A Review of Mechano-Biochemical Models for Testing Composite Restorations.

Due to the severe mechano-biochemical conditions in the oral cavity, many dental restorations will degrade and eventually fail. For teeth restored with resin composite, the major modes of failure are secondary caries and fracture of the tooth or restoration. While clinical studies can answer some of the more practical questions, such as the rate of failure, fundamental understanding on the failure mechanism can be obtained from laboratory studies using simplified models more effectively. Reviewed in this article are the 4 main types of models used to study the degradation of resin-composite restorations, namely, animal, human in vivo or in situ, in vitro biofilm, and in vitro chemical models. The characteristics, advantages, and disadvantages of these models are discussed and compared. The tooth-restoration interface is widely considered the weakest link in a resin composite restoration. To account for the different types of degradation that can occur (i.e., demineralization, resin hydrolysis, and collagen degradation), enzymes such as esterase and collagenase found in the oral environment are used, in addition to acids, to form biochemical models to test resin-composite restorations in conjunction with mechanical loading. Furthermore, laboratory tests are usually performed in an accelerated manner to save time. It is argued that, for an accelerated multicomponent model to be representative and predictive in terms of both the mode and the speed of degradation, the individual components must be synchronized in their rates of action and be calibrated with clinical data. The process of calibrating the in vitro models against clinical data is briefly described. To achieve representative and predictive in vitro models, more comparative studies of in vivo and in vitro models are required to calibrate the laboratory studies.

Androgenic and estrogenic regulation of Atrogin-1, MuRF1 and myostatin expression in different muscle types of male mice.

PURPOSE: The molecular factors targeted by androgens and estrogens on muscle mass are not fully understood. The current study aimed to explore gene and protein expression of Atrogin-1, MuRF1, and myostatin in an androgen deprivation-induced muscle atrophy model. METHODS: We examined the effects of Orx either with or without testosterone (T) or estradiol (E2) administration on Atrogin-1 gene expression, and MuRF1 and myostatin gene and protein expression. Measurements were made in soleus (SOL), extensor digitorum longus (EDL) and levator ani/bulbocavernosus (LA/BC) of male C57BL/6 mice. RESULTS: Thirty days of Orx resulted in a reduction in weight gain and muscle mass. These effects were prevented by T. In LA/BC, Atrogin-1 and MuRF1 mRNA was increased throughout 30 days of Orx, which was fully reversed by T and partially by E2 administration. In EDL and SOL, a less pronounced upregulation of both genes was only detectable at the early stages of Orx. Myostatin mRNA levels were downregulated in LA/BC and upregulated in EDL following Orx. T, but not E2, reversed these effects. No changes in protein levels of MuRF1 and myostatin were found in EDL at any time point following Orx. CONCLUSIONS: The atrophy in SOL and EDL in response to androgen deprivation, and its restoration by T, is accompanied by only minimal changes in atrogenes and myostatin gene expression. The marked differences in muscle atrophy and atrogene and myostatin mRNA between LA/BC and the locomotor muscles suggest that the murine LA/BC is not an optimal model to study Orx-induced muscle atrophy.

Endothelium-dependent hyperpolarization in small gastric arteries.

OBJECTIVE: In many blood vessels, stimulation of the endothelium with various vasoactive substances induces, besides the nitric oxide (NO) and prostacyclin pathways, a third mechanism evoking dilatation. It is based on hyperpolarization of the vascular smooth muscle cell membrane. In the present study, we investigated the existence of endothelium-dependent hyperpolarization in small gastric arteries of the rat and explored its underlying mechanism. METHODS: Membrane potentials were recorded by conventional microelectrode techniques in isolated segments of small gastric arteries, the normalized diameter of which was determined from the passive wall tension-internal circumference characteristics as measured with a myograph. RESULTS: After blocking NO and prostaglandin synthesis, application of acetylcholine (3 x 10(-7) M) resulted in a membrane hyperpolarization in endothelium intact but not in endothelium-denuded arteries. This membrane potential change was increased by pre-exposure to a low concentration (30 microM) of Ba2+, which selectively inhibits inward rectifying potassium channels. Moreover, the acetylcholine-induced hyperpolarization was unaffected by additional pre-exposure to high concentrations (0.5 mM) of the Na/K-ATPase inhibitor ouabain, which by itself caused a secondary slow endothelium-independent hyperpolarization after an initial peak depolarization. CONCLUSIONS: We conclude that acetylcholine produces endothelium-dependent hyperpolarization in gastric small arteries, which does not rely on activation of smooth muscle cell inward rectifying K+ channels or Na/K pumps, and might prove to be another important regulator of gastric mucosal blood flow.

