Endothelium-denuded Rat Aorta Research Articles

Characterization of the contractile 5-hydroxytryptamine receptor in the renal artery of the normotensive rat.

Our goal was to characterize the 5-hydroxytryptamine (5-HT) receptor(s) mediating contraction in the isolated right renal artery, testing the hypothesis that the 5-HT(2A) receptor would be the primary and likely only 5-HT receptor involved in contraction. Contraction of arteries was investigated in isolated tissue baths, and expression of 5-HT receptors was measured using immunohistochemical and Western analyses. Compared with endothelium-denuded rat aorta, a tissue with an established 5-HT(2A) receptor, endothelium-denuded renal artery contracted to 5-HT with a 10-fold greater potency. Surprisingly, the 5-HT(2B) receptor agonist alpha-methyl-5-(2-thienylmethoxy)-1H-indole-3-ethanamine hydrochloride (BW723C86) caused a concentration-dependent contraction that was antagonized by the 5-HT(2B) receptor antagonist 6-methyl-1,2,3,4-tetrahydro-1-[3,4-dimethoxyphenyl) methyl-9H-pyrido[3,4b]indole] hydrochloride (LY272015) and nonselective 5-HT(2) receptor antagonist 6-methyl-1-(1-methylethyl)-ergoline-8b-carboxylic acid 2-hydroxy-1-methylpropyl ester maleate (LY53857). Correlation of -log EC(50) values with binding affinities (pK(i)) indicated that contraction of the renal artery elicited by 13 different agonists was likely consistent with activation of a 5-HT(2A) (r = 0.928) and 5-HT(2B) (r = 0.843) receptor. 5-HT-induced contraction was shifted by the 5-HT(2A) receptor antagonist ketanserin (3 and 10 nM) and the 5-HT(2B) receptor antagonist LY272015 (10 and 50 nM). Higher than expected concentrations of the 5-HT(2A)/5-HT(2B) receptor antagonist LY53857 were needed to antagonize 5-HT-induced contraction and the 5-HT(2B) receptor antagonist 2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine (RS127445) was virtually inactive. Western and immunohistochemical analyses of the renal artery validated the presence of 5-HT(2A) and 5-HT(2B) receptor protein. These results suggest that the renal artery possesses a complex 5-HT receptor population, including ketanserin- and LY272015-sensitive receptors. This unique pharmacology may reflect differences in 5-HT receptor coupling between tissues or heterogeneity in the subtype(s) of 5-HT receptors expressed in the renal artery.

Lysophosphatidylcholine potentiates vascular contractile responses in rat aorta via activation of tyrosine kinase.

We previously reported that while lysophosphatidylcholine (LPC) does not itself produce contraction, it significantly potentiates the contractile responses induced by high-K(+), UK14,304 (a selective alpha(2)-adrenoceptor agonist) and phorbol ester in the endothelium-denuded rat aorta. To further investigate this phenomenon, we examined the effects of genistein and tyrphostin B42 (both tyrosine kinase inhibitors) on the LPC-induced potentiation of the contractile responses to high-K(+) and UK14,304 in the endothelium-denuded rat aorta. Although genistein (3 x 10(-6) M, 10(-5) M) did not affect the high-K(+)-induced contractile response, it selectively inhibited the potentiating effect of LPC on the contraction and it strongly inhibited the LPC-induced augmentation of the associated increases in [Ca(2+)](i). Genistein also attenuated the LPC-induced augmentation effects on both the increase in [Ca(2+)](i) and contractile response induced by the UK14,304. In contrast, daidzein (10(-5) M) did not inhibit the potentiating effect of LPC. Tyrphostin B42 (3 x 10(-5) M) attenuated the potentiating effect of LPC on high K(+)-induced contractions. Western blot analysis showed that LPC increased the tyrosine phosphorylation of a number of proteins, including 42 and 44 kDa proteins and 53 - 64 kDa proteins. These protein phosphorylations were inhibited by genistein. Sodium orthovanadate (10(-4) M), a tyrosine phosphatase inhibitor, also markedly enhanced the high-K(+)-induced contractile responses. This enhancing effect was attenuated by genistein. These results suggest that the LPC-induced augmentation of contractile responses in the rat aorta is due to activation of tyrosine kinase, which in turn regulates Ca(2+) influx.

