Effect of overexpression of human aldehyde dehydrogenase 2 in LLC-PK1 cells on glyceryl trinitrate biotransformation and cGMP accumulation.

BACKGROUND AND PURPOSE: Recent studies suggest a primary role for aldehyde dehydrogenase 2 (ALDH2) in mediating the biotransformation of organic nitrates, such as glyceryl trinitrate (GTN), to the proximal activator of soluble guanylyl cyclase (sGC), resulting in increased cGMP accumulation and vasodilation. Our objective was to assess the role of ALDH2 in organic nitrate action using a cell culture model. EXPERIMENTAL APPROACH: Porcine renal epithelial (LLC-PK1) cells possess an intact NO-sGC-cGMP signaling system, and can be used as a biochemical model of organic nitrate action. We used a pcDNA3.1-human ALDH2 expression vector to establish a stably transfected cell line (PK1(ALDH2)) that overexpressed ALDH2, or small interfering RNA (siRNA) to deplete endogenous ALDH2, and assessed GTN biotransformation and GTN-induced cGMP formation. KEY RESULTS: ALDH2 activity in the stably transfected cells was approximately sevenfold higher than wild-type cells or cells stably transfected with empty vector (PK1(vector)); and protein expression, as assessed by immunoblot analysis, was markedly increased. In PK1(ALDH2), GTN biotransformation was significantly increased as a result of increased glyceryl-1,2-dinitrate formation compared to wild-type or PK1(vector). However, the incubation of PK1(ALDH2) with 1 or 10 µM GTN did not alter GTN-induced cGMP accumulation compared with wild-type or PK1(vector) cells. Furthermore, siRNA-mediated depletion of ALDH2 had no effect on GTN-induced cGMP formation. CONCLUSIONS AND IMPLICATIONS: In an intact cell system, neither overexpression nor depletion of ALDH2 affects GTN-induced cGMP formation, indicating that ALDH2 does not mediate the mechanism-based biotransformation of GTN to an activator of sGC.

Changes in aldehyde dehydrogenase 2 expression in rat blood vessels during glyceryl trinitrate tolerance development and reversal.

BACKGROUND AND PURPOSE: Recent studies have suggested an essential role for aldehyde dehydrogenase 2 (ALDH2) in the bioactivation of organic nitrates such as glyceryl trinitrate (GTN). In the present study, we utilized an in vivo GTN tolerance model to further investigate the role of ALDH2 in GTN bioactivation and tolerance. EXPERIMENTAL APPROACH: We assessed changes in aortic ALDH activity, and in ALDH2 protein expression in various rat blood vessels (aorta, vena cava, femoral artery and femoral vein) during continuous GTN exposure (0.4 mg·h⁻¹ for 6, 12, 24 or 48 h) or after a 1-, 3- or 5-day drug-free period following a 48 h exposure to GTN, in relation to changes in vasodilator responses to GTN and in vascular GTN biotransformation. KEY RESULTS: A decrease was observed in both ALDH2 protein expression (80% in tolerant veins and 30% in tolerant arteries after 48 h exposure to GTN) and aortic ALDH activity, concomitant with decreased vasodilator responses to GTN and decreased aortic GTN biotransformation. However, after a 24 h drug-free period following 48 h of GTN exposure, vasodilator responses to GTN and aortic GTN biotransformation activity had returned to control values, whereas vascular ALDH2 expression and aortic ALDH activity were still significantly depressed, and remained so for 3-5 days following cessation of GTN exposure. CONCLUSIONS AND IMPLICATIONS: The dissociation of reduced ALDH activity and ALDH2 expression from the duration of the impaired vasodilator and biotransformation responses to GTN in nitrate-tolerant blood vessels, suggests that factors other than changes in ALDH2-mediated GTN bioactivation contribute to nitrate tolerance.

Chronic prenatal ethanol exposure increases apoptosis in the hippocampus of the term fetal guinea pig.