Regional differences in anandamide- and methanandamide-induced membrane potential changes in rat mesenteric arteries.

The possibility that anandamide is an endothelium-derived hyperpolarizing factor was explored in the rat mesenteric vasculature by use of conventional microelectrode techniques. In the main mesenteric artery, anandamide and its more stable analog methanandamide hardly caused a measurable change in membrane potential of the smooth muscle cells, which promptly hyperpolarized to EDHF liberated by acetylcholine. Inhibition of endogenous anandamide breakdown by phenylmethylsulfonyl fluoride did not increase membrane responses to acetylcholine. The CB(1) receptor antagonist SR141716 did not significantly influence EDHF-mediated hyperpolarization except at extremely high concentrations. Smooth muscle cells of third to fourth order branches of the mesenteric artery, which have a more negative resting membrane potential and show smaller responses to acetylcholine, hyperpolarized by about 6 mV to both anandamide and methanandamide, whereas another CB(1) receptor agonist, WIN 55,212-2, had no effect. Mechanical endothelium removal or pre-exposure to SR141716A did not affect anandamide- and methanandamide-induced hyperpolarizations. However, in the presence of capsazepine, a selective vanilloid receptor antagonist, these membrane potential changes were reversed to a small depolarization, whereas EDHF-induced hyperpolarizations were not affected. Pretreating small vessels with capsaicin, causing desensitization of vanilloid receptors and/or depletion of sensory neurotransmitter, completely blocked methanandamide-induced hyperpolarizations. These findings show that anandamide cannot be EDHF. In smooth muscle cells of small arteries, anandamide-induced changes in membrane potential are mediated by vanilloid receptors on capsaicin-sensitive sensory nerves. The different membrane response to the cannabinoids between the main mesenteric artery and its daughter branches might be explained by the different density of perivascular innervation.

Influence of cannabinoids on the delayed rectifier in freshly dissociated smooth muscle cells of the rat aorta.

The influence of the cannabinoids anandamide, methanandamide and WIN 55212-2 on the delayed rectifier K(+) current (I(K(V))) in rat arterial myocytes was investigated. Anandamide caused a concentration-dependent reduction of total peak and late K(+) current (I(K)). The maximal effect (about 50% inhibition of I(K)) was reached with 3 microM, and half-maximal current block was observed at 0.6 microM. Blockade was voltage-independent. Inhibition of I(K) by the cannabinoid was associated with a characteristic increase in the rate of current relaxation. Methanandamide (10 microM), a metabolically more stable analogue of anandamide, decreased I(K) with a similar time course. Current traces in the presence of the drug also showed an acceleration of inactivation. The presence of TEA did not impair the inhibition by anandamide or methanandamide, but inhibition was prevented by pre-exposure to 4-AP, showing that both cannabinoids inhibited I(K(V)) while having no influence on Ca(2+)-dependent K(+) current (I(K(Ca))). The CB(1) receptor antagonist SR141716A (10 microM) did not influence the action of anandamide or methanandamide. Arachidonic acid (1 microM) increased I(K) considerably. However, in the presence of TEA it caused a decrease of I(K(V)) with a characteristic increase in the rate of current relaxation. WIN 55212-2 (20 microM) caused similar inhibition of I(K). Internally applied anandamide (10 microM) or methanandamide (10 microM) was ineffective at influencing I(K). In the dialyzed cells, the additional external application of a cannabinoid promptly initiated inhibition. The results show that anandamide, methanandamide and WIN 55212-2 affect I(K(V)) in a cannabinoid receptor-independent way similar to that of arachidonic acid, which, unlike the cannabinoids, additionally increases a Ca(2+)-activated K(+) current. It is suggested that cannabinoids might bind to an external site on or near the K(v) channel of the vascular smooth muscle cells.