Interleukin-1beta, Src- and non-Src tyrosine kinases, and nitric oxide synthase induction in rat aorta in vitro.

We studied the potential roles for endogenous interleukin-1beta (IL-1beta) and for several signaling pathways in the spontaneous induction in vitro of inducible nitric oxide synthase (iNOS) in endothelium-denuded rat aorta rings. Added IL-1beta augmented, whereas the IL-1beta receptor antagonist IL-1ra blocked, spontaneous iNOS induction. Furthermore, increases in IL-1beta mRNA preceded those of iNOS mRNA. Mitogen-activated protein kinase kinase and phosphatidyl inositol 3' kinase inhibition did not block iNOS induction, whereas nuclear factor kappaB inhibition did. The sarcoma virus tyrosine kinase (Src) family-selective inhibitor 4-amino-5(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) blocked the upregulation of IL-1beta mRNA and the subsequent induction of iNOS but not the induction of iNOS stimulated by exogenously added IL-1beta. In contrast, the non-Src inhibitors TP 47/AG 213 and genistein and the tyrosine phosphatase inhibitor vanadate did not affect the spontaneous upregulation of IL-1beta mRNA but blocked both the IL-1beta-mediated and spontaneous induction of iNOS. We conclude that 1) the upregulation of tissue IL-1beta, via a signaling pathway involving a Src family kinase, plays a key role in rat vascular iNOS induction and 2) non-Src tyrosine kinases play roles downstream from IL-1beta for iNOS induction.

.ALPHA.-Adrenoceptor Agonists Produce Ca2+ Oscillations in Isolated Rat Aorta: Role of Protein Kinase C.

We investigated the relationship between tension development and the cytosolic free Ca2+ level ([Ca2+]i) in responses to norepinephrine (NE) and selective alpha2-adrenoceptor agonist, UK14,304 of the endothelium-denuded rat aorta loaded with fura PE-3. NE (3 x 10(-8) M) evoked a rapid increase in [Ca2+]i followed by slight decreasing to a steady state level and produced a contraction. After the NE-induced increase in [Ca2+]i had reached a maximum, the [Ca2+]i showed persistent oscillations. The Ca2+ oscillations were superimposed on the sustained increase in [Ca2+]i. UK14,304 (3 x 10(-6) M) also evoked an increase in [Ca2+]i and produced a contraction. However, the UK14,304-induced effect on [Ca2+]i was characterized by pronounced oscillations, and the amplitude of the sustained increase in [Ca2+]i was less than that seen with NE. Protein kinase C inhibitor, Ro31-8220 (3 x 10(-6) M) and verapamil (10(-5) M) abolished both NE and UK14,304-evoked Ca2+ oscillations. UK14,304-induced contractions were also strongly inhibited by Ro31-8220 and verapamil. However, NE induced contractions were partly inhibited by these inhibitors. The sustained increases in [Ca2+]i evoked NE and UK14,304 were not significantly inhibited by Ro31-8220 and verapamil. These results suggest that NE and UK14,304 produce Ca2+ oscillations during sustained contractions in rat aorta. The alpha2 adrenoceptor agonist, UK14,304-induced sustained contraction and Ca2+ oscillations may be due to PKC activation and opening of voltage-dependent L type Ca2+ channels.

Potential functional significance of brain-type and muscle-type nitric oxide synthase I expressed in adventitia and media of rat aorta.

