbol esters.

Stimulation of phosphatidylinositol metabolism by neurotransmitters produces diacylglycerol, an activator of protein kinase C, which may be involved in hormone-mediated contractions. We studied the effect of a tumor-promoting phorbol ester, 12-deoxyphorbol 13-isobutyrate 20-acetate (DPBA), on contraction of caudal artery rings of Wistar-Kyoto control (WKY) and spontaneously hypertensive rats (SHR) in order to examine whether protein kinase C-mediated mechanisms are increased in SHR. Although DPBA alone did not produce contractions of either WKY or SHR caudal artery rings, it greatly potentiated the contractions evoked by norepinephrine, norepinephrine, vasopressin, potassium, and calcium ionophore A23187. The potentiation of contractile response to these agents by DPBA was dependent on extracellular calcium. The DPBA potentiation of contractions evoked by norepinephrine, vasopressin, and potassium was significantly greater (p less than 0.05) in SHR than in WKY, while no differences were observed between strains for the contractions evoked by calcium ionophore A23187. These results indicate that the protein kinase C-mediated responses are increased in SHR caudal artery rings, and this effect appears to be due to increased calcium influx through cell membrane calcium channels.

Contractile responses to adrenergic nerve stimulation are enhanced with removal of endothelium in rat caudal artery

Removal of the endothelium from isolated perfused rat caudal arteries produced a two fold increase in the contractile response to transmural nerve stimulation. Pretreatment with 6-hydroxydopamine eliminated the contractile response to adrenergic nerve stimulation but failed to uncover any vasodilatory effect of electrical stimulation, either directly on smooth muscle or via non-adrenergic nerves. Endothelial removal also produced two and four fold enhancement of the contractile responses to the selective α 1-and α 2-adrenoceptor agonists methoxamine and B-HT 920. However, pK B values for prazosin and yohimbine versus both agonists indicate that both methoxamine and B-HT 920 are acting primarily at α 1-adrenoceptors in this tissue. These results provide evidence that endothelial factors released either at basal levels or by the stimulation of agonists play a significant physiological role in modifying the contractile responses of blood vessels.

Increased 45Ca Influx in Response to α1-Adrenoceptor Stimulation in Spontaneously Hypertensive Rat Caudal Artery

The isometric tension development and 45Ca influx in response to norepinephrine (NE) and methoxamine stimulation were investigated in caudal arteries of spontaneously hypertensive rats (SHR) and Wistar Kyoto normotensive rats (WKY). The maximum isometric tension developed as well as 45Ca influx in response to NE and methoxamine stimulation were significantly increased (p less than 0.05) in SHR caudal arteries as compared with WKY. On the other hand, neither the isometric tension developed nor the 45Ca influx in response to K+ depolarization were different between WKY and SHR caudal arteries. Estimation of [3H]prazosin binding to the membranes isolated from caudal artery of WKY and SHR showed a single class of high-affinity binding sites with Kd values for SHR 128 +/- 14 pM and for WKY 141 +/- 19 pM, and Bmax values for SHR 108 +/- 14 fmol/mg protein and for WKY 113 +/- 21 fmol/mg protein. From these results, we conclude: (a) Increased contractile response of SHR caudal artery rings to alpha 1-adrenoceptor stimulation appears at least in part to be due to an increased Ca2+ influx through receptor-operated Ca2+ channels; (b) the affinity or density of alpha 1-adrenoceptors estimated by [3H]prazosin binding is not altered in the SHR caudal artery.

Effect of locally generated angiotensin II on noradrenergic neuroeffector function in the rat isolated caudal artery.

The effects of angiotensin II and its precursors angiotensin I and tetradecapeptide renin substrate were investigated in isolated segments of the rat caudal artery. Each peptide constricted the rat caudal artery and also enhanced vasoconstrictor responses to sympathetic nerve stimulation (0.5 Hz, 10 s). The threshold concentrations for each peptide in enhancing sympathetic vasoconstrictor responses were lower than those required to produce vasoconstriction. Tetradecapeptide renin substrate was the least potent of the three peptides and had the slowest onset of action. Angiotensin II and angiotensin I each enhanced noradrenergic transmission to the same degree, whether perfused through the lumen or added to the adventitial surface of the artery. In contrast, tetradecapeptide renin substrate was more potent when applied to the adventitial surface. The effects of angiotensin I were blocked by the converting enzyme inhibitor enalaprilat, whereas the effects of tetradecapeptide renin substrate were unaltered by enalaprilat, or by the renin inhibitors pepstatin or a decapeptide renin inhibitor. These findings suggest that tetradecapeptide renin substrate and angiotensin I may be converted to angiotensin II within the rat caudal artery, with subsequent enhancement of noradrenergic neuroeffector function. However, the enzyme responsible for the conversion of tetradecapeptide renin substrate cannot be determined from the present findings.

Direct augmentation by cyclosporin A of the vascular contractile response to nerve stimulation.

Cyclosporin A administration is associated with an increased incidence of hypertension. To evaluate the direct effects of the drug on the contractile responses of vascular tissue to adrenergic stimuli, rat caudal artery ring segments were studied before and after the addition of cyclosporin A or its ethanol vehicle in vitro. In a dose-related manner, cyclosporin A augmented the contractile response to transmural nerve stimulation, with a highly significant (p less than 0.001 relative to that produced by the vehicle) lowering of the stimulation rate, a 50% of maximum contractile response (ED50) that elicited. The difference between pretreatment and treatment maximal responses to transmural nerve stimulation was also significantly greater (p less than 0.01) in the cyclosporin A-treated preparations than in those receiving the vehicle. In similar experiments, the responses to exogenous norepinephrine were not significantly affected. The effect of cyclosporin A on transmural nerve stimulation was demonstrated at several extracellular calcium concentrations. The results suggest that cyclosporin A enhances nerve stimulation responses by a presynaptic mechanism.

Release of endogenous ATP from rat caudal artery.

Electrical field stimulation of the rat caudal artery (0.5-ms pulses at 8 Hz for 3 min) results in the release of norepinephrine (quantified by HPLC-electrochemical detection) and adenyl purines including ATP, ADP and AMP (quantified by HPLC-fluorescence detection). The amount of ATP released from the tissue exceeded the amount of norepinephrine. Because postjunctional alpha 1-adrenoceptor stimulation with methoxamine also causes release of ATP, both neuronal and extraneuronal sites may contribute to the overflow of ATP. Results with the alpha 1-adrenoceptor antagonist prazosin lend support to this notion. Prazosin (10(-6) M) completely blocked the release of ATP by methoxamine but only partially reduced the release of ATP by field stimulation.

Felodipine Actions on Vascular Muscle Ca2+ Channels

Felodipine is a potent, but relatively slow, relaxation agent in rat caudal artery, decreasing 30 mM K+ contractions by 39% at a concentration of 1 nM and 88% at 10 nM. Membrane potential at rest was unchanged, but 10 nM felodipine caused a 5 mV hyperpolarization of vascular muscle cells previously depolarized by 30 mM K+. Felodipine selectively decreased the sustained, but not the transient, type of calcium current in a whole-cell voltage clamp of azygos venous muscle cells. Relaxation by felodipine was additive to that caused by 10 microM trifluoperazine, a calmodulin antagonist, arguing against an action of felodipine via calmodulin. The vascular muscle relaxant effects of felodipine appear to be explainable by blockade of the sustained type of calcium channels.

Spontaneous arterial lesions: Their possible role in arteriosclerosis

Spontaneous lesions of the arterial wall involving the internal elastic lamellae and variable amounts of the intima are described in the spontaneously hypertensive rat caudal, renal, and mesenteric arteries. A simple model for producing similar circumferential lesions in rat and rabbit carotid arteries has been developed and the subsequent repair of these lesions is described. Two types of circumferential lesion can be produced by the application of 50–160 g of longitudinally applied tension. Small lesions can be up to 400 μm in length and are characterized by the loss of a small area of endothelium and rupture of the internal elastic lamellae. No demonstrable damage to the media is detected in these lesions. Larger lesions can be up to 1 mm in length and are characterized by the loss of endothelium and rupture of the internal as well as a variable number of medial elastic lamellae. Little, if any, damage to the medial smooth muscle cells is observed although the extracellular matrix is often disrupted. Small lesions are completely reendothelialized within 24 hr and larger lesions within 7–10 days. Both large and small lesions repair without the formation of an intimal thickening of smooth muscle cells, despite quite marked damage to the media of the larger lesions.