It is hypothesized that chronic prenatal ethanol exposure (CPEE), via maternal ethanol administration, increases mitochondrial-directed apoptosis in the hippocampus of the term fetus that precedes loss of hippocampal CA1 pyramidal cells. To test this hypothesis, timed pregnant guinea pigs received chronic oral administration of: 4 g ethanol/kg maternal body weight/day, isocaloric-sucrose/pair-feeding or water throughout gestation. At gestational day 65 (term fetus) and postnatal day 0 (neonate), individual offspring were euthanized, and the brain was excised and dissected. CPEE, compared with the isocaloric-sucrose/pair-fed and water control groups, decreased the brain weight of the term fetus and neonate. CPEE did not alter the density of CA1 pyramidal cells in the hippocampus of the term fetus and neonate. In the term fetus, CPEE increased cytochrome c content in the cytosolic fraction of the hippocampus, altered the mitochondrial localization of cytochrome c in cells of the dorsal hippocampus, and increased the percentage of cells in the dorsal hippocampus containing activated caspase-3 and cleaved poly(ADP-ribose) polymerase. The data indicate that CPEE increases neuroapoptosis in the hippocampus of term fetus, which appears to occur via an intrinsic, mitochondrial-directed mechanism initiated by leakage of pro-apoptotic cytochrome c from mitochondria into the cytoplasm.

Induction of c-jun and TGF-beta 1 in Fischer 344 rats during amiodarone-induced pulmonary fibrosis.

Amiodarone (AM) is an antidysrhythmic agent with a propensity to cause pulmonary toxicity, including potentially fatal fibrosis. In the present study, the potential roles of c-Jun and transforming growth factor (TGF)-beta 1 in AM-induced inflammation and fibrogenesis were examined after intratracheal administration of AM (1.83 micromol/day on days 0 and 2) or an equivalent volume (0.4 ml) of distilled water to male Fischer 344 rats. Northern and immunoblot analyses demonstrated that lung TGF-beta 1 (mRNA and protein) expression was increased 1.5- to 1.8-fold relative to control during the early inflammation period and 1 day, 1 wk, and 2 wk post-AM treatment. Lung c-Jun protein expression was increased concomitantly with evidence of AM-induced fibrosis; at 5 wk post-AM treatment, c-Jun protein was increased 3.3-fold relative to control. The results indicate a role for induction of c-jun and TGF-beta 1 expression in the development of AM-induced pulmonary fibrosis in the Fischer 344 rat and provide potential targets for therapeutic intervention.

In vitro activation of soluble guanylyl cyclase and nitric oxide release: a comparison of NO donors and NO mimetics.

Nitric oxide (NO) performs a central role in biological systems, binding to the heme site of soluble guanylyl cyclase (sGC), leading to enzyme activation and elevation of intracellular levels of cGMP. Organic nitrates, in particular, nitroglycerin (GTN), are clinically important nitrovasodilators that function as NO-mimetics in biological systems. Comparison of sGC activation data with electrochemically measured rates of NO release for genuine NO donors, NONOates and nitrosothiols, yields an excellent correlation between the EC(50) for sGC activation and the rate constant for NO release, k(NO). However, activation of sGC by GTN and the nitrates has very different characteristics, including the requirement for specific added thiols, for example, cysteine. The reaction of GTN with cysteine in anaerobic solution yields NO slowly, and NO release, measured by chemiluminescence detection, is quenched by added metal ion chelator. The generation of NO under aerobic conditions is 100-fold slower than the anaerobic reaction. Furthermore, NO release from the reaction of GTN with cysteine in phosphate buffer is too slow to account for sGC activation by GTN/cysteine. The slow rate of the chemical reaction to release NO suggests that nitrates can activate sGC by an NO-independent mechanism. In contrast to the genuine NO donors, GTN behaves as a partial agonist with respect to sGC activation, but in the presence of the allosteric sGC activator, YC-1, GTN exhibits full agonist activity.

A novel nitrate ester reverses the cognitive impairment caused by scopolamine in the Morris water maze.

The objective of this study was to test the hypothesis that activation of soluble guanylyl cyclase and increased cGMP formation in the brain would improve task acquisition in cognitively impaired animals. We evaluated the effects of a novel nitrate ester, GT 715 (2,3-dinitrooxy-(2,3-bis-nitrooxypropyldisulfanyl)-propane), in scopolamine-induced impairment of task acquisition in the Morris water maze. GT 715 improved task acquisition in scopolamine-pretreated animals in a time- and dose-dependent manner, whereas the prototypical nitrate ester, glyceryl trinitrate (GTN), was ineffective. GT 715 also was more effective and more potent than GTN for activation of hippocampal guanylyl cyclase. The results of this study therefore suggest that stimulation of cerebral soluble guanylyl cyclase activity may be an effective strategy to improve learning and memory performance in individuals in whom cognitive abilities are impaired by injury, disease, or ageing.