EDHF-mediated relaxation in rat gastric small arteries: influence of ouabain/Ba2+ and relation to potassium ions.

In several blood vessels, endothelium-dependent vasorelaxation is in part mediated by an endothelium-derived hyperpolarizing factor (EDHF), the nature of which is as yet unknown. Experiments were performed to investigate whether the recently raised hypothesis that EDHF might be identified as the potassium ion, released by activation of endothelial K(Ca) channels and inducing relaxation by stimulation of Na+/K+-pump and the inward rectifier K+ conductance, might be valid for small rat gastric arteries. EDHF-induced relaxation (assessed as the nitro-L-arginine/indomethacin resistant component of acetylcholine-induced relaxation), but not nitroprus-side-induced relaxation is strongly inhibited in the presence of ouabain (0.5 mM)/Ba2+ (30 microM), ouabain being responsible for the greater part of the inhibition. This inhibition is reversible. Application of increasing concentrations of K+ elicits transient relaxations in some preparations, but in a greater part of the preparations, no or only small relaxations. In membrane potential measurements, it was found that increasing concentrations of extracellular K+ consistently depolarized smooth muscle cells, whereas acetylcholine elicits hyperpolarization. The K(Ca) channel openers NS 1619 and 1-EBIO elicit relaxation effects that are not diminished after removal of the endothelium and are not inhibited by ouabain/Ba2+. It is concluded that EDHF-mediated relaxation is sensitive to inhibition by ouabain/Ba2+, but that the relation of this inhibitory influence to an action of K+ as EDHF is uncertain.

Influence of some phospholipase A2 and cytochrome P450 inhibitors on rat arterial smooth muscle K+ currents.

The hyperpolarizing factor that is liberated by vascular endothelial cells in response to various agonists, and known to induce relaxation by opening of smooth muscle K+ channels, has been suggested to be a product of cytochrome P450 dependent arachidonic acid metabolism. In this study, the direct influence of two phospholipase A2 inhibitors and of five structurally and mechanistically different cytochrome P450 inhibitors on K+ currents in freshly isolated vascular smooth muscle cells from the rat aorta was investigated. On stepping the cell membrane potential from -70 mV to a series of depolarized test potentials, a noisy outward current developed at test potentials > +10 mV, which showed no appreciable inactivation during the voltage pulse. It was largely abolished by 3 mM external tetraethylammonium chloride (TEA), suggesting that it predominantly consisted of current through large-conductance Ca(2+)-activated K+ channels. The phospholipase A2 inhibitor quinacrine considerably inhibited this TEA-sensitive current, while 4-bromophenacylbromide exerted no effect. The cytochrome P450 inhibitors proadifen and miconazole reversibly decreased the amplitude of I(K), while clotrimazole and 1-aminobenzotriazole had no effect. Conversely, 17-octadecynoic acid increased whole-cell I(K). These results show that some phospholipase A2 and cytochrome P450 inhibitors may interfere with K+ channel activation in the rat arterial smooth muscle cell. The relevance of these findings to studies on the involvement of cytochrome P450 dependent metabolism in the generation of the endothelium-derived hyperpolarizing factor in intact arteries is discussed.

Barium decreases endothelium-dependent smooth muscle responses to transient but not to more prolonged acetylcholine applications.

The influence of inhibiting the inward rectifier and Na/K pump on endothelium-dependent hyperpolarizations in smooth muscle cells of the mesenteric artery was investigated. Membrane potential was measured with microelectrodes, and the influence of low concentrations of Ba2+ (30 microM) and of high concentrations of ouabain (0.5 mM) on smooth muscle hyperpolarization elicited by prolonged or by transient exposure to acetylcholine (ACh, 3x10(-7) M) was assessed in the continuous presence of NG-nitro-L-arginine (100 microM) and indomethacin (50 microM). Pre-exposure to Ba2+ did not inhibit the magnitude of smooth muscle cell hyperpolarization induced by ACh superfusion, but significantly slowed its onset and time course. The membrane potential response to transient ACh applications, however, was impaired. After combined Ba2+ and ouabain pre-exposure, peak hyperpolarizations to ACh superfusion were somewhat decreased but not abolished. In addition, 4-5 mM increases of the extracellular K+ concentration consistently depolarized smooth muscle cells. These findings argue against the idea that smooth muscle inward rectifier K+ channels and Na/K pumping play a role in the ACh-induced endothelium-dependent hyperpolarization of this preparation. Moreover, the slowing of smooth muscle membrane hyperpolarization by Ba2+ is discussed in terms of the influence of this ion on the release of hyperpolarizing factor.