Skeletal muscle and myocardium express microNOS I, an elongated splice variant of neuronal-type nitric oxide (NO) synthase (NOS I), and NOS III, endothelial-type NO synthase, respectively. This study was designed to elucidate whether vascular smooth muscle also contains a constitutively expressed NO synthase isoform. In the rat, microNOS I contains an insert of 102 nucleotides after nucleotide 2865 of the cDNA, yielding a protein of 164 kd. Reverse transcription-polymerase chain reaction with primers flanking this insert and with insert-specific primers indicated that endothelium-denuded rat aorta expresses both brain-type NOS I and microNOS I. RNase protection analyses with an antisense RNA probe overlapping the microNOS I insert detected significant amounts of NOS I mRNA and lesser amounts of microNOS I mRNA in endothelium-denuded aorta. Western blots using a specific polyclonal antibody recognizing NOS I and microNOS I showed a major band of the 160-kd NOS I and a lesser band of a slightly larger protein in endothelium-denuded aorta. Immunohistochemistry demonstrated low levels of NOS I/microNOS I immunoreactivity in the medial layer of rat aorta, whereas the endothelium expressed only NOS III immunoreactivity. When the adventitia also was removed, NOS I and microNOS I mRNA decreased markedly but remained detectable in the medial layer. In functional experiments with endothelium-denuded rat aortic rings (that contained no NOS III), contractions induced by KCl were markedly increased in the presence of the NOS inhibitor N(G)-nitro-L-arginine. These data demonstrate that 2 subforms of NOS I are expressed in nonendothelial components of rat aorta: NOS I and lesser amounts of microNOS I. Under certain conditions, this NOS I/microNOS I expression could serve as a backup system to the functionally predominant NOS III.

L-NAME inhibits Mg(2+)-induced rat aortic relaxation in the absence of endothelium.

1 L-NG-nitro-arginine methyl ester (L-NAME; 100 microM), a nitric oxide synthase (NOS) inhibitor, reversed the relaxation induced by 3 microM acetylcholine (ACh) and 2-10 mM Mg2+ in endothelium-intact (+E) rat aortic rings precontracted with 1 microM phenylephrine (PE). In PE-precontracted endothelium-denuded (-E) rat aorta, 3 microM ACh did not, but Mg2+ caused relaxation which was reversed by L-NAME, but not by D-NAME. 2 The concentration response profiles of L-NAME in reversing the equipotent relaxation induced by 5 mM Mg2+ and 0.2 microM ACh were not significantly different. 3 L-NAME (100 microM) also reversed Mg(2+)-relaxation of -E aorta pre-contracted with 20 mM KCl or 10 microM prostaglandin F2alpha (PGF2alpha). L-NG-monomethyl-arginine (L-NMMA; 100 microM) was also effective in reversing the Mg(2+)-relaxation. 4 Addition of 0.2 mM Ni2+, like Mg2+, caused relaxation of PE-pre-contracted -E aorta, which was subsequently reversed by 100 microM L-NAME. 5 Reversal of the Mg(2+)-relaxation by 100 microM L-NAME in PE-precontracted -E aorta persisted following pre-incubation with 1 microM dexamethasone or 300 microM aminoguanidine (to inhibit the inducible form of NOS, iNOS). 6 Pretreatment of either +E or -E aortic rings with 100 microM L-NAME caused elevation of contractile responses to Ca2+ in the presence of 1 microM PE. 7 Our results suggest that L-NAME exerts a direct action on, as yet, unidentified vascular smooth muscle plasma membrane protein(s), thus affecting its reactivity to divalent cations leading to the reversal of relaxation. Such an effect of L-NAME is unrelated to the inhibition of endothelial NOS or the inducible NOS.

Atypical β-adrenoceptors of rat thoracic aorta

The possible existence of atypical β-adrenoceptors in vascular smooth muscle of rat isolated thoracic aorta was investigated. Isoprenaline (10 −8–10 −4 M) produced concentration-dependent relaxation of phenylephrine (10 −5 M) precontracted rings of endothelium-denuded rat aorta in vitro. Isoprenaline-induced relaxation was resistant to blockade by atenolol (10 −6 M). But, propranolol (2 × 10 −7 M) caused a non-competitive inhibition and SR 59230A (6.6 × 10 −6 M), a β 3-adrenoceptor selective antagonist, failed to produce additional antagonism in presence of propranolol. BRL 37344 (10 −8–10 −4 M), a β 3-selective agonist, did not relax ring segments precontracted with phenylephrine (10 −5 M) in the absence of endothelium. The non-conventional partial agonist (–)-cyanopindolol (5 × 10 −6–10 −4 M) induced a marked relaxation in phenylephrine (10 −5 M) precontracted aortic rings without endothelium. This vasodilation was resistant to blockade by propranolol (2 × 10 −7 M) and SR 59230A (10 −5 M). Salbutamol (10 −8–10 −4 M) produced concentration-dependent relaxation in isolated endothelium-denuded aortic rings precontracted with phenylephrine (10 −5 M). Propranolol (2 × 10 −7 M), but not atenolol (10 −6 M), inhibited this relaxant response. It is concluded that in endothelium-denuded thoracic aorta, salbutamol acts through β 2-adrenoceptors whereas isoprenaline seems to activate both β 2-adrenoceptors and an atypical β-adrenergic receptor. This atypical β-adrenoceptor is distinct from putative β 3-adrenoceptor and maybe resembles the reported fourth cardiac β-adrenoceptor.

