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Because macrophages play a major role in atherosclerotic plaque destabilization, selective removal of macrophages represents a promising approach to stabilize plaques. We showed recently that the protein synthesis inhibitor cycloheximide, in contrast to puromycin, selectively depleted macrophages in rabbit atherosclerotic plaques without affecting smooth muscle cells (SMCs). The mechanism of action of these two translation inhibitors is dissimilar and could account for the differential effects on SMC viability. It is not known whether selective depletion of macrophages is confined to cycloheximide or whether it can also be achieved with translation inhibitors that have a similar mechanism of action. Therefore, in the present study, we investigated the effect of anisomycin, a translation inhibitor with a mechanism of action similar to cycloheximide, on macrophage and SMC viability. In vitro, anisomycin induced apoptosis of macrophages in a concentration-dependent manner, whereas SMCs were only affected at higher concentrations. In vivo, anisomycin selectively decreased the macrophage content of rabbit atherosclerotic plaques through apoptosis. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole] prevented anisomycin-induced macrophage death, without affecting SMC viability. SB202190 decreased anisomycin-induced p38 MAPK phosphorylation, did not alter c-Jun NH(2)-terminal kinase (JNK) phosphorylation, and increased extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. The latter effect was abolished by the mitogen-activated protein kinase kinase 1/2 inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene ethanolate], although the prevention of anisomycin-induced macrophage death by SB202190 remained unchanged. The JNK phosphorylation inhibitor SP600125 did not affect anisomycin-induced macrophage or SMC death. In conclusion, anisomycin selectively decreased the macrophage content in rabbit atherosclerotic plaques, indicating that this effect is not confined to cycloheximide. p38 MAPK, but not ERK1/2 or JNK, plays a major role in anisomycin-induced macrophage death.

Recent evidence indicates that the protein synthesis inhibitor cycloheximide triggers selective macrophage death in rabbit atheroma-like lesions without affecting smooth muscle cells (SMCs) or the endothelium, thereby favoring a stable plaque phenotype. In this study, we report that puromycin, a protein synthesis inhibitor with a different mode of action but with similar ability to inhibit de novo protein synthesis, did not reveal plaque-stabilizing effects. The macrophage and the SMC content readily decreased in puromycin-treated atheroma-like lesions in rabbit carotid arteries. Moreover, puromycin induced apoptosis in macrophages and SMCs in vitro. Puromycin-treated SMCs showed signs of endoplasmic reticulum (ER) stress, as demonstrated by CCAAT/enhancer-binding protein homologous protein (CHOP) protein expression, splicing of X-box-binding protein 1 mRNA, and phosphorylation of eukaryotic translation initiation factor 2alpha. The ER stress inducer thapsigargin up-regulated CHOP protein expression in SMCs without affecting their viability, indicating that ER stress not necessarily results in cell death. Puromycin, but not thapsigargin, activated the ER stress-related caspase-12. Treatment of SMCs with a combination of cycloheximide and puromycin inhibited ER stress and partially improved SMC viability. In addition, puromycin, but not cycloheximide or thapsigargin, induced intracellular accumulation of polyubiquitinated proteins in SMCs, whereas the proteasome function was not affected. Taken together, puromycin, in contrast to cycloheximide, induces SMC apoptosis, thereby favoring an unstable plaque phenotype. SMC death upon puromycin treatment could only be partially prevented by cycloheximide, which completely blocked ER stress. However, other or additional mechanisms, such as increased polyubiquitination of proteins, might be involved in puromycin-induced SMC death.

Macrophages are an essential component of unstable atherosclerotic plaques and play a pivotal role in the destabilization process. We have demonstrated previously that local delivery of the mammalian target of rapamycin (mTOR) inhibitor everolimus selectively clears macrophages in rabbit plaques. Because mTOR controls mRNA translation, inhibition of protein synthesis might induce selective macrophage cell death. We therefore investigated in the present study the effect of the protein synthesis inhibitor cycloheximide on macrophage and smooth muscle cell (SMC) viability. In vitro studies with cultured macrophages and SMCs showed that cycloheximide induced selective apoptosis of macrophages in a concentration- and time-dependent manner. Moreover, macrophages could be selectively depleted in rabbit carotid artery rings with collar-induced atherosclerotic plaques after in vitro treatment with cycloheximide. Local in vivo administration of cycloheximide via osmotic minipumps to rabbit carotid arteries with collar-induced atherosclerotic plaques significantly reduced the macrophage but not the SMC content. Cycloheximide-treated plaques showed signs of apoptosis (increased terminal deoxynucleotidyl transferase end labeling and fluorescein isothiocyanate-Val-Ala-dl-Asp(O-methyl)-fluoromethylketone labeling) that did not colocalize with SMCs. Organ chamber studies demonstrated that the functionality of SMCs and the endothelium were not influenced by cycloheximide treatment. All together, these findings demonstrate that cycloheximide decreases the macrophage load in atherosclerotic plaques by induction of apoptosis without changing SMC content or contractility.

