Reduced sarco/endoplasmic reticulum Ca(2+) uptake activity can account for the reduced response to NO, but not sodium nitroprusside, in hypercholesterolemic rabbit aorta.

BACKGROUND: Hypercholesterolemia (HC) impairs acetylcholine-induced relaxation but has little effect on that caused by the NO donor sodium nitroprusside (SNP), suggesting that acetylcholine releases less NO from the endothelium in HC. The relaxation to authentic NO gas, however, is also impaired in HC aortic smooth muscle, indicating an abnormal smooth muscle response. NO relaxes arteries by both cGMP-dependent and -independent mechanisms, and the response involves calcium (Ca(2+)) store refilling via the sarco/endoplasmic reticulum calcium ATPase (SERCA). We studied the involvement of cGMP and SERCA in the smooth muscle response to NO and SNP in HC rabbit aorta. METHODS AND RESULTS: A selective guanylyl cyclase inhibitor, 1H-[1,2,4]-oxadiazole-[4,3-a]quinoxalin-1-one, eliminated SNP-induced relaxation but only partially blocked NO-induced relaxation in both normal and HC aorta. The residual relaxation to NO was still less in HC and, in both normal and HC aorta, was abolished by concomitant administration of the SERCA inhibitor cyclopiazonic acid (CPA). In contrast, CPA did not affect SNP-induced relaxation in either normal or HC aorta. SERCA activity measured by (45)Ca(2+) uptake was markedly decreased in HC, although SERCA2 protein expression did not change significantly. CONCLUSIONS: These data suggest that NO-induced relaxation but not that to SNP is partially mediated by cGMP-independent Ca(2+) uptake into sarco/endoplasmic reticulum and that reduced sarco/endoplasmic reticulum Ca(2+) pump function can account for the impaired response to NO in HC.

Properties of a native cation channel activated by Ca2+ store depletion in vascular smooth muscle cells.

Depletion of intracellular Ca(2+) stores activates capacitative Ca(2+) influx in smooth muscle cells, but the native store-operated channels that mediate such influx remain unidentified. Recently we demonstrated that calcium influx factor produced by yeast and human platelets with depleted Ca(2+) stores activates small conductance cation channels in excised membrane patches from vascular smooth muscle cells (SMC). Here we characterize these channels in intact cells and present evidence that they belong to the class of store-operated channels, which are activated upon passive depletion of Ca(2+) stores. Application of thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca(2+) ATPase, to individual SMC activated single 3-pS cation channels in cell-attached membrane patches. Channels remained active when inside-out membrane patches were excised from the cells. Excision of membrane patches from resting SMC did not by itself activate the channels. Loading SMC with BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), which slowly depletes Ca(2+) stores without a rise in intracellular Ca(2+), activated the same 3-pS channels in cell-attached membrane patches as well as whole cell nonselective cation currents in SMC. TG- and BAPTA-activated 3-pS channels were cation-selective but poorly discriminated among Ca(2+), Sr(2+), Ba(2+), Na(+), K(+), and Cs(+). Open channel probability did not change at negative membrane potentials but increased significantly at high positive potentials. Activation of 3-pS channels did not depend on intracellular Ca(2+) concentration. Neither TG nor a variety of second messengers (including Ca(2+), InsP3, InsP4, GTPgammaS, cyclic AMP, cyclic GMP, ATP, and ADP) activated 3-pS channels in inside-out membrane patches. Thus, 3-pS nonselective cation channels are present and activated by TG or BAPTA-induced depletion of intracellular Ca(2+) stores in intact SMC. These native store-operated cation channels can account for capacitative Ca(2+) influx in SMC and can play an important role in regulation of vascular tone.

Mechanism of nitric oxide-induced vasodilatation: refilling of intracellular stores by sarcoplasmic reticulum Ca2+ ATPase and inhibition of store-operated Ca2+ influx.

The precise mechanisms by which nitric oxide (NO) decreases free [Ca2+]i, inhibits Ca2+ influx, and relaxes vascular smooth muscle are poorly understood. In rabbit and mouse aorta, agonist-induced contractions and increases in [Ca2+]i were resistant to nifedipine, suggesting Ca2+ entry through non-L-type Ca2+ channels. Relaxations to NO were inhibited by thapsigargin (TG) or cyclopiazonic acid (CPA) indicating the involvement of sarcoplasmic reticulum ATPase (SERCA). Studies of the effect of NO on [Ca2+]i and the rate of Mn2+ influx with fura-2 fluorometry in rabbit aortic smooth muscle cells in primary culture were designed to test how SERCA is involved in mediating the response to NO. When cells were stimulated with angiotensin II (AII), NO accelerated the removal of Ca2+ from the cytoplasm, decreased [Ca2+]i, and inhibited Ca2+ and Mn2+ influx. Inhibition of SERCA abolished all the effects of NO. In contrast, inhibition of the Na+/Ca2+exchanger or the plasma membrane Ca2+ ATPase had no influence on the ability of NO to decrease [Ca2+]i. NO maximally decreased [Ca2+]i within 5 s, whereas significant inhibition of AII-induced Ca2+ and Mn2+ influx required more than 15 s. The inhibition of cation influx strictly depended on [Ca2+]o and functional SERCA, suggesting that during the delay before NO inhibits Ca2+ influx, the influx of Ca2+ and the uptake into intracellular stores are required. In the absence of [Ca2+]o, NO diminished the AII-induced [Ca2+]i transient by a SERCA-dependent mechanism and increased the amount of Ca2+ in the stores subsequently released by ionomycin. The present study indicates that the initial rapid decrease in [Ca2+]i caused by NO in vascular smooth muscle is accounted for by the uptake of Ca2+ by SERCA into intracellular stores. It is proposed that the refilling of the stores inhibits store-operated Ca2+ influx through non-L-type Ca2+ conducting ion channels and that this maintains the decrease in [Ca2+]i and NO-induced relaxation.

Evidence that additional mechanisms to cyclic GMP mediate the decrease in intracellular calcium and relaxation of rabbit aortic smooth muscle to nitric oxide.

1. The role of cyclic GMP in the ability of nitric oxide (NO) to decrease intracellular free calcium concentration [Ca2+]i and divalent cation influx was studied in rabbit aortic smooth muscle cells in primary culture. In cells stimulated with angiotensin II (AII, 10(-1) M), NO (10(-10) - 10(-6) M) increased cyclic GMP levels measured by radioimmunoassay and decreased [Ca2+]i and cation influx as indicated by fura-2 fluorimetry. 2. Zaprinast (10(-4) M), increased NO-stimulated levels of cyclic GMP by 3-20 fold. Although the phosphodiesterase inhibitor lowered the level of [Ca2+]i reached after administration of NO, the initial decreases in [Ca2+]i initiated by NO were not significantly different in magnitude or duration from those that occurred in the absence of zaprinast. 3. The guanylyl cyclase inhibitor, H-(1,2,4) oxadiazolo(4,3-a) quinoxallin-1-one (ODQ, 10(-5) M), blocked cyclic GMP accumulation and activation of protein kinase G, as measured by back phosphorylation of the inositol trisphosphate receptor. ODQ and Rp-8-Br-cyclic GMPS, a protein kinase G inhibitor, decreased the effects of NO, 10(-10) - 10(-8) M, but the decrease in [Ca2+]i or cation influx caused by higher concentrations of NO (10(-7) - 10(-6) M) were unaffected. Relaxation of intact rabbit aorta rings to NO (10(-7) - 10(-5) M) also persisted in the presence of ODQ without a significant increase in cyclic GMP. Rp-8-Br-cyclic GMPS blocked the decreases in cation influx caused by a cell permeable cyclic GMP analog, but ODQ and/or the protein kinase G inhibitor had no significant effect on the decrease caused by NO. 4. Although inhibitors of cyclic GMP, protein kinase G and phosphodiesterase can be shown to affect the decrease in [Ca2+]i and cation influx via protein kinase G, these studies indicate that when these mechanisms are blocked, cyclic GMP-independent mechanisms also contribute significantly to the decrease in [Ca2+]i and smooth muscle relaxation to NO.

Reduced responsiveness of hypercholesterolemic rabbit aortic smooth muscle cells to nitric oxide.

The response to nitric oxide of intracellular free Ca2+ levels, measured by fura 2 fluorimetry, and cyclic GMP, measured by RIA, was evaluated on smooth muscle cells of the thoracic aorta in primary culture from normal and cholesterol-fed rabbits. Relaxation to acetylcholine and nitric oxide was also determined in isolated rings of aorta. After 10 weeks of high-cholesterol diet, the intact aorta relaxed less to both acetylcholine and nitric oxide. In cultured cells from hypercholesterolemic rabbits, intracellular Ca2+ oscillated, and the mean Ca2+ levels were approximately twofold greater than in normal aortic cells. Nitric oxide failed to affect basal Ca2+ in either cell type. The peak and sustained rise in intracellular Ca2+ induced by angiotensin II (10(-7) mol/L) were similar in the two cell types. However, nitric oxide (10(-10) to 10(-6) mol/L) decreased the sustained Ca2+ levels to a significantly smaller extent in cells from cholesterol-fed rabbits. In addition, in cells from hypercholesterolemic rabbits, nitric oxide added before angiotensin II inhibited to a smaller degree the transient increase in intracellular free Ca2+ caused by angiotensin II in the nominal absence of extracellular Ca2+, as well as the increase in Ca2+ associated with the addition of extracellular Ca2+. Measurements of fura 2 quenching caused by Mn2+ influx confirmed that nitric oxide inhibited the entry of extracellular divalent cations significantly less in cells from hypercholesterolemic rabbits. Basal levels of cyclic GMP were significantly less than normal, and nitric oxide increased levels of cyclic GMP to a significantly smaller degree in cells from cholesterol-fed rabbits. These data indicate a substantial resistance to nitric oxide action in aortic smooth muscle cells of cholesterol-fed rabbits. This observation is consistent with the notion that resistance of smooth muscle cells to nitric oxide contributes to abnormal endothelium-dependent vasodilation during hypercholesterolemia and can play a role in the pathogenesis of atherosclerosis.

Vasodilatory properties of recombinant maxadilan.

Maxadilan is a peptide from the salivary gland of the sand fly Lutzomyia longipalpis, a vector for leishmaniasis. Cutaneous injection of femtomolar quantities of maxadilan produces long-lasting erythema, making it the most potent vasodilator known. Isolated rabbit thoracic and abdominal aorta, carotid artery, and iliac artery demonstrated dose-dependent arterial relaxation in response to maxadilan with a mean effective concentration (EC50) of 2.7 +/- 1.5, 2.1 +/- 0.5, 2.6 +/- 0.4, and 1.9 +/- 0.5 nM, respectively. Maxadilan proved to be at least sevenfold more potent than nitroglycerin in each arterial bed (EC50 = 25 +/- 12, 32 +/- 9, 37 +/- 10, and 22 +/- 13 nM, respectively; P < 0.05 for each vs. maxadilan). Arterial relaxation to maxadilan was independent of endothelium and was equipotent in the thoracic and abdominal aorta, carotid artery, and iliac artery. Arterial relaxation to maxadilan was not inhibited by K(+)-channel antagonists, methylene blue, quinacrine, or ouabain. Maxadilan-mediated arterial relaxation was found to be adenosine 3',5'-cyclic monophosphate (cAMP) dependent, as it was potentiated by the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine and theophylline, and it was inhibited by the protein kinase A inhibitor H-89. Consistent with this observation, incubation of thoracic aorta with maxadilan (0.1 microM) produced a time-dependent increase in arterial cAMP content coincident with arterial relaxation. Using rabbit aortic smooth muscle cells, we also observed a time-dependent reduction in intracellular calcium in response to maxadilan. Thus these data indicate that maxadilan, a peptide from the sand fly salivary gland, is a potent vasodilator that reduces intracellular calcium through a cAMP-dependent mechanism.

Effect of elevated glucose on cyclic GMP and eicosanoids produced by porcine aortic endothelium.

The short-term effects of elevated glucose on cyclic GMP (cGMP) and eicosanoid production in pig aortic endothelial cell monolayers was determined by incubating cells in 5.5 mM or 44 mM glucose for 6 hours. Bradykinin- or A23187-stimulated cGMP production was significantly reduced in cells incubated in 44 mM glucose compared with 5.5 mM glucose. Stimulation of cGMP levels with exogenously added nitric oxide (NO) was also decreased to a similar extent in cells exposed to 44 mM glucose. These data suggest that NO production stimulated by bradykinin or A23187 was unchanged by elevated glucose. Assayed eicosanoids, including 6-ketoprostaglandin (PG) F1 alpha, PGE2 alpha, and 15(S)-hydroxy-(5Z, 8Z, 11Z, 13E)-eicosatetraenoic acid, stimulated by bradykinin or A23187, were increased in cells exposed to 44 mM glucose. These eicosanoid products formed from exogenously added arachidonic acid did not differ between cells incubated in 5.5 mM or 44 mM glucose. Hyperosmolar concentrations of mannose or sucrose had no effect on cGMP levels but did mimic the effect of elevated glucose on eicosanoid production. These data suggest that hyperglycemia in diabetes may interfere with NO-induced guanylate cyclase activation but not NO production in the endothelium and that increased phospholipase activity, secondary to hyperosmolarity, may account for elevated eicosanoid levels.

Aldose reductase inhibition restores endothelial cell function in diabetic rabbit aorta.

A possible relationship between increased aldose reductase activity and abnormal endothelium-dependent relaxation was examined in aorta from alloxan-induced diabetic rabbits. Isolated aorta of diabetic rabbits, contracted submaximally with phenylephrine, showed significantly decreased endothelium-dependent relaxations induced by acetylcholine or adenosine diphosphate compared to those from normal rabbits. Basal and acetylcholine-stimulated levels of cyclic GMP and the relaxations in response to an endothelium-independent vasodilator, sodium nitroprusside, were not significantly different between diabetic and normal rabbits, indicating that nitric oxide release and action on the vascular smooth muscle were unchanged. The release of thromboxane A2 from diabetic vessels was increased, as previously demonstrated. Treatment with an aldose reductase inhibitor, zopolrestat, normalized the elevated red blood cell sorbitol levels in diabetic rabbits. Zopolrestat also restored the abnormal acetylcholine- and adenosine diphosphate-induced relaxations of the aorta. The aldose reductase inhibitor had no effect on the levels of cyclic GMP or on the increased release of thromboxane A2 in diabetic aorta. These findings suggest that increased activity of the aldose reductase pathway in hyperglycemia is responsible for the abnormal endothelium-dependent relaxation in diabetic blood vessels. Significant alterations in endothelial production of neither nitric oxide nor vasoconstrictor prostanoids could be directly implicated in the improvement caused by the drug, suggesting another mechanism of action.

Cyclic GMP modulators on vascular adrenergic neurotransmission.

The presence of the endothelium reduced the sensitivity of isolated rabbit carotid artery to endogenous norepinephrine released by electrical stimulation of adrenergic nerves or displaced by tyramine and to exogenously applied norepinephrine, phenylephrine and UK 14304. The maximal contractions induced by the selective alpha 2-agonist UK 14304 were much more profoundly depressed in arteries with endothelium than those induced by the nonselective alpha-adrenoceptor agonist norepinephrine or by the selective alpha 1-agonist. LY 83583, a cyclic-guanosine-monophosphate (GMP)-lowering agent, abolished the endothelium-dependent depression of tone induced by the agonists and converted the sensitivity of arteries with endothelium to that of endothelium-denuded preparations. M & B 22948, a selective cyclic GMP phosphodiesterase inhibitor, significantly inhibited contractions caused by electrical stimulation of adrenergic nerves, tyramine, norepinephrine and UK 14304 in rings with, but not in those without, endothelium. Yohimbine, an alpha 2-adrenoceptor antagonist, increased contractions caused by UK 14304 in rings with endothelium only, but had no significant effect on the contractions caused by exogenously applied norepinephrine or phenylephrine. In the presence of prazosin, an alpha 1-blocker, UK 14304 caused minimal relaxation (about 20%) in rings with endothelium only which were inhibited by yohimbine, suggesting a minor role of direct endothelial cell alpha 2-mediated release of relaxing factors. The over-flow of endogenous norepinephrine caused by electrical stimulation was not affected by treatment with LY 83583 or M & B 22948, suggesting that altering cyclic GMP levels has no major role in prejunctional modulation of norepinephrine release. These findings support the notion that intrinsic levels of cyclic GMP may act as a regulator of adrenergic neurotransmission due primarily to endothelium-derived relaxing factor which is released basally, and to a lesser extent by an activation of endothelial cell alpha 2-adrenoceptors.

Adrenergic denervation in rabbits with diabetes mellitus.

The influence of alloxan-induced diabetes mellitus on the sympathetic neuroeffector junction of the rabbit carotid artery denuded of endothelium was studied. Six weeks of diabetes resulted in a neuropathy characterized by a 38% reduction in the arterial content of norepinephrine. Norepinephrine release from the nerves measured from electrically stimulated superfused arterial segments was decreased. The cocaine-sensitive accumulation of [3H]-norepinephrine (NE) was also reduced, reflecting decreased neuronal uptake. The consequences of these prejunctional changes were studied by measuring isometric contractions of arterial rings caused by electrical nerve stimulation or by exogenous norepinephrine. Despite the reduced release of norepinephrine, neurogenic contractions were normal, suggesting an increased sensitivity of the smooth muscle. After neuronal uptake was blocked, the neurogenic contractions of diabetic arteries were less than normal, reflecting the reduction in transmitter release. The sensitivity of diabetic arteries to exogenous norepinephrine was increased under control conditions; maximal contractions were unchanged. Blockade of norepinephrine uptake increased norepinephrine sensitivity more in normal than in diabetic arteries, and there was no longer a significant difference in sensitivity. Thus, under control conditions, neurogenic contractions of the partially denervated diabetic rabbit carotid artery are paradoxically normalized by increased alpha-adrenergic sensitivity of the smooth muscle. The increased sensitivity caused by reduced neuronal uptake can thus preserve neurogenic vasoconstriction and cause supersensitivity to exogenous catecholamines in the sympathetic neuropathy caused by diabetes mellitus.

The endothelium inhibits activation by calcium of vascular neurotransmission.

The role of calcium in the inhibition by the endothelium of adrenergic neurotransmission was studied in isolated rabbit carotid artery. Contractions induced by transmural electrical field stimulation (0.5-8 Hz), norepinephrine (10(-8)-3 X 10(-5) M), potassium depolarization (15-30 mM), or by readdition of calcium (0.15-2.4 mM) to a calcium-free medium containing potassium (15 mM) were significantly smaller in rings with compared with rings without endothelium. The voltage-dependent calcium channel activator, BAY K 8644 (10(-6) M), increased contractions to all contractile stimuli in rings with more than in rings without endothelium and thereby abolished the inhibitory influence of the endothelium. The inhibition of neurogenic contractions by the endothelium was also, in part, prejunctional, as indicated by decreased overflow of endogenous norepinephrine from superfused segments with compared with segments without endothelium evoked by electrical stimulation (2 Hz) or by reinfusion of calcium (2.5 mM) to calcium-free medium containing potassium (80 mM). BAY K 8644 (10(-6) M) enhanced the overflow of norepinephrine evoked by electrical stimulation or calcium from segments with more than from segments without endothelium and abolished the difference. Thus the endothelium inhibits activation by extracellular calcium of adrenergic nerves and vascular smooth muscle. The action of the endothelium is overcome by BAY K 8644, suggesting that voltage-dependent calcium channels are important in the inhibitory role of the endothelium in both adrenergic nerves and smooth muscle cells.

Augmented adrenergic contractions of carotid arteries from cholesterol-fed rabbits due to endothelial cell dysfunction.

Transmural electrical stimulation was used to elicit frequency-dependent adrenergic neurogenic contractions in isolated carotid arteries from cholesterol-fed and control rabbits. In rings with endothelium, responses to adrenergic nerve stimulation were significantly greater in arteries from cholesterol-fed as compared with those from control rabbits. Responses to adrenergic nerve stimulation of rings without endothelium were not different between the two groups. Methylene blue, a guanylate cyclase inhibitor, increased contractions of rings with endothelium and abolished the difference between the responses of arteries from cholesterol-fed and control rabbits. Methylene blue had no significant effect on arteries without endothelium. The overflow of endogenous norepinephrine (NE) caused by transmural electrical stimulation was not different between segments of arteries from cholesterol-fed and control rabbits. In control rabbits, exogenously applied NE contracted arteries with endothelium less than arteries without endothelium, whereas in cholesterol-fed rabbits the contractions caused by NE were not different between arteries with and without endothelium. Acetylcholine-induced relaxations were not different between rings with endothelium from cholesterol-fed and control rabbits. These results suggest that hypercholesterolemia selectively impairs the inhibitory influence of the endothelium on adrenergic contractions.

Endothelium inhibits norepinephrine release from adrenergic nerves of rabbit carotid artery.

The overflow of endogenous norepinephrine caused by transmural electrical stimulation or depolarization with potassium was smaller in superfused segments of the rabbit carotid artery with intact endothelium than in segments denuded of endothelium. In segments preincubated with [3H]norepinephrine, the lesser overflow was found to be partially due to metabolism by the endothelium of the neurotransmitter. Even after treatment to block the disposition of norepinephrine, the endothelium acted as a partial physical barrier to the overflow of norepinephrine into the lumen of arteries superfused and perfused selectively. However, a lesser overflow of norepinephrine to the adventitia of the artery accounted for the majority of the difference in overflow between segments with and without endothelium. The inhibition by the endothelium of the overflow of norepinephrine from adrenergic nerves was unaffected by blocking prejunctional alpha 2-adrenoceptors, prostaglandin synthesis, free radicals, or guanylate cyclase. Vasodilators released from the endothelium of a donor artery inhibited contractions caused by adrenergic nerve stimulation of a bioassay artery but failed to inhibit norepinephrine release. These observations indicate that the endothelium 1) metabolizes norepinephrine, 2) acts as a physical barrier to its overflow into the blood vessel lumen, and 3) inhibits the release of the adrenergic transmitter from adrenergic nerves.

Influence of the endothelium on tone and the response of isolated pig coronary artery to norepinephrine.

The influence of the endothelium on smooth muscle tone and the response of the pig right coronary artery to norepinephrine (NE) was studied. Isolated rings of artery with and without endothelium were stretched in the presence of nitroprusside to a tension previously determined to be optimal for contraction. During wash out of the nitroprusside, rings without endothelium spontaneously generated tone representing 24% of the contraction caused by potassium (120 mM); in rings with endothelium no significant spontaneous tone was observed. Relaxations were caused by NE in rings with endothelium contracted with prostaglandin F2 alpha (PGF2 alpha). In rings without endothelium, NE relaxed spontaneously generated tone as well as that produced by PGF2 alpha. Independent of the mode or degree of contraction, rings with endothelium were more sensitive to NE than rings without endothelium. The difference in sensitivity to NE between rings with and without endothelium was likely due to endothelial cell alpha-2 adrenoceptors, inasmuch as the difference was abolished by rauwolscine. In the presence of propranolol and prazosin, endothelium-dependent relaxations were observed which were also inhibited by rauwolscine. Nevertheless, beta adrenoceptors are the predominant mediator of the relaxation to NE of pig coronary smooth muscle, because propranolol caused a greater shift to the right of the relaxation induced by NE compared to that caused by endothelium removal. Accordingly, under resting conditions, NE caused contractions only in the presence of propranolol. These contractions were attenuated by prazosin or rauwolscine, but blocked only by a combination of both alpha adrenoceptor antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of 5-hydroxytryptamine leads to dysfunction of adrenergic nerves in canine coronary artery following intimal damage in vivo.

Previous in vitro studies have demonstrated that coronary artery adrenergic nerves are a principal site of accumulation of 5-hydroxytryptamine released from aggregating platelets. The purpose of this study was to determine whether 5-hydroxytryptamine is accumulated by adrenergic nerves at sites of endothelial damage and platelet aggregation in vivo. Coronary artery 5-hydroxytryptamine content and response to in vitro adrenergic nerve stimulation were studied in dogs 24 hours following balloon catheter-induced intimal injury. 5-Hydroxytryptamine content was significantly increased in the catheter-damaged arteries, and there was a coincident decrease in the content of norepinephrine. The relaxation caused by acetylcholine was abolished in the catheter-injured arteries, indicating loss of this endothelial cell-mediated function. The normal beta-adrenergic relaxation caused by nerve stimulation was inhibited, and in some cases, contractions resulted; these effects were prevented by serotonergic receptor antagonists. The sensitivity to exogenously added norepinephrine was unchanged, indicating that the changes in the response to nerve stimulation were not due to an altered smooth muscle response to the native neurotransmitter. These observations indicate that following intimal damage, which produces platelet aggregation on the luminal surface of the blood vessel, 5-hydroxytryptamine can assume a transmitter role in coronary artery adrenergic nerves and thereby cause their dysfunction.

Endothelium inhibits responses of rabbit carotid artery to adrenergic nerve stimulation.

Transmural electrical stimulation of isolated ring segments of the rabbit carotid artery caused frequency-dependent contractions; these were blocked by tetrodotoxin or prazosin. Mechanical or chemical removal of the endothelium markedly augmented responses to electrical stimulation. Inhibition of norepinephrine uptake and metabolism with cocaine, hydrocortisone, and pargyline increased contractions in rings with endothelium more than those without endothelium, but responses remained greater in rings denuded of endothelium. Methylene blue, an inhibitor of guanylate cyclase, enhanced responses to electrical stimulation of rings with intact endothelium only. Combined inhibition of guanylate cyclase and norepinephrine disposition increased the contractions and abolished the difference between the responses of rings with and without endothelium. In a perfusion-cascade system, the perfusate of donor segments with endothelium relaxed a bioassay ring without endothelium. Electrical stimulation of the segment caused no further relaxation of the bioassay ring. However, contractions caused by electrically stimulating the bioassay ring were depressed during superfusion with the perfusate of segments with, but not without, endothelium, indicating that vasodilators spontaneously released from the endothelium inhibit responses to nerve stimulation. These observations suggest that inhibition by the endothelium of the response to adrenergic nerve stimulation results from 1) spontaneous release of endothelium-derived vasodilators and 2) disposition of norepinephrine by the endothelial cells.