Carbachol, an acetylcholine receptor agonist, enhances production in rat aorta of 2-arachidonoyl glycerol, a hypotensive endocannabinoid

The production of 2-arachidonoyl glycerol, an endogenous cannabinoid, is enhanced in normal, but not in endothelium-denuded rat aorta on stimulation with carbachol, an acetylcholine receptor agonist. 2-Arachidonoyl glycerol potently reduces blood pressure in rats and may represent an endothelium-derived hypotensive factor.

Lysophosphatidylcholine potentiates vascular contractile responses by enhancing vasoconstrictor-induced increase in cytosolic free Ca 2+ in rat aorta

We investigated the effects of palmitoyl- l-α-lysophosphatidylcholine on the contractile responses of the endothelium-denuded rat aorta to high K +, noradrenaline, UK14,304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline) (a selective α 2 adrenoceptor agonist) and phorbol 12-myristate 13-acetate (PMA). Lysophosphatidylcholine at concentrations from 10 −6 M to 10 −4 M did not contract aortic strips. However, lysophosphatidylcholine strongly potentiated the UK14,304-induced contraction. High K +- and PMA-induced contractions were also potentiated. In contrast, the noradrenaline-induced contraction was only slightly potentiated by 10 −5 M lysophosphatidylcholine. In fura PE-3-loaded aortic strips, lysophosphatidylcholine (10 −5 M) markedly augmented the increase in both cytosolic free Ca 2+ ([Ca 2+] i) and contractile tension induced by UK14,304, high K + and PMA. Nicardipine (10 −7 M) and 10 −6 M Ro-31-8220 ({1-[3-(amidinothio)propyl-1H-indoyl-3-yl]-3-(1-methyl-1H-indoyl-3-yl)-maleimide-methane sulfate) strongly inhibited the increase in [Ca 2+] i and contractile tension induced by UK14,304 and in the presence of these inhibitors, the enhancing effects of lysophosphatidylcholine were attenuated. However, the enhancing effect on high K +-induced contraction was not affected by Ro-31-8220. These results suggest that lysophosphatidylcholine may cause an augmentation of the increase in [Ca 2+] i induced by UK14,304 which response is depend on protein kinase C activation and in this way potentiate contractile responses in the rat aorta. Protein kinase C independent mechanisms may also be involved in the enhancing effect of lysophosphatidylcholine on smooth muscle contraction.

Effects of the selective cyclooxygenase-2 inhibitor nimesulide on vascular contractions in endothelium-denuded rat aorta

We have examined the effects of the selective cyclooxygenase-2 inhibitor nimesulide and the non-selective cyclooxygenase inhibitor indomethacin on vascular responsiveness of endothelium-denuded rat aorta. Isometric contractions were obtained to the α-adrenoceptor agonists phenylephrine (full agonist) and clonidine (partial agonist relative to phenylephrine) and to endothelin-1 and KCl. Maximum contractile responses to the partial agonist clonidine were significantly reduced by nimesulide (10 μM) and by indomethacin (10 μM) to 60.8±8.5% ( n=8) and 69.0±9.6% ( n=12) of control, respectively, as compared with the effects of vehicle (99.0±5.8%; n=17). The inhibitors had lesser effects against contractions to phenylephrine: nimesulide had no significant effect, whereas indomethacin caused a small but significant reduction in the maximum contraction to phenylephrine to 90.3±5.0% ( n=12) of control (vehicle: 108.0±5.2%, n=15 nimesulide: 111.8±5.9%, n=5). Neither nimesulide nor indomethacin had any effect on contractions to endothelin-1 or KCl. These actions differed from the effects of the Ca 2+ entry blocker nifedipine, which significantly reduced contractions to clonidine and KCl to a similar extent. The maximum contraction to clonidine was also significantly reduced by the thromboxane receptor antagonist SQ 29548 (1 μM) to 83.4±6.4% of control ( n=7) (vehicle 115.5±7.5%, n=7). It is concluded that the cyclooxygenase inhibitors nimesulide or indomethacin reduce vascular responsiveness to α-adrenoceptor agonists in endothelium-denuded rat aorta, presumably by preventing the formation of vasoconstrictor prostaglandins in aortic smooth muscle by cyclooxygenase-2. This reduced vascular responsiveness was most clearly seen with the partial agonist clonidine.