Endothelin receptor antagonism does not prevent the development of in vivo glyceryl trinitrate tolerance in the rat.

There is evidence that increased endothelial production of endothelin-1 (ET-1) may contribute to glyceryl trinitrate (GTN) tolerance. We used the competitive ET(A) receptor antagonist ZD2574 to determine whether chronic ET(A) receptor blockade affected the biochemical and functional responses to GTN during the development of GTN tolerance in vivo. Tolerance induced using transdermal GTN patches resulted in a 5.3 +/- 1.2-fold increase in the EC(50) value for GTN relaxation in isolated aorta from GTN-tolerant rats. Coadministration of ZD2574 (100 mg kg(-1) t.i.d. for 3 days) during tolerance induction had no effect on GTN-induced relaxation. This dose of ZD2574 markedly blunted the pressor response to ET-1, indicating effective blockade of ET(A) receptors, and also abolished the initial transient depressor response to ET-1, indicating that blockade of endothelial ET(B) receptors also occurred using this dosage regimen for ZD2574. Consistent with the relaxation data, coadministration of ZD2574 had no effect on the decrease in GTN-induced cGMP accumulation or on the decrease in GTN biotransformation that occurred in aortae from GTN-tolerant animals. Radioimmunoassay data indicated that the GTN tolerance induction protocol caused a 2.3 +/- 0.4-fold and a 2.2 +/- 0.5-fold increase in total tissue ET-1 levels in tolerant aorta and vena cava, respectively. These data suggest that chronic inhibition of ET receptors by ZD2574 was not sufficient to prevent or diminish the tolerance-inducing effects of GTN, and that the increase in ET-1 levels observed in tolerant tissues may occur as a consequence of the vascular changes that occur during chronic GTN exposure.

Effects of the flavoprotein inhibitor, diphenyleneiodonium sulfate, on ex vivo organic nitrate tolerance in the rat.

The flavoprotein inhibitor, diphenyleneiodonium (DPI), inhibits the action of glyceryl trinitrate (GTN) and the D-enantiomer of isoidide dinitrate (IIDN), but not the L-enantiomer (L-IIDN), in isolated rat aorta via inhibition of the bioactivation of these prodrugs. Paradoxically, a vascular NAD(P)H oxidase, which also is inhibited by DPI, has been proposed to generate superoxide that quenches nitric oxide (NO) produced during GTN biotransformation, and increased oxidase levels are proposed to contribute to the phenomenon of organic nitrate tolerance. We examined the effect of DPI on isolated rat aorta using an in vivo model of organic nitrate tolerance. The EC(50) values for GTN-, D-IIDN-, and L-IIDN-induced relaxation of aorta from GTN-tolerant rats were increased 4.5- to 7.5-fold. Treatment of blood vessels with DPI (0.3 microM) increased the EC(50) values for GTN and D-IIDN by the same magnitude in control and tolerant aortae, a result that would not be predicted if DPI and GTN tolerance affected common targets. The expression of NADPH-cytochrome P450 reductase (CPR) during in vivo tolerance was assessed by NADPH-dependent cytochrome c reductase activity of aortic microsomes, immunoblotting, and Northern analysis. By all three determinants, CPR expression was unchanged in aorta from GTN-tolerant rats. Superoxide dismutase-inhibitable NADPH-dependent cytochrome c reductase activity (a measure of superoxide generation) of tolerant rat aortic microsomes was not different from that of controls. Superoxide dismutase-inhibitable NADH-dependent cytochrome c reductase activity was detected only in microsomes from tolerant animals. DPI caused a modest increase in the sensitivity for relaxation by the NO donor DEA NONOate to an equal extent in tolerant and nontolerant tissues, whereas the superoxide scavenger, 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), had no effect on the sensitivity for relaxation by GTN. These results would not be expected if tolerance-induced increases in superoxide were a causative factor for the reduced relaxation response in tolerance. We conclude that neither reduced flavoprotein-dependent metabolic activation of organic nitrates, such as that mediated by CPR, nor increased superoxide due to increased NAD(P)H oxidase activity can account for the development of in vivo tolerance to GTN.

Enantioselective inhibition of the biotransformation and pharmacological actions of isoidide dinitrate by diphenyleneiodonium sulphate.