Several lines of evidence suggest that macrophages play a key role in atherosclerotic plaque destabilization and rupture. Therefore, selective removal of macrophages from plaques via pharmacological therapy could represent a promising approach to stabilize "vulnerable," rupture-prone lesions. Yet, how macrophages can be eliminated from plaques without influencing other cell types, including smooth muscle cells (SMCs), is unknown. In the present study, we report that benzyloxycarbonyl-Val-Ala-DL-Asp(O-methyl)-fluoromethylketone (z-VAD-fmk), a caspase inhibitor with broad specificity, induces nonapoptotic cell death of J774A.1 and RAW264.7 macrophages but not of SMCs. Cell death was characterized by bulk degradation of long-lived proteins, processing of microtubule-associated protein light chain 3, and cytoplasmic vacuolization, which are all markers of autophagy. However, necrosis also occurred, and the number of necrotic cells rapidly increased during z-VAD-fmk treatment. Primary mouse peritoneal macrophages were resistant to z-VAD-fmk-mediated cell death, but unlike SMCs, they underwent z-VAD-fmk-mediated necrosis after pretreatment with interferon-gamma. Further evidence indicated that the expression level of receptor-interacting protein 1 (RIP1) mediates the sensitivity to z-VAD-fmk. Importantly, upon z-VAD-fmk treatment, J774A.1 macrophages overexpressed and secreted several chemokines and cytokines, including tumor necrosis factor (TNF) alpha. The combination of z-VAD-fmk and TNFalpha, but not TNFalpha alone, induced SMCs necrosis via a mechanism that required RIP1 expression. These results suggest that z-VAD-fmk, despite its selective cell death inducing capacity, would be detrimental for the stability of atherosclerotic plaques due to enlargement of the necrotic core, stimulation of inflammatory responses, and indirect induction of SMC death.

Gastrointestinal (GI) smooth muscle responses to stimulation of the nonadrenergic noncholinergic inhibitory nerves have been suggested to be mediated by polypeptides, ATP, or another unidentified neurotransmitter. The discovery of nitric-oxide (NO) synthase inhibitors greatly contributed to our understanding of mechanisms involved in these responses, leading to the novel hypothesis that NO, an inorganic, gaseous molecule, acts as an inhibitory neurotransmitter. The nerves whose transmitter function depends on the NO release are called "nitrergic", and such nerves are recognized to play major roles in the control of smooth muscle tone and motility and of fluid secretion in the GI tract. Endothelium-derived relaxing factor, discovered by Furchgott and Zawadzki, has been identified to be NO that is biosynthesized from l-arginine by the constitutive NO synthase in endothelial cells and neurons. NO as a mediator or transmitter activates soluble guanylyl cyclase and produces cyclic GMP in smooth muscle cells, resulting in relaxation of the vasculature. On the other hand, NO-induced GI smooth muscle relaxation is mediated, not only by cyclic GMP directly or indirectly via hyperpolarization, but also by cyclic GMP-independent mechanisms. Numerous cotransmitters and cross talk of autonomic efferent nerves make the neural control of GI functions complicated. However, the findingsrelated to the nitrergic innervation may provide us a new way of understanding GI tract physiology and pathophysiology and might result in the development of new therapies of GI diseases. This review article covers the discovery of nitrergic nerves, their functional roles, and pathological implications in the GI tract.

Although five muscarinic receptor subtypes have now been cloned, only three receptor subtypes have been demonstrated pharmacologically in bronchial tissue (designated M1, M2 and M3). By using drugs that show selectivity for these receptor subtypes, in combination with a sensitive and specific high-pressure liquid chromatography method that enables the direct measurement of acetylcholine (ACh) release, the existence and function of these muscarinic receptor subtypes was investigated in rabbit trachea in vitro. Atropine and ipratropium bromide, nonselective antimuscarinic agents, dose-dependently suppressed contraction of rabbit trachea induced by transmural electrical stimulation, but at the same time enhanced the release of ACh, suggesting the presence of an autoregulatory feed-back system. The M2/M4 receptor antagonists methoctramine, 11-((2-[(diethylamino)methyl]-1-piperidinyl)acetyl)-5, 11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one, 5,11-dihydro-11-([(2-(2-[(dipropylamino)methyl]-1-piperidinyl)ethyl) amino]carbonyl)-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one and 11-((4-[4-(diethylamino)butyl]-1-piperidinyl)acetyl)-5, 11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one also dose-dependently increased ACh release during electrical stimulation, indicating that the powerful negative feedback system is mediated by presynaptic autoreceptors of the M2 or M4 type.(ABSTRACT TRUNCATED AT 250 WORDS)