Thrombin receptor expression is increased by angiotensin II in cultured and native vascular smooth muscle cells

The factors involved in restenosis after balloon angioplasty are poorly characterized but the local concentration of the potent mitogens angiotensin II (AII) and thrombin is known to be increased at sites of vascular injury. We investigated the possibility of a synergistic interaction between AII and thrombin by studying the effects of AII on the expression of the thrombin receptor in rat aortic smooth muscle cells (VMSC). Thrombin receptor mRNA expression was studied by Northern blot analysis and RT-PCR using total RNA extracted from VMSC or from endothelium-denuded rat aortae. As a measure of thrombin receptor protein expression, we assessed either the thrombin-stimulated release of 6-keto-prostaglandin F1 alpha from VMSC or the contraction of endothelium-denuded rat aortic rings. The thrombin receptor mRNA was expressed at a low level in both cultured and native VMSC. AII concentration- and time-dependently increased expression of thrombin receptor mRNA in VMSC and augmented the thrombin-induced release of 6-keto-prostaglandin F1 alpha as well as the thrombin induced contraction. Blockade of the angiotensin subtype 1 (AT1) receptor by EXP3174 or D8731 prevented the AII-mediated increase in thrombin receptor expression. The effect of AII on the increase in thrombin receptor mRNA expression was enhanced by the protein kinase C inhibitor Ro 31-8220, but was unaffected by prolonged incubation with phorbol myristate acetate or the tyrosine kinase inhibitors genistein and erbstatin A. These results demonstrate that AII enhances the expression of thrombin receptor in cultured and native VMSC. In cultured cells, this effect is mediated by the activation of the AT1 receptor subtype. This synergistic effect between AII and the thrombin receptor may promote the extensive proliferation of smooth muscle cells in response to vascular injury.

17 beta-Estradiol reduces vasoconstriction in endothelium-denuded rat aortas through inducible NOS.

Estrogen produces vasodilatation through the induction of nitric oxide synthase (NOS) in the endothelium, but there are many reports of endothelium-independent effects. In the present study, these processes were investigated in rat aortas isolated from ovariectomized rats. Long-term in vitro treatment with 17 beta-estradiol (10 nM for 24 h) in an organ culture system slightly reduced acetylcholine-mediated vasorelaxation in endothelium-intact aortic rings. 17 beta-Estradiol (1 and 10 nM for 24 h) also attenuated the phenylephrine-induced constriction in endothelium-denuded aortas, and this effect was inhibited by the NOS inhibitor L-N5-(1-iminoethyl)ornithine hydrochloride, as well as the estrogen receptor antagonist ICI-182,780. Furthermore, 17 beta-estradiol treatment (1 and 10 nM for 24 h) increased nitric oxide production as assessed by the conversion of [3H]arginine to [3H]citrulline in endothelium-denuded rat aortas. These effects were prevented by the protein synthesis inhibitor cycloheximide. 17 beta-Estradiol (10 nM for 24 h) treatment also induced the formation of inducible NOS (iNOS) protein in aortas. The results indicate that 17 beta-estradiol can attenuate the vasoconstrictor effect of phenylephrine by a process that involves induction of iNOS in nonendothelial cells of the aorta. We suggest that long-term estrogen therapy may induce a partial hyporesponsiveness in vascular smooth muscle via a small but sustained nitric oxide production.

Pranidipine, a new 1,4-dihydropyridine calcium channel blocker, enhances cyclic GMP-independent nitric oxide-induced relaxation of the rat aorta.

Pranidipine, a new calcium channel blocker, prolonged endothelium-dependent relaxation induced by acetylcholine in an aortic ring preparation, contracted with prostaglandin F2alpha. This action was not shared by amlodipine. The effect was not modified by indomethacin, suggesting that the action of pranidipine does not involve prostanoid metabolism. N(G)-nitro-L-arginine completely prevented the action of Pranidipine. The drug affected neither nitric oxide (NO) synthase activity nor the level of cyclic GMP in the vessel. Pranidipine did not affect the sensitivity of the contractile proteins to calcium. Pranidipine also did not alter cyclic GMP-induced relaxation in alpha-toxin-skinned vascular preparations. Pranidipine also prolonged glyceryl trinitrate-induced relaxation in the endothelium denuded rat aorta. Furthermore, pranidipine enhanced relaxation of the aorta induced by glyceryl trinitrate even in the presence of methylene blue, a guanylyl cyclase inhibitor. This action was not modified by iberiotoxin or by charybdotoxin, two inhibitors of the calcium-activated potassium channel. The results strongly suggest that pranidipine enhances cyclic GMP-independent NO-induced relaxation of smooth muscle by a mechanism other than through NO-induced hyperpolarization. These effects were in direct contrast to amlodipine, another new 1,4-dihydropyridine calcium antagonist.

Effects of Several Class I Antiarrhythmic Drugs on Isolated Rat Aortic Vascular Smooth Muscle

1. The vasorelaxant effects of seven NA + channel blockers (i.e., class I antiarrhythmic agents), quinidine, disopyramide, imipramine, lidocaine, mexiletine, flecainide, and desipramine, were investigated in isolated endothelium-denuded rat aorta. 2. All drugs induced a concentration-dependent relaxation in aorta precontracted with either 80 mM KCl or 10 −5 M noradrenaline and, with the exception of mexiletine, they were more potent in inhibiting KCl-induced contractions. 3. The degree of inhibition of high KCl–induced contractions produced by quinidine and desipramine increased with the time of depolarization. Furthermore, the inhibitory effect of quinidine also increased in aorta preincubated in 40 mM KCl, whereas the inhibitory effects of other antiarrhythmics were almost similar in 5 or 40 mM KCl solution. 4. In conclusion, all these class I antiarrhythmic drugs inhibited Ca 2+ entry through voltage- and receptor-gated channels as well as Ca 2+ release from intracellular stores. As a consequence, they decrease the availability of intracellular free Ca 2+ required for vascular smooth muscle contraction.

Effect of capsaicin on membrane currents in cultured vascular smooth muscle cells of rat aorta

The application of capsaicin (1 μM) produced a minor relaxant effect in endothelium-denuded rat aortae. However, capsaicin caused a greater relaxation of blood vessels precontracted with high K + or phenylephrine. The effects of capsaicin on the ionic currents were also examined in A7r5 vascular smooth muscle cells. The tight-seal whole-cell voltage clamp technique was used. Capsaicin inhibited the Ba 2+ inward current (I Ba) through the voltage-dependent L-type Ca 2+ channel in a concentration-dependent fashion, whereas calcitonin gene-related peptide and phenylephrine produced a minor increase in I Ba. Capsaicin did not alter the overall shape of current-voltage relationship of I Ba. However, capsaicin (3 μM) shifted the quasi-steady-state inactivation curve of I Ba to more negative membrane potential by about 5 mV. These effects of capsaicin on I Ba were reversible. In addition, capsaicin had inhibitory effects on voltage dependent K + currents. These results suggest that inhibition of the voltage-dependent L-type Ca 2+ channel is involved in the capsaicin-induced relaxation of the vascular smooth muscle, whereas capsaicin-induced inhibition of voltage-dependent K + channels might produced an increase in cell excitability.