1. We have shown previously that the D- and L- enantiomers of isoidide dinitrate (D-IIDN and L-IIDN) exhibit a potency difference for relaxation and cyclic GMP accumulation in isolated rat aorta and that this is related to preferential biotransformation of the more potent enantiomer (D-IIDN). The objective of the current study was to examine the effect of the flavoprotein inhibitor, diphenyleneiodonium sulphate (DPI), on the enantioselectivity of IIDN action. 2. In isolated rat aortic strip preparations, exposure to 0.3 microM DPI resulted in a 3.6 fold increase in the EC50 value for D-IIDN-induced relaxation, but had no effect on L-IIDN-induced relaxation. 3. Incubation of aortic strips with 2 microM D- or L-IIDN for 5 min resulted in significantly more D-isoidide mononitrate formed (5.0 +/- 1.5 pmol mg protein(-1)) than L-isoidide mononitrate (2.1 +/- 0.7 pmol mg protein(-1)) and this difference was abolished by pretreatment of tissues with 0.3 microM DPI. DPI had no effect on glutathione S-transferase (GST) activity or GSH-dependent biotransformation of D- or L-IIDN in the 105,000 x g supernatant fraction of rat aorta. 4. Consistent with both the relaxation and biotransformation data, treatment of tissues with 0.3 microM DPI significantly inhibited D-IIDN-induced cyclic GMP accumulation, but had no effect on L-IIDN-induced cyclic GMP accumulation. 5. In the intact animal, 2 mg kg(-1) DPI significantly inhibited the pharmacokinetic and haemodynamic properties of D-IIDN, but had no effect L-IIDN. 6. These data suggest that the basis for the potency difference for relaxation by the two enantiomers is preferential biotransformation of D-IIDN to NO, by an enzyme that is inhibited by DPI. Given that DPI binds to and inhibits NADPH-cytochrome P450 reductase, the data are consistent with a role for the cytochromes P450-NADPH-cytochrome P450 reductase system in this enantioselective biotransformation process.

Inhibition of NADPH-cytochrome P450 reductase and glyceryl trinitrate biotransformation by diphenyleneiodonium sulfate.

We reported previously that the flavoprotein inhibitor diphenyleneiodonium sulfate (DPI) irreversibly inhibited the metabolic activation of glyceryl trinitrate (GTN) in isolated aorta, possibly through inhibition of vascular NADPH-cytochrome P450 reductase (CPR). We report that the content of CPR represents 0.03 to 0.1% of aortic microsomal protein and that DPI caused a concentration- and time-dependent inhibition of purified cDNA-expressed rat liver CPR and of aortic and hepatic microsomal NADPH-cytochrome c reductase activity. Purified CPR incubated with NADPH and GTN under anaerobic, but not aerobic conditions formed the GTN metabolites glyceryl-1,3-dinitrate (1,3-GDN) and glyceryl-1,2-dinitrate (1,2-GDN). GTN biotransformation by purified CPR and by aortic and hepatic microsomes was inhibited > 90% after treatment with DPI and NADPH. DPI treatment also inhibited the production of activators of guanylyl cyclase formed by hepatic microsomes. We also tested the effect of DPI on the hemodynamic-pharmacokinetic properties of GTN in conscious rats. Pretreatment with DPI (2 mg/kg) significantly inhibited the blood pressure lowering effect of GTN and inhibited the initial appearance of 1,2-GDN (1-5 min) and the clearance of 1,3-GDN. These data suggest that the rapid initial formation of 1,2-GDN is related to mechanism-based GTN biotransformation and to enzyme systems sensitive to DPI inhibition. We conclude that vascular CPR is a site of action for the inhibition by DPI of the metabolic activation of GTN, and that vascular CPR is a novel site of GTN biotransformation that should be considered when investigating the mechanism of GTN action in vascular tissue.

Isoform-specific biotransformation of glyceryl trinitrate by rat aortic glutathione S-transferases.

The objective of this study was to purify and characterize rat aortic glutathione S-transferases (GSTs) and to assess their role in the biotransformation of the nitrovasodilator, glyceryl trinitrate (GTN). Two class alpha GSTs (Ya and Yc) a class mu GST (Yb2) and a class pi GST (Yp) were identified in rat aortic cytosol. Partial purification of three of these (Yb2, Yc and Yp) was achieved by affinity chromatography with S-hexylglutathione agarose. Further purification by cation- and anion-exchange chromatography resulted in the purification of GST Yc and GST Yb2/Yp to apparent homogeneity, a purification of 200- and 110-fold, respectively. Purified GST Yc and Yb2/Yp mediated GTN biotransformation with similar rates. GST activity and GTN biotransformation by rat aortic cytosol and affinity-purified GSTs were highly sensitive to inhibition by the class mu selective inhibitors Basilen Blue and bromosulfophthalein. Removal of GST Yb2 from rat aortic cytosol by immunoprecipitation resulted in marked inhibition of GST activity and GTN biotransformation. We conclude that the GSTs account for the major portion of GTN biotransformation in rat aortic cytosol, and that this is primarily attributable to the GST Yb2 isoform.