Mechanisms involved in the vasorelaxing influence of histamine on isolated human subcutaneous resistance arteries.

The effects of histamine were analysed on human subcutaneous small arteries. No effect was seen on non-precontracted preparations. After precontraction (norepinephrine 1 microM and K+ 30 mM) histamine potently relaxed the arteries (EC50 = 0.3 microM; max. effect = 95% relaxation). The histamine H1 receptor antagonist, pyrilamine (10 microM), had only a small, non-significant inhibitory influence on histamine-induced relaxation while the histamine H2 receptor antagonist, cimetidine (0.1 mM), had a significant inhibitory influence. Relaxation was completely blocked in the presence of both antagonists. Both 2-pyridylethylamine (histamine H1 receptor agonist) and dimaprit (histamine H2 receptor agonist) elicited relaxation. Removal of endothelium reduced the relaxation effects of histamine and 2-pyridylethylamine, but not of dimaprit. Inhibition of nitric oxide synthesis by nitro-L-arginine significantly inhibited histamine-induced relaxation and even more clearly the cimetidine-resistant component. We conclude that histamine potently relaxes human subcutaneous arterioles, and that most probably both muscular histamine H2 receptors and endothelial histamine H1 receptors, thus activating nitric oxide release, contribute to the relaxation.

Evidence against the involvement of cytochrome P450 metabolites in endothelium-dependent hyperpolarization of the rat main mesenteric artery.

1. The influence of different inhibitors of cytochrome P450 mono-oxygenase on the endothelium-dependent and -independent hyperpolarization in the isolated rat main mesenteric artery was investigated. 2. Application of acetylcholine (ACh; 1 microM) for 10 min evoked an endothelium-dependent peak hyperpolarization of about 18 mV followed by a partial recovery to a level 7 mV more negative than the resting value (-50.2 +/- 0.5 mV). 3. Proadifen (30 microM) completely and reversibly inhibited the ACh-induced hyperpolarization. Conversely, the imidazole antimycotics clotrimazole (30 microM) and miconazole (100 microM) had less effect on the peak endothelium-dependent hyperpolarization. The suicide substrate inhibitors 17-octadecynoic acid (17-ODYA; 5 microM) and 1-aminobenzotriazole (1-ABT; 2 mM) did not significantly influence endothelium-dependent hyperpolarization. 4. The endothelium-independent hyperpolarization (16 mV) evoked by leveromakalim (300 nM) was completely inhibited by proadifen as well as by clotrimazole and miconazole but was not affected by 17-ODYA or 1-ABT. 5. These results do not support the view that the ACh-induced endothelium-dependent hyperpolarization in the rat mesenteric artery is mediated by cytochrome P450 mono-oxygenase metabolites. Proadifen and imidazole antimycotics impair the activation of ATP-regulated K+ channels in mesenteric artery cells, rendering non-specific inhibition of smooth muscle K+ channel activation an alternative explanation for the inhibitory influence of some (but not all) P450 inhibitors on endothelium-dependent hyperpolarization in this preparation.

Influence of cytochrome P-450 inhibitors on endothelium-dependent nitro-L-arginine-resistant relaxation and cromakalim-induced relaxation in rat mesenteric arteries.

In several blood vessels, endothelium-dependent vasorelaxation is in part mediated by an endothelium-derived hyperpolarizing factor (EDHF), the nature of which is as yet unknown. However, some evidence suggests that EDHF might be a cytochrome P-450-dependent monooxygenase metabolite of arachidonic acid. By using isometric tension measurements on rat main mesenteric arteries, the influence of four structurally and mechanistically different cytochrome P-450 inhibitors (proadifen, miconazole, 1-amino-benzotriazole, and 17-octadecynoic acid) was investigated on relaxations elicited by EDHF, assessed as the nitro-L-arginine-resistant component of acetylcholine-induced relaxation, and on relaxations provoked by the endothelium-independent potassium channel opener cromakalim. Proadifen (30 microM) inhibited the EDHF- as well as the cromakalim-induced relaxation, but not that elicited by nitroprusside. Also miconazole (30 microM) inhibited both the EDHF and the cromakalim-induced relaxation. On the other hand, 17-octadecynoic acid (5 microM) had no influence, and 1-aminobenzotriazole (1 mM) even potentiated EDHF- and cromakalim-induced relaxations. We conclude that the EDHF, released from the rat mesenteric artery by acetylcholine, is unlikely to be a cytochrome P-450-dependent monooxygenase metabolite of arachidonic acid and that proadifen and miconazole interfere with the action of cromakalim.

The influence of pregnancy on endothelium-derived nitric oxide mediated relaxations in isolated human resistance vessels.

Pregnancy is associated with drastic hemodynamic adaptations, including a decrease in peripheral resistance. Vascular resistance is substantially influenced by endothelium-derived nitric oxide (NO). This study was designed to investigate whether pregnancy might influence endothelium-derived NO-mediated relaxations in human resistance arteries. Reactivity of isolated human subcutaneous arteries, dissected out of abdominal fat from women who underwent a laparotomy or cesarean section, was studied using a small vessel myograph. Addition of acetylcholine (1 nM-10 microM) or bradykinin (1 nM-10 microM) to precontracted preparations elicited concentration-dependent relaxation responses that were dependent on the presence of the endothelium and were partially inhibited by the NO-synthase inhibitor nitro-L-arginine (0.1 mM). The relaxations to acetylcholine and bradykinin were similar in vessels isolated from pregnant and non-pregnant women. Nitro-L-arginine (0.1 mM) had no influence on basal tone and had a similar inhibitory influence on the endothelium-mediated relaxations in vessels from non-pregnant and pregnant women. These results indicate that the influence of endothelium-derived NO in human subcutaneous resistance arteries is not altered at the end of pregnancy.

Nitric oxide induced membrane hyperpolarization in the rat aorta is not mediated by glibenclamide-sensitive potassium channels.

Using conventional intracellular microelectrode techniques, the membrane potential (Em) of vascular smooth muscle cells in isolated segments of thoracic rat aorta was measured. The influence of exogenous and of endothelium-derived nitric oxide on the Em was assessed, and the involvement of glibenclamide-sensitive channels in the observed membrane hyperpolarization was investigated. Exposures of the aorta strips to sodium nitroprusside (10(-8)-10(-5) M) caused a concentration-dependent and endothelium-independent hyperpolarization. Maximal hyperpolarization (5.7 +/- 0.4 mV) was obtained with 10(-5) M sodium nitroprusside. Acetylcholine (10(-8)-10(-5) M produced endothelium-dependent hyperpolarizations. At low concentrations, a slow Em change was elicited, which was sustained in the presence of the vasodilator. Higher concentrations of acetylcholine caused hyperpolarizations consisting of an initial transient peak followed by a more sustained component. Pre-exposure to Ng-nitro-L-arginine (L-NNA, 2 x 10(-4) M), which depolarized Em by 2.5 +/- 0.7 mV, significantly attenuated the later component of hyperpolarizations, indicating that it is NO dependent. Glibenclamide (10-5 M) did not significantly affect the hyperpolarization induced by 10(-6)-10(-5) M sodium nitroprusside nor the maintained component of hyperpolarization induced by 10(-5) M acetylcholine. It is concluded that the hyperpolarization caused by exogenous or endogenous NO in the rat aorta is not mediated by activation of glibenclamide-sensitive potassium channels.

Differential influence of extracellular and intracellular pH on K+ accumulation in ischaemic mammalian cardiac tissue.