Voltage‐ and time‐dependent inhibitory effects on rat aortic and porcine coronary artery contraction induced by propafenone and quinidine

1. Class I antiarrhythmic drugs (e.g. Na+ channel blockers) such as propafenone and quinidine also inhibit voltage-gated Ca2+ and K+ channels. In the present paper the voltage- and time-dependent inhibitory effects of propafenone and quinidine were studied on depolarization-induced vascular contractions and 45Ca2+ uptake in isolated endothelium denuded rat aorta and pig left descending coronary artery. 2. Quinidine and propafenone (10(-7) M -5 x 10(-5) M) produced a concentration-dependent relaxation of the contractions induced by 80 mM KCl. Propafenone was significantly more potent (P < 0.05) than quinidine in both rat aorta and pig coronary arteries but both drugs more potent (P < 0.05) in relaxing rat aorta than pig coronary arteries. In rat aortic rings, the relaxant effects of propafenone were unaffected by pretreatment with the Na+ channel blocker, tetrodotoxin. 3. The degree of inhibition produced after prolonged exposure (40 min) to propafenone and quinidine differed as the time of depolarization with 80 mM KCl was increased. Quinidine (3 x 10(-6) M, 10(-5) M and 3 x 10(-5) M) not only produced an inhibition at the very early stage of contraction, but also a time-dependent inhibition was observed. In contrast, propafenone (10(-6) M, 3 x 10(-6) M and 10(-5) M) produced a more marked concentration-dependent early block but only a mild time-dependent inhibition.4. The voltage-dependence of propafenone- and quinidine-induced inhibition, was studied in rat aorta and coronary arteries which had been incubated in 5 or 40mM KCl Ca2+-free solution and then contracted by changing the bath solution to 100 mM KCI and 2 mM CaCl2 solution. The inhibitor effects of quinidine were significantly enhanced (P <0.05) when the preparations were preincubated in 40 mMKCl (depolarizing) solution. In contrast, the effects of propafenone were quite similar in 5 or in 40 mMKCI solution.5. Quinidine, 10-5 M, produced a greater inhibition (P<0.05) of 100 mM KCl-stimulated 45Ca2+ uptake in aortic rings preincubated in depolarizing as compared to normal solution. In contrast, the inhibition produced by 3 x 10-6 M propafenone was similar in aortic rings incubated in 5 or 40 mM KCl solution.6.It is concluded that both quinidine and propafenone inhibited vascular smooth muscle contraction which could be attributed to reduced Ca2+ entry. The voltage- and time-dependent inhibitory effects of quinidine may reflect an increased binding of the drug to Ca2+ channels at depolarized potentials.

Evidence for preservation of epithelial function in cryopreserved porcine and human bronchi

Porcine and human bronchi have been investigated in vitro without or after storage at -196 degrees C in Krebs-Henseleit solution containing 2.0 M dimethyl sulphoxide and 0.1 M sucrose as cryoprotectants. In bronchi from both species maximal post-thaw contractile responses to acetylcholine (ACh) were reduced by about 25-30% compared to unfrozen bronchi. To assess the viability of bronchi and endothelium-denuded rat aortic strips, was employed. The release of an epithelium-derived inhibitory factor (EpDIF) was induced by ACh and assessed in terms of concentration-dependent relaxation of the endothelium-denuded rat aortae. Following removal of bronchial epithelium, ACh failed to elicit any relaxation of rat aorta. With cryopreserved bronchi from both pig and human about 5 and 30 times higher concentrations of ACh, respectively, were required to elicit the same relaxant response as with unfrozen bronchi. The results suggest that after the freezing-thawing process both smooth muscle and epithelial function is largely preserved and provide support for the use of cryopreservation for storage of airway preparations for pharmacological studies.