Inhibition of the pharmacological actions of glyceryl trinitrate after the electroporetic delivery of a glutathione S-transferase inhibitor.

It is generally accepted that the biotransformation of organic nitrates to an activator of soluble guanylyl cyclase (presumably NO) is a prerequisite for their vasodilator actions. The glutathione S-transferases (GSTs) mediate glyceryl trinitrate (GTN) biotransformation, but whether this results in guanylyl cyclase activation and relaxation of vascular smooth muscle is equivocal. We used electroporation of adherent cultured cells to deliver the membrane-impermeable GST inhibitor basilen blue (BB) into porcine kidney epithelial cells. This resulted in significant inhibition of GTN biotransformation because of a reduction in the formation of glyceryl-1,2-dinitrate, but not glyceryl-1, 3-dinitrate. In the 105,000 x g supernatant fraction of porcine kidney epithelial cells, BB significantly inhibited the formation of both GTN metabolites. Electroporation of porcine kidney epithelial cells with BB also inhibited GTN-induced cyclic GMP accumulation. This was caused in part by inhibition of soluble guanylyl cyclase by BB. To differentiate BB-mediated inhibition of the bioactivation of GTN from its inhibitory effect on guanylyl cyclase, inhibition of cyclic GMP accumulation induced by GTN and that induced by the spontaneous NO-releasing compound, t-butyl-S-nitrosothiol were compared. Maximum inhibition of cyclic GMP accumulation by BB was 80% and 40% with GTN and t-butyl-S-nitrosothiol as the stimulating compounds, respectively. These data suggest that GSTs mediate the biotransformation of GTN to an activator of guanylyl cyclase and support the contention that vascular GSTs participate in mediating the relaxant effects of organic nitrates.

Effect of dexamethasone treatment on the biotransformation of glyceryl trinitrate: cytochrome P450 3A1 mediated activation of rat aortic guanylyl cyclase by glyceryl trinitrate.

It is generally accepted that organic nitrates act via vascular biotransformation to an activator of guanylyl cyclase (presumably NO), resulting in increased cyclic GMP accumulation and vascular smooth muscle relaxation. Previously, we have shown that cytochrome P450 can mediate the biotransformation of glyceryl trinitrate (GTN) and that at least a portion of this biotransformation results in the formation of an activator of guanylyl cyclase. To assess the role of the cytochrome P450 3A subfamily in this phenomenon, we treated male and female rats with dexamethasone (DEX) (150 mg/kg, i.p., daily for 3 days). Under anerobic conditions, hepatic microsomal biotransformation of GTN was increased three-fold in DEX-treated male rats compared with all other treatment groups. Incubation of aortic 100,000 x g supernatant fraction from untreated rats (as a source of guanylyl cyclase) with GTN and hepatic microsomes from all groups resulted in concentration-dependent increases in guanylyl cyclase activation. Microsomes from DEX-treated male and female rats demonstrated a significantly greater activation of guanylyl cyclase compared with microsomes from untreated males and females. Furthermore, GTN-induced guanylyl cyclase activation mediated by microsomes from DEX-treated male and female rats was markedly inhibited by a polyclonal antibody raised to rat CYP3A1. Since CYP3A2 is absent or very low in hepatic microsomes from DEX-treated adult female rats, this identifies CYP3A1 as an isoform capable of biotransforming GTN to an activator of guanylyl cyclase. Similarly, CYP2C11 was identified as an isoform capable of biotransforming GTN to an activator of guanylyl cyclase, since monoclonal antibody to CYP2C11 inhibited GTN-induced activation of guanylyl cyclase mediated by microsomes from control male rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the biotransformation and pharmacological actions of glyceryl trinitrate by the flavoprotein inhibitor, diphenyleneiodonium sulfate.

Recent studies suggest a role for the vascular cytochrome P450-NADPH cytochrome P450 reductase system in mediating the biotransformation of glyceryl trinitrate (GTN) to nitric oxide (or some closely related species), resulting in increased cyclic GMP accumulation and vasodilation. In this study we tested the effect of the flavoprotein inhibitor, diphenyleneiodonium sulfate (DPI) on GTN action in isolated rat aorta. Exposure of phenylephrine-contracted tissues to DPI (10 nM-0.3 microM) resulted in 3- to 10-fold increases in the EC50 values for GTN-induced relaxation in both endothelium-intact tissues and endothelium-denuded tissues, whereas the vasodilator response to sodium nitroprusside was unaffected. Consistent with the relaxation data, cyclic GMP accumulation induced by 0.3 or 2 microM GTN was inhibited by 0.3 microM DPI in both endothelium-intact and endothelium-denuded aortic strips, whereas cyclic GMP accumulation induced by 0.1 microM sodium nitroprusside was unaffected. The regioselective formation of glyceryl-1,2-dinitrate observed during the aortic biotransformation of GTN was inhibited markedly by DPI. In tissues incubated with 0.3 or 10 microM DPI for 30 min followed by washout for 60 min, the EC50 values for GTN-induced relaxation were increased 2-fold, and both GTN-induced cyclic GMP accumulation and vascular GTN biotransformation were decreased. This suggests an irreversible component to the inhibitory action of DPI. Together, these data provide evidence for the involvement of a flavoprotein (e.g., NADPH cytochrome P450 reductase) in the metabolic activation of GTN required for expression of its vasodilator activity.

Isoproterenol exerts cyclic AMP independent effects on DNA synthesis in cultured adventitial fibroblasts from spontaneously hypertensive and Wistar-Kyoto rats.

Understanding the mechanism behind the growth response evident in the vasculature of the spontaneously hypertensive rat (SHR) remains elusive. Fibroblasts from the aortic adventitial layer of the SHR manifest the heightened proliferative rate in vitro relative to Wistar-Kyoto (WKY) rats that is conspicuous in cultured aortic smooth muscle cells. The adenylylcyclase/cyclic AMP signal transduction pathway is believed to be altered in hypertensive people and animals such that responses to beta-adrenoceptor activation are blunted. The present study examined the effects of beta-adrenoceptor-mediated versus direct activation of adenylylcyclase on intracellular cyclic AMP accumulation and subsequent DNA synthesis in cultured aortic fibroblasts. We hypothesized that elevation of cyclic AMP levels by both isoproterenol and forskolin would normalize the proliferative capacity of SHR fibroblasts. Forskolin increased intracellular cyclic AMP levels and inhibited epidermal growth factor stimulated thymidine incorporation in an equivalent manner in both SHR and WKY adventitial fibroblasts, implying that there is no difference in adenylylcyclase activity. Isoproterenol elevated cyclic AMP levels to a significantly greater degree in the SHR than did forskolin, and yet, relative to forskolin, attenuated growth factor induced DNA synthesis to a lesser extent. These data suggest that isoproterenol, via beta-adrenoceptor activation, exhibits both cyclic AMP dependent and cyclic AMP independent effects in adventitial fibroblasts. The cyclic AMP independent effects of isoproterenol oppose the expected observations due to cyclic AMP and may offer an explanation to the blunted responses to beta-adrenoceptor activation evident both in vitro and in vivo.

Biotransformation of organic nitrates and vascular smooth muscle cell function.

The organic nitrates are interesting examples of drugs that undergo biotransformation at their site of action to generate the active form of the drug. Furthermore, tolerance to the vasodilator effects of organic nitrates is associated with impairment of this metabolic activation process. Despite considerable research effort, the intracellular processes and the chemical reaction pathways by which organic nitrates are converted to their active form are still unresolved. This review by Brian Bennett and colleagues summarizes the characteristics of organic-nitrate biotransformation in vascular smooth muscle, the difficulties encountered when assessing this biotransformation, and the evidence for the role of two identified vascular biotransformation systems (glutathione-S-transferases and the cytochrome P450 system) in the metabolic activation of organic nitrates.

Stimulation of postsynaptic and inhibition of presynaptic adenylyl cyclase activity by metabotropic glutamate receptor activation.

To determine the subcellular distribution of cyclic AMP-coupled metabotropic glutamate receptors (mGluRs), the effects of glutamate agonists on adenylyl cyclase activity were examined using two hippocampal membrane preparations. These were synaptosomes (SY), which are composed of presynaptic terminals, and synaptoneurosomes (SN), which are composed of both pre- and postsynaptic elements. In SY, a water-soluble analogue of forskolin (7 beta-forskolin) increased enzyme activity approximately 10-fold at the highest concentration tested. The selective metabotropic receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) inhibited enzyme activity as did glutamate and quisqualate. L-Amino-4-phosphobutanoate (L-AP4) had no effect on enzyme activity at any concentration tested. The metabotropic receptor antagonist L-2-amino-3-phosphopropionic acid (L-AP3) was not effective in the SY in antagonizing the agonist-induced decreases in adenylyl cyclase activity by glutamate or 1S,3R-ACPD. It was, however, effective at antagonizing quisqualate-induced decreases in enzyme activity. In SN, at the highest concentration tested, 7 beta-forskolin produced a 60-fold increase in adenylyl cyclase activity. As was observed in SY, glutamate decreased adenylyl cyclase activity in SN. In contrast, 1S,3R-ACPD, quisqualate, and L-AP4 increased adenylyl cyclase activity. In the SN, L-AP3 was ineffective in antagonizing any agonist-induced increases (1S,3R-ACPD, L-AP4, and quisqualate) or decreases (glutamate) in adenylyl cyclase activity. The data suggest that postsynaptic metabotropic glutamate receptor activation results in stimulation of adenylyl cyclase activity, whereas inhibition of this enzyme appears to be mediated at least partly through presynaptic mechanisms.

Forskolin and isoproterenol effect discrete responses on epidermal growth factor induced DNA synthesis in aortic smooth muscle cells.

Defining the mechanisms regulating the proliferation of vascular smooth muscle is necessary to better understand the pathogenesis of atherosclerosis and hypertension. In the present investigation, we examined the effects of incubation with forskolin or isoproterenol on the proliferation of cultured rat aortic smooth muscle cells. Forskolin, a direct activator of adenylate cyclase, and isoproterenol, a beta-adrenergic agonist, increased intracellular cyclic AMP levels in a concentration-dependent manner, subsequent to a 5-min exposure. Isobutylmethylxanthine at 100 microM attenuated epidermal growth factor stimulated DNA synthesis by 35% without affecting intracellular cyclic AMP levels. Forskolin dose-dependently augmented this inhibition. In contrast, a 24-h exposure of cells to isoproterenol resulted in a biphasic effect on growth factor stimulated thymidine incorporation. Both forskolin and isoproterenol attenuated thymidine incorporation to the same degree up to 12 h poststimulation, the onset of S phase. By 16 h poststimulation, [3H]thymidine incorporation in smooth muscle cells treated with isoproterenol was significantly enhanced by 50%, whereas forskolin treatment continued to attenuate DNA synthesis by 40%. Somewhat surprisingly, this disparity in effect on DNA synthesis was evident in spite of heterologous desensitization to rechallenge by either forskolin or isoproterenol subsequent to a 24-h incubation with either drug. These results suggest that the isoproterenol enhancement of epidermal growth factor stimulated DNA synthesis in rat aortic smooth muscle cells may be cyclic AMP independent.

Comparison of liposome fusion and electroporation for the intracellular delivery of nonpermeant molecules to adherent cultured cells.

Intracellular delivery of nonpermeant molecules to cultured cells in situ can be problematic. This work is a comparison between two methods of accomplishing this delivery; liposome-mediated delivery and electroporation. The final goal was to examine the effects of the glutathione S-transferase (GST) inhibitor Basilen Blue (BB) on glyceryl trinitrate biotransformation in porcine kidney epithelial (PK1) cells after intracellular delivery. Initial evaluation used the fluorescent markers carboxyfluorescein and lucifer yellow (LY). This was followed by biochemical analysis of glyceryl trinitrate biotransformation. Liposome-mediated delivery proved ineffective in spite of variations in the lipid composition of the liposomes and the use of an agglutinin and a fusogen. In contrast, electroporation was a very effective method for intracellular delivery of both lucifer yellow and basilen blue to the PK1 cells. The results show that in cells where Basilen Blue was introduced, there was a decrease in both the glyceryl trinitrate (GTN) biotransformation and ratio of glyceryl-1,2-dinitrate (1,2-GDN) to glyceryl-1,3-dinitrate (1,3-GDN) formed. The technique of in situ electroporation shows great promise for the assessment of a variety of nonpermeant molecules of pharmacological interest.