The possible role of nitric oxide (NO) as inhibitory nonadrenergic noncholinergic (NANC) neurotransmitter was studied in the rat gastric fundus. NO induced tetrodotoxin-resistant NANC relaxations in longitudinal muscle strips similar to those induced by electrical stimulation. Incubation with the stereospecific inhibitors of the NO biosynthesis NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (L-NNA) resulted in an increase of the basal tension which was reversed partly by L-arginine, but not by D-arginine. L-NMMA and L-NNA inhibited the relaxations to electrical stimulation, but not those induced by ATP, vasoactive intestinal polypeptide (VIP), norepinephrine or NO. This inhibitory effect was prevented by L-arginine, but not by D-arginine. In a second series of experiments, the gastric fundus served as donor tissue in a superfusion bioassay with de-endothelialized rings of rabbit aorta as detector tissue. The fundus released a labile factor with vasodilator activity upon electrical stimulation. This release was inhibited by tetrodotoxin and L-NNA, whereas it was increased by L-arginine. The biological activity was enhanced by superoxide dismutase and eliminated by hemoglobin. Our results indicate that NO is formed and released upon brief stimulation of the NANC nerves in the rat gastric fundus which is essential for the transient relaxations in this preparation. Therefore, we suggest NO or a NO releasing substance as inhibitory NANC transmitter in the rat gastric fundus.

The 15-hydroperoxy metabolite of arachidonic acid (15-hydroperoxyeicosatetraenoic acid; 15-HPETE) and its hydroxyderivative 15-hydroxyeicosatetraenoic acid (15-HETE) are known to evoke contractions in a variety of isolated blood vessels. In this study, segments of isolated canine coronary, splenic, femoral and renal arteries were exposed to 15-HETE and 15-HPETE; both metabolites induced small basal relaxations followed by contractions at higher concentrations. The contractions were augmented by indomethacin and could be blocked by the thromboxane A2 receptor antagonists BM13177 and BM13505. In vessels in which the tone was raised with prostaglandin F2 alpha, both 15-lipoxygenase metabolites evoked marked relaxations, which were in part dependent on the presence of the endothelium. When the segments were contracted with norepinephrine or increased KCl concentration, 15-HETE and 15-HPETE induced relaxations followed by additional contractions. The relaxations to the fatty acid derivatives were not inhibited by BM13505. In tissues without endothelium, the relaxations to 15-HETE and 15-HPETE were completely blocked by indomethacin; in tissues with endothelium, indomethacin only partly inhibited the relaxations to 15-HETE, whereas the drug did not interfere with the relaxing effects of 15-HPETE. Our experiments indicate that in isolated canine arteries 15-lipoxygenase metabolites of arachidonic acid can 1) induce contractions, most likely by direct activation of thromboxane A2 receptors on smooth muscle cells, and 2) evoke relaxations that are in part endothelium dependent; the endothelium-independent part of the relaxations was inhibited by indomethacin. Thus, the relaxations to these metabolites seem to occur via the release of an endothelium-derived relaxing factor and via production of a cyclooxygenase metabolite.

The effects of 5-hydroxytryptamine (5-HT), the 5-HT1-like receptor agonist 5-carboxamidotryptamine and the 5-HT3 receptor agonist 2-methyl-5-hydroxytryptamine were studied on circular muscle strips of the canine terminal ileum and ileocolonic junction. Serial administration of 5-HT or of 5-carboxamidotryptamine induced slow tonic contractions that at higher concentrations of 5-HT (10(-4)-3 x 10(-4] were preceded by an initial relaxation and a fast phasic contraction. The concentration-response curves to both agonists were competitively shifted to the right by the mixed 5-HT1/5-HT2 receptor antagonist methysergide. The initial relaxation and fast phasic contraction were inhibited by the 5-HT3 receptor antagonist ICS 205-930 and tetrodotoxin. Atropine blocked the fast phasic contraction, but enhanced the relaxation. During acetylcholine-induced contractions, 5-HT and 2-methyl-5-hydroxytryptamine (greater than or equal to 10(-5) M), but not 5-carboxamidotryptamine, evoked relaxations that were blocked by ICS 205-930 and tetrodotoxin, but not by adrenoceptor antagonists. Thus, in the canine terminal ileum and ileocolonic junction, 5-HT stimulates neuronal 5-HT3 receptors and excitatory 5-HT1-like receptors located on smooth muscle. Stimulation of the 5-HT3 receptors results in an acetylcholine-mediated contraction and a relaxation mediated by an as yet unknown nonadrenergic noncholinergic neurotransmitter.

The nonadrenergic noncholinergic (NANC) relaxations in response to electrical stimulation and acetylcholine in the canine terminal ileum and ileocolonic junction were further characterized and the possible involvement of the putative NANC neurotransmitter ATP was investigated. During a norepinephrine-induced contraction, noncumulative administration of ATP and the nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP) induced concentration-dependent relaxations in both the terminal ileum and ileocolonic junction; these responses were not inhibited by atropine, timolol or guanethidine. Desensitization to ATP blocked the ATP-induced relaxations, but not those to electrical stimulation or acetylcholine. All relaxations were abolished by tetrodotoxin. Theophylline or dipyridamole had no effect. Hexamethonium and desensitization to DMPP blocked the relaxations to acetylcholine and DMPP, but not those to electrical stimulation or ATP. The relaxations to acetylcholine, ATP and DMPP were inhibited by lidocaine. Thus, in the canine terminal ileum and ileocolonic junction, ATP induces NANC relaxations of neuronal origin, but ATP is unlikely to be the final NANC neurotransmitter mediating the relaxations to electrical stimulation or acetylcholine. These data also extend our previous observations on the presence of an inhibitory nicotinic innervation in these tissues.

A comparative study of cholinergic and adrenergic mechanisms was performed on isolated circular muscle strips of the canine ileum, ileocolonic junction and colon. Cholinergic agonists evoked atropine sensitive contractions, which were most prominent in the colon. The presence of neostigmine to inhibit acetylcholinesterase did not alter the relative sensitivities of the tissues to acetylcholine. The different maximal contraction to cholinergic agents discriminates the colon from the ileum and the ileocolonic junction. Alpha adrenoceptor agonists produced greater contractions in the ileocolonic junction and only marginal contractions in the colon. Phentolamine completely and prazosin and yohimbine partially blocked the response of the ileocolonic junction to norepinephrine, suggesting the involvement of both alpha-1 and alpha-2 adrenoceptors. As in other sphincteric gut smooth muscle, the contractile effects of alpha adrenergic agents are more important than the inhibitory ones. The different effects of cholinergic and adrenergic agents on the ileocolonic junction, as compared sphincteric properties in the canine ileocolonic junction. sphincteric properties in the canine ileocolonic junction.

The effects of tertatolol, a new and powerful beta adrenoceptor blocking drug, on post- and prejunctional beta receptors were investigated; canine vascular tissues (saphenous veins, coronary arteries and splenic arteries) and guinea-pig trachea and atria were used. At concentrations below 10(-5) M, tertatolol did not alter basal tension or contractile responses to electrical stimulation, norepinephrine, K+ or prostaglandin F2 alpha; at doses at or above 10(-5) M the drug-evoked contractions which were reduced by phentolamine and were absent in denervated veins. Tertatolol at 10(-5) M and 3 X 10(-5) M augmented the basal efflux of [3H] norepinephrine in saphenous veins labeled with the 3H-transmitter. In veins, 10(-5) M of tertatolol depressed the contractions caused by electrical stimulation without affecting those to exogenous norepinephrine; this concentration of the drug also inhibited the stimulation-induced overflow of [3H]norepinephrine. The major part of the present study was designed to test the beta receptor blocking properties of tertatolol and to compare its effects with those of propranolol. Tertatolol inhibited, in a concentration-dependent manner, the relaxations caused by isoproterenol in saphenous veins, splenic arteries and coronary arteries and the relaxations evoked by norepinephrine and epinephrine in coronary arteries; the potency of tertatolol was higher than that of propranolol. In trachea and right atria of the guinea-pig, tertatolol inhibited, in a concentration-dependent manner, the dose-response curves to isoproterenol; the relative potency of tertatolol was higher than that of propranolol. In dog saphenous veins, previously incubated with [3H]norepinephrine, tertatolol (10(-7)M) blocked the increased stimulation-evoked overflow of the 3H-transmitter induced by isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)