The separate and the combined influence of lowering extracellular and intracellular pH on the extracellular accumulation of K+ ions in ischaemic mammalian cardiac tissue was investigated. Isolated guinea-pig papillary muscles were superfused in vitro while micro-electrode measurements of the transmembrane potentials and of the extracellular pH and K+ activity and of the intracellular pH and Na+ activity were performed. Muscles were reversibly subjected to spaced episodes of simulated ischaemia, and ischaemic K+ accumulation was measured. During normal superfusion, acidification of the intracellular pH (pHi approximately 6.8) effected by transient exposure to NH+4 containing superfusate was associated with an amiloride-sensitive rise of the intracellular Na+ activity (aiNa) from 5.1 +/- 0.5 to 19.5 +/- 2.4 mM. This increase was associated with a transient hyperpolarization of the membrane potential and shortening of the action potential duration from 190 +/- 7 to 111 +/- 16 ms. When a similar intracellular acidosis was induced 3 to 5 min before imposing ischaemia, extracellular K+ accumulation was increased compared to ischaemia alone. Surface K+ activity (asK) measured after 10 min of ischaemia was 9.6 +/- 0.8 mM following intracellular acidification v 6.4 +/- 0.4 mM following control superfusion. When the extracellular pH (pHo) was decreased to 6.85 prior to the ischaemic insult, extracellular K+ accumulation was not different from that observed during ischaemia after control superfusion (5.1 +/- 0.5 v 4.7 +/- 0.3 mM, respectively, after 10 min of ischaemia). When combined intracellular and extracellular acidosis was produced before ischaemia, the enhancing influence of lowered pHi on K+ accumulation was not observed. In these acid loading conditions, aiNa rose to the same level as observed following NH+4 withdrawal without simultaneous external acidification (26.9 +/- 2.0 mM v 23.9 +/- 2.0 mM, respectively). The results indicate that decreasing pHi before ischaemia accelerates the ischaemic increase of extracellular K+. On the other hand, although prior extracellular acidification by itself does not directly influence ischaemic K+ accumulation, it is in some way protective by reducing the intracellular proton-stimulated K+ efflux. This reduction seems not to be due to decreased cellular Na+ loading.

Heterogenous effects of histamine on isolated rat coronary arteries.

The influence of increasing concentrations of histamine (0.1 microM-1 mM) was studied on proximal and distal ring segments of left anterior descendens coronary arteries isolated from rats. Addition of histamine to prostaglandin F2 alpha (10 microM)-precontracted proximal segments elicited a further contraction. This effect was endothelium-independent and mediated by a histamine H1 receptor mechanism since it was blocked by the histamine H1 receptor antagonist, mepyramine (10 microM), and not by the histamine H2 receptor antagonist, cimetidine (100 microM), and since it was mimicked by the histamine H1 receptor agonist, 2-pyridylethylamine, and not by the histamine H2 receptor agonist, dimaprit. Addition of histamine to prostaglandin F2 alpha-precontracted distal segments elicited concentration-dependent relaxation. This relaxation is histamine H2 receptor-mediated since it was blocked by cimetidine (100 microM) and not by mepyramine (10 microM) and since dimaprit but not 2-pyridylethylamine elicited relaxation. The relaxation was not due to the release of endothelial NO, prostaglandins or activation of ATP-regulated K+ channels since it was not inhibited by NG-nitro-L-arginine methyl ester (100 microM) or NG-nitro-L-arginine (100 microM), indomethacin (10 microM) or glibenclamide (10 microM). Our results show that the effects of histamine on rat left anterior descendens coronary arteries are heterogenous, depending on the relative location within the coronary vasculature and possibly, the relative preponderance of histamine H1 or H2 receptors on the smooth muscle cells.

The basal endothelial inhibitory influence on vascular tone is not affected in nitroglycerin-tolerant rat aorta.

Prolonged exposure to nitrovasodilators produces tolerance and dependence. Nitrovasodilators exert their action on vascular smooth muscle cells by activation of guanylyl cyclase. Nitrates share this mechanism with endothelial NO, which exerts a continuous inhibitory influence on vascular tone. Whether the basal inhibitory endothelial influence might be affected in rat aorta exposed in vitro to a tolerance-inducing concentration of nitroglycerin was investigated in this study. It was found that the basal inhibitory influence, assessed as its inhibitory influence on norepinephrine-induced contraction, and as the contractile effect of N-nitro-L-arginine methyl ester or methylene blue, was the same in nitroglycerin-tolerant and control aortic rings. Our results give an indication that changes in basal inhibitory endothelial influence are not involved in the phenomena of nitrate tolerance and nitrate dependence.