NMDA causes release of nitric oxide from rat spinal cord in vitro

Anatomic studies have localized nitric oxide synthase (NOS) activity in the rat spinal cord dorsal horn and intermediolateral cell column. Behavioral and electrophysiologic studies suggest that N-methyl- d-aspartate (NMDA) stimulates nitric oxide synthesis in the dorsal horn. This report describes a novel bioassay to determine directly in vitro whether NMDA causes release of nitric oxide from rat spinal cord. Modified Krebs-Henseleit solution at 26°C was perfused over spinal cord slices from adult male rats, then dropped onto a ring of endothelium-denuded rat aorta. Following preconstriction with phenylephrine, NMDA (10 −10 to 10 −3 M) alone or with other drugs was added to the perfusion solution and vascular tension measured. NMDA-containing solutions applied directly on the preconstricted vessels without exposure to spinal cord tissue had no effect on vessel tone. In contrast, NMDA via the spinal cord perfusion caused concentration-dependent vascular relaxation, which was blocked by MK-801, hemoglobin, methylene blue, and several arginine analogues which inhibit NOS. [ 14C]citrulline assay suggested NOS in rat spinal cord was non-endothelial in nature. NMDA perfusion of spinal cord slices in vitro causes vascular relaxation in this bioassay due to actions on NMDA receptors and which is consistent with release of nitric oxide. These results support previous anatomical, behavioral, and electrophysiologic studies in rat spinal cord and describe a novel, sensitive, and simple bioassay for nitric oxide release from neural tissue in vitro.

An analysis of the inhibitory effects of prazosin on the phenylephrine response curves of the rat aorta.

On the endothelium-intact rat aorta some studies have shown prazosin to cause nonparallel rightward shifts of alpha 1-adrenoceptor agonist response curves. The aim of the present study was to analyze the inhibitory effect of prazosin on the phenylephrine responses of the endothelium-intact and endothelium-denuded rat aorta. Firstly I used phenoxybenzamine treatment to characterize the phenylephrine responses. The KA values for phenylephrine were 0.13-0.18 microM and 0.07-0.16 microM in the endothelium-intact and endothelium-denuded rat aorta, respectively. In order to produce maximal responses of the endothelium-intact or--denuded preparation, phenylephrine had to occupy 95-99% of the alpha 1-adrenoceptors. Secondly I compared the inhibitory effects of phentolamine and prazosin on the endothelium-intact rat aorta. Phentolamine at 0.1 and 1 microM caused parallel rightward shifts of phenylephrine response curves with no effect on phenylephrine maximal responses (phentolamine pA2 = 7.9). The inhibitory effects of phentolamine were readily reversible. Prazosin at 0.1-10 nM caused nonparallel rightward shifts of the phenylephrine response curves with a depression of the maximal response. These inhibitory effects of prazosin were either irreversible or only very slowly reversible in drug-free solution and slowly reversible in the presence of phentolamine. Ninety min was required for the inhibitory effect of prazosin to reach equilibrium whereas phentolamine was at equilibrium after 45 min. Finally I have characterized the inhibitory actions of prazosin on the endothelium-denuded rat aorta. Prazosin caused parallel rightward shifts of phenylephrine response curves with no effect on phenylephrine maximal responses. The inhibitory effects of prazosin were at equilibrium after 45 min and were readily reversible.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasorelaxation of rat thoracic aorta caused by osthole isolated from Angelica pubescens

The pharmacological effects of osthole on isolated rat thoracic aorta were examined. Osthole inhibited norepinephrine (NE, 3 μM)-induced phasic and tonic contractions in rat thoracic aorta in a concentration-dependent manner (40–200 μM). The tonic contraction elicited by NE was also relaxed by the addition of osthole. This relaxing effect of osthole was not affected by indomethacin (20 μM) and was still observed in endothelium-denuded rat aorta. Methylene blue (50 μM) partially antagonized this relaxing effect of osthole. In high-K + medium (80 mM), the Ca 2+ (0.03–3 mM)-induced vasocontraction was inhibited concentration dependently by osthole (20–100 μM). Addition of osthole (100 μM) at the plateau of the K + (80 mM)-induced contraction caused relaxation. Methylene blue (50 μM) did not antagonize this relaxation. In Ca 2+-free medium, the caffeine (10 mM)-induced phasic contraction was also suppressed by osthole in a concentration-dependent manner. Although the cAMP level was not changed by osthole, the cGMP level of rat aorta was increased by osthole in a concentration-dependent manner. The increase in cGMP level caused by osthole was completely blocked by methylene blue. [ 3H]Inositol monophosphate formation caused by NE was not affected by osthole at a concentration of 200 μM. The 45Ca 2+ influx elicited by either NE or high K + was inhibited by osthole in a concentration-dependent manner. It is concluded that osthole relaxes rat thoracic aorta by virtue of its Ca 2+-channel blocking properties and by elevating cGMP levels in vascular smooth muscle.