Experiments were performed to examine whether isolated canine blood vessels have the ability to accumulate [3H]-5-hydroxytryptamine ([3H]-5-HT) and if so, whether the 3H-amine can be released from the tissues by stimuli known to evoke release of norepinephrine. Helical strips of saphenous veins and cerebral arteries obtained from dogs were incubated with [3H]-5-HT and mounted for superfusion. Both blood vessels take up [3H]-5-HT in a concentration-dependent manner; this accumulation is markedly reduced in veins and arteries denervated with 6-hydroxydopamine and in veins pretreated with cocaine. In saphenous veins and cerebral arteries labeled with [3H]-5-HT, a basal efflux of 3H, consisting mainly of [3H]-5-hydroxyindole-acetic acid, was observed. Electrical stimulation of the preparations evoked a frequency-dependent overflow of tritium due in part to an overflow of intact [3H]-5-HT. This stimulation-induced 3H-overflow was inhibited by tetrodotoxin and was nearly absent in denervated tissues and in veins pretreated with cocaine. In saphenous veins the overflow of 3H evoked by electrical stimulation was markedly augmented by phentolamine but not influenced by methiothepin. In the veins, both K+ and tyramine caused an overflow of [3H]-5-HT which was absent after denervation or after pretreatment of the preparations with cocaine. Our results show that the major part of the [3H]-5-HT which is taken up by the isolated blood vessels accumulates in the adrenergic nerves and that the indoleamine can be released from these nerves as a "false" transmitter, together with norepinephrine.

Serotonin can accumulate in the adrenergic nerves of vascular tissues. We have determined whether in the isolated perfused dog saphenous vein 1) luminal administration of serotonin can result in its accumulation in the adrenergic nerves and 2) endothelium can interfere with the transport of the amine into the vessel wall. Saphenous veins were perfused with medium containing [3H]serotonin, [3H]norepinephrine or [3H]epinephrine; after washout, significant amounts of 3H were detected in the veins. The 3H-accumulation was augmented when the endothelium was removed mechanically; the augmented accumulation was only observed when the [3H]amines reached the tissues from the intimal side. In coronary arteries perfused with [3H]serotonin, similar results were obtained. No increased 3H-accumulation was noted in veins without endothelium perfused in the presence of cocaine. Nerve stimulation of veins labeled with [3H]serotonin caused an augmented release of 3H from the tissues without endothelium. Pargyline augmented the accumulation of [3H]serotonin and [3H]norepinephrine and decreased the difference between tissues with or without endothelium only for norepinephrine. Perfusion of venous segments with platelets, labeled with [3H]serotonin, resulted in a 3H-content which was significantly higher in the veins without endothelium. Our experiments show that serotonin and other amines, applied luminally to perfused blood vessels, can accumulate in the adrenergic nerves and that the endothelium can reduce this accumulation. Serotonin, originating from aggregating platelets, can penetrate the vessel wall much easier at sites of endothelial denudation and this serotonin also can enter the adrenergic nerves.

The activity of the beta adrenoceptor antagonist tertatolol on renal vasoconstrictions was investigated. Infusion of increasing concentrations of tertatolol (10(-8) to 10(-5) M) progressively inhibited the constrictor responses to bolus injections of norepinephrine and to electrical stimulation in isolated perfused kidneys of both normotensive and spontaneously hypertensive rats. Also, in kidneys of normotensive rats the vasoconstrictions caused by serotonin and barium chloride were inhibited by tertatolol. During sustained vasoconstrictions induced by infusion of norepinephrine (6 X 10(-7) M) increasing doses of tertatolol (2.5 X 10(-7) g to 2 X 10(-5) g) caused rapid, reversible dilatations in the rat kidneys. The inhibitory responses caused by tertatolol were not antagonized by propranolol, atropine, hexamethonium, SCH23390, metoclopramide, mepyramine, cimetidine, naloxone, cocaine or indomethacin. During constrictions caused by norepinephrine, methylene blue significantly inhibited the renal vasodilatations caused by tertatolol, acetylcholine, papaverine and nitroglycerin but not those caused by atrial natriuretic factor. Unlike the other vasodilators, tertatolol did not inhibit the constrictions induced by prostaglandin F2 alpha (5 X 10(-6) M) in the rat kidneys. In canine renal arteries with endothelium, tertatolol (10(-9) to 10(-5) M) did not cause relaxations during contractions induced by norepinephrine, electrical stimulation or prostaglandin F2 alpha. Our data illustrate that tertatolol has potent vasodilator properties in the isolated perfused vasoconstricted rat kidney. The dilator response to the beta blocker cannot be inhibited by a variety of classical receptor blockers but ultimately seems to depend on the formation of cyclic GMP.