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We report the in vitro assessment of pharmacotoxicity for the high-affinity GHB receptor ligand, NCS-382, using neuronal stem cells derived from mice with a targeted deletion of the aldehyde dehydrogenase 5a1 gene (succinic semialdehyde dehydrogenase(SSADH)-deficient mice). These animals represent a phenocopy of the human disorder of GABA metabolism, SSADH deficiency, that metabolically features accumulation of both GABA and the GABA-analog γ-hydroxybutyric acid in conjunction with a nonspecific neurological phenotype. We demonstrate for the first time using MDCK cells that NCS-382 is actively transported and capable of inhibiting GHB transport. Following these in vitro assays with in vivo studies in aldh5a1-/- mice, we found the ratio of brain/liver GHB to be unaffected by chronic NCS-382 administration (300mg/kg; 7 consecutive days). Employing a variety of cellular parameters (reactive oxygen and superoxide species, ATP production and decay, mitochondrial and lysosomal number, cellular viability and necrosis), we demonstrate that up to 1mM NCS-382 shows minimal evidence of pharmacotoxicity. As well, studies at the molecular level indicate that the effects of NCS-382 at 0.5mM are minimally toxic as evaluated using gene expression assay. The cumulative data provides increasing confidence that NCS-382 could eventually be considered in the therapeutic armament for heritable SSADH deficiency.

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Gamma-hydroxybutyrate (GHB) is a drug of abuse, an approved therapeutic for narcolepsy, an agent employed for facilitation of sexual assault, as well as a biomarker of succinic semialdehyde dehydrogenase deficiency (SSADHD). Our laboratory seeks to identify surrogate biomarkers in SSADHD that can shed light on the developmental course of this neurometabolic disease. Since GHB may be quantified in hair as a potential surrogate to identify victims of drug-related assault, we have opted to examine its level in SSADHD. We quantified GHB in hair derived from ten patients with SSADHD, and documented a significant negative age correlation. These findings are consistent with recent results in patient biological fluids, including plasma and red blood cells. These findings may provide additional insight into the developmental course of SSADHD (Jansen et al., J Inherit Metab Dis 39:795-800, 2016).

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γ-Hydroxybutyric acid (GHB), a minor metabolite of the inhibitory neurotransmitter GABA, can accumulate to significant concentrations in the heritable disorder of GABA degradation, succinic semialdehyde dehydrogenase (SSADH) deficiency (SSADHD). Moreover, GHB may be employed in therapeutic settings (treatment of narcolepsy), as well as instances of illicit activity, including acquaintance sexual assault and the induction of euphoria. High-affinity binding sites for GHB in the brain have been identified, although the absolute identity of these receptors remains unclear. Pharmacological antagonism of GHB binding may have multiple instances of therapeutic relevance. The high affinity GHB receptor antagonist, NCS-382 (6,7,8,9-tetrahydro-5-hydroxy-5H-benzo-cyclohept-6-ylideneacetic acid) has not been piloted in humans. To address the potential clinical utility of NCS-382, we have piloted initial studies of its toxicology in HepG2 and primary hepatocyte cells. At high dose (0.5mM), NCS-382 showed no capacity for inhibition of microsomal CYPs (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and minimal potential for activation of xenobiotic nuclear receptors. Additional cellular integrity and functional assays (viability, oxidative stress, apoptosis, ATP production) revealed little evidence for cytotoxicity, and a low degree of dysregulation of >370 genes actively engaged in the mediation of cellular toxicity. In vitro testing indicates a low probability of cellular toxicity associated with NCS-382.

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We hypothesized that blood levels of γ-aminobutyric acid (GABA) and γ-hydroxybutyric acid (GHB), biomarkers of succinic semialdehyde dehydrogenase deficiency (SSADHD), would correlate with age. GABA and GHB were quantified in plasma and red blood cells (RBCs) from 18 patients (age range 5-41 years; median 8). Both metabolites negatively correlated with age (P < 0.05). Plasma and RBC GHB declined with age, reaching a nadir and approximate steady state by 10 years. Declining plasma GABA achieved this approximate steady state at 30-40 years of age. These biomarker relationships may reflect further GABA- and GHB-ergic neurotransmission imbalances that correlate with the onset of adolescent/adulthood neuropsychiatric morbidity and epilepsy in SSADHD.

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Discovered some 35 years ago, succinic semialdehyde dehydrogenase deficiency (SSADHD) represents a rare, autosomal recessively-inherited defect in the second step of the GABA degradative pathway. Some 200 patients have been reported, with broad phenotypic and genotypic heterogeneity. SSADHD represents an unusual neurometabolic disorder in which two neuromodulatory agents, GABA (and the GABA analogue, 4-hydroxybutyrate), accumulate to supraphysiological levels. The unexpected occurrence of epilepsy in several patients is counterintuitive in view of the hyperGABAergic state, in which sedation might be expected. However, the epileptic status of some patients is most likely represented by broader imbalances of GABAergic and glutamatergic neurotransmission. Cumulative research encompassing decades of basic and clinical study of SSADHD reveal a monogenic disease with broad pathophysiological and clinical phenotypes. Numerous metabolic perturbations unmasked in SSADHD include alterations in oxidative stress parameters, dysregulation of autophagy and mitophagy, dysregulation of both inhibitory and excitatory neurotransmitters and gene expression, and unique subsets of SNP alterations of the SSADH gene (so-called ALDH5A1, or aldehyde dehydrogenase 5A1 gene) on the 6p22 chromosomal arm. While seemingly difficult to collate and interpret, these anomalies have continued to open novel pathways for pharmacotherapeutic considerations. Here, we present an update on selected aspects of SSADHD, the ALDH5A1 gene, and future avenues for research on this rare disorder of GABA metabolism.

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HEALTHY was a 3-year middle school-based primary prevention trial to reduce modifiable risk factors for type 2 diabetes in youth. The study was conducted at seven centers across the country. This paper describes the recruitment and retention activities employed in the study. Schools and students were the focus of recruitment and retention. Each center was responsible for the recruitment of six schools; eligibility was based on ability to enroll a sufficient number of predominately minority and lower socioeconomic status students. Study staff met with district superintendents and school principals to verify the eligibility of schools, and to ascertain how appropriate the school would be for conducting the trial. Sixth grade students were recruited employing a variety of techniques; students and their parents did not know whether their school was randomized to the intervention or control arm. This cohort was followed through sixth, seventh and eighth grades. In the eighth grade, an additional sample of students who were not originally enrolled in the study was recruited in a similar manner to participate in data collection to allow for cross-sectional and dose-response secondary analyses. Parents signed informed consent forms and children signed informed assent forms, as per the needs of the local Institutional Review Board. Parents received a letter describing the results of the health screening for their children after data collection in sixth and eighth grades. Retention of schools and students was critical for the success of the study and was encouraged through the use of financial incentives and other strategies. To a large extent, student withdrawal due to out-migration (transfer and geographical relocation) was beyond the ability of the study to control. A multi-level approach that proactively addressed school and parent concerns was crucial for the success of recruitment and retention in the HEALTHY study.

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Maximal coronary conductance with adenosine in anaemic fetal sheep is twice that of non-anaemic fetuses. To investigate whether this increase in conductance persists into adulthood we studied twin sheep as fetuses and again as adults. Nine anaemic fetuses (118 days gestation) underwent isovolaemic haemorrhage for 18.0 +/- 4.6 days (means +/- S.D.) during which time the haematocrit was reduced from 39.9 +/- 5.2 % to 16.3 +/- 3.4 % and oxygen content from 8.6 +/- 1.3 to 2.3 +/- 0.2 ml dl-1. At 138 days the anaemic fetuses were transfused; at delivery the haematocrit was 29.3 +/- 6.8 % compared to nine control fetuses in which the haematocrit was 38.5 +/- 4.3 %. The weight at delivery was 3.5 +/- 0.36 kg in the anaemic fetuses vs. 4.2 +/- 0.83 kg in controls. Twenty-eight weeks later, we placed an occluder on the descending thoracic aorta and inferior vena cava, a flow probe around the proximal left circumflex coronary artery, and catheters in the left atrial appendage, jugular and carotid vessels. Maximal coronary conductance was determined in the adults by recording coronary blood flow as driving pressure was altered by inflating the occluders while adenosine was infused into the left atrium. Right atrial, left atrial, systolic and mean arterial pressures, systemic vascular resistance and haematocrit were not different between 'in utero anaemic' and control adults. The adults that were anaemic in utero weighed less than the controls 39.4 +/- 4.6 kg vs. 45.0 +/- 5.6 kg. Maximal conductance was greater in the adults that were anaemic in utero: 11.2 +/- 4.0 ml min(-1) (100 g)(-1) mmHg-1 as compared to 6.1 +/- 1.8 ml min(-1) (100 g)(-1) mmHg(-1) in the controls. Vascular reactivity of the mesenteric arteries was not different. These data suggest that coronary conductance can be modified in utero by anaemia (high flow and hypoxaemia) and that the remodelled coronary tree persists to adulthood.

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The renal cell line mIMCD3 exhibits markedly upregulated phosphorylation of the extracellular signal-regulated kinase (ERK) 1 and 2 in response to urea treatment (200 mM for 5 min). Previous data have suggested the involvement of a classical protein kinase C (cPKC)-dependent pathway in downstream events related to urea signaling. We now show that urea-inducible ERK activation requires extracellular calcium; unexpectedly, it occurs independently of activation of cPKC isoforms. Pharmacological inhibitors of known intracellular calcium release pathways and extracellular calcium entry pathways fail to inhibit ERK activation by urea. Fura 2 ratiometry was used to assess the effect of urea treatment on intracellular calcium mobilization. In single-cell analyses using subconfluent monolayers and in population-wide analyses using both confluent monolayers and cells in suspension, urea failed to increase intracellular calcium concentration. Taken together, these data indicate that urea-inducible ERK activation requires calcium action but not calcium entry. Although direct evidence is lacking, one possible explanation could include involvement of a calcium-dependent extracellular moiety of a cell surface-associated protein.

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beta-Adrenergic receptor (beta-AR)-mediated (cAMP-dependent) vasorelaxation declines with advancing age. It has been shown that angiotensin II (ANG II), a potent vasoconstrictor, enhances cAMP-mediated vasorelaxation. Therefore, we questioned whether ANG II could reverse age-related, impaired beta-AR-mediated vasorelaxation and cAMP production. Pretreatment of aortic rings from 6-wk-old or 6-mo-old male Fischer 344 rats with ANG II significantly enhanced vasorelaxation induced by isoproterenol (Iso), a beta-AR agonist, and forskolin, a direct activator of adenylyl cyclase, but not dibutyryl-cAMP or isobutylmethylxanthine. The ANG II effect was blocked by losartan but not PD-123319 and was not observed in the aortas from 12- and 24-mo-old animals. Iso-stimulated cAMP production in the aorta was enhanced in the presence of ANG II in the 6-wk-old and 6-mo-old age groups only. Results suggest ANG II cannot reverse the age-related impairment in beta-AR-dependent vasorelaxation. We conclude aging may affect a factor common to both ANG II-receptors and beta-AR signaling pathways or aging may impair cross-talk between these two receptor pathways.

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The modulation of presynaptic voltage-dependent calcium channels by classical second messenger molecules such as protein kinase C and G protein betagamma subunits is well established and considered a key factor for the regulation of neurotransmitter release. However, little is known of other endogenous mechanisms that control the activity of these channels. Here, we demonstrate a unique modulation of N-type calcium channels by farnesol, a dephosphorylated intermediate of the mammalian mevalonate pathway. At micromolar concentrations, farnesol acts as a relatively non-discriminatory rapid open channel blocker of all types of high voltage-activated calcium channels, with a mild specificity for L-type channels. However, at 250 nM, farnesol induces an N-type channel-specific hyperpolarizing shift in channel availability that results in approximately 50% inhibition at a typical neuronal resting potential. Additional experiments demonstrated the presence of farnesol in the brain (rodents and humans) at physiologically relevant concentrations (100-800 pmol/g (wet weight)). Altogether, our results indicate that farnesol is a selective, high affinity inhibitor of N-type Ca(2+) channels and raise the possibility that endogenous farnesol and the mevalonate pathway are implicated in neurotransmitter release through regulation of presynaptic voltage-gated Ca(2+) channels.

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BACKGROUND: FK506 is widely used in organ transplantation and causes hypertension. However, little is known about the impact of the drug on the cardiovascular system. METHODS: We therefore investigated the effect of FK506 on resistance artery and blood pressure responsiveness to vasoconstrictors and vasodilators. Studies were conducted in vitro using human and murine resistance artery, ex vivo in resistance artery isolated from rats treated with FK506 (6 mg/kg/day), and in vivo in conscious, treated animals. RESULTS: In vitro exposure (24 hr) of human and rat resistance artery to FK506 (1000 ng/ml) increased the sensitivity to norepinephrine (NE) and impaired the response to acetylcholine (Ach) and sodium nitroprusside (SNp). In contrast, arteries isolated from rats given FK506 for eight days showed a reduced sensitivity to NE (P < 0.05) and a normal endothelium-dependent relaxation. Their incubation with L-arginine caused a significant reduction in Ach sensitivity in the FK506 group (P < 0.05) but not in controls, suggesting enhancement of nitric oxide production by the drug. The sensitivity to SNp was reduced, as in the in vitro experiments (P < 0.05). Rats given FK506 for eight days presented blood pressure similar to that in controls but also presented signs of a compensatory response to excess vasodilation: tachycardia (P < 0.01), reduced blood pressure sensitivity to NE and Ach, blunted heart rate response to both agonists, and exaggerated hypotension at high doses of Ach. After 21 days of treatment, blood pressure remained similar to that in controls, but resistance artery showed further functional deterioration, with significant impairment of the maximum responses to Ach and to SNp. CONCLUSION: FK506 presents significant vascular toxicity affecting mainly smooth muscle relaxation and alters vascular hemodynamics. The data suggest that similar cardiovascular changes may occur in transplant patients and represent the forerunner of hypertension often seen with more prolonged use of the drug.

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We recently demonstrated that farnesol, a 15-carbon isoprenoid, blocks L-type Ca2+ channels in vascular smooth muscle cells. To elucidate farnesol's mechanism of action, we performed whole-cell and perforated-patch clamp experiments in rat aortic A7r5 cells and in Chinese hamster ovary (CHO) C9 cells expressing smooth muscle Ca2+ channel alpha 1C subunits. Farnesol dose-dependently and voltage-independently inhibited Ba2+ currents in both A7r5 and CHOC9 cells, with similar half-maximal inhibitions at 2.6 and 4.3 micromol/L, [corrected] respectively (P=NS). In both cell lines, current inhibition by farnesol was prominent over the whole voltage range without changes in the current-voltage relationship peaks. Neither intracellular infusion of the stable GDP analogue guanosine-5'-O-(2-thiodiphosphate) (100 micromol/L) [corrected] via the patch pipette nor strong conditioning membrane depolarization prevented the inhibitory effect of farnesol, which indicates G protein-independent inhibition of Ca2+ channels. In an analysis of the steady-state inactivation curve for voltage dependence, farnesol induced a significant, negative shift ( approximately 10 mV) of the potential causing 50% channel inactivation in both cell lines (P<0. 001). In contrast, the steepness factor characterizing the voltage sensitivity of the channels was unaffected. Unlike pharmacological Ca2+ channel blockers, farnesol blocked Ca2+ currents in the resting state: initial block was 63+/-8% in A7r5 cells and 50+/-9% in CHOC9 cells at a holding potential of -80 mV. We then gave 500 mg/kg body weight farnesol by gavage to Sabra hypertensive and normotensive rats and found that farnesol reduced blood pressure significantly in the hypertensive strain for at least 48 hours. We conclude that farnesol may represent an endogenous smooth muscle L-type Ca2+ channel antagonist. Because farnesol is active in cells expressing only the pore-forming alpha1 subunit, the data further suggest that this subunit represents the molecular target for farnesol binding and principal action. Finally, farnesol has a blood pressure-lowering action that may be relevant in vivo.

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OBJECTIVES: Carboxyl methylation is a reversible post-translational event which regulates the function of several cellular proteins. Because the human Na+-H+ antiporter (NHE-1) possesses a C-terminal consensus sequence for carboxyl methylation, we examined the role of protein carboxyl methylation in the regulation of intracellular pH homeostasis. DESIGN: Experiments were conducted using human platelets and N-acetyl-S-trans,trans-farnesyl-L cysteine (AFC), a specific prenylcysteine methyltransferase inhibitor. The effect of AFC on both basal intracellular pH (pHi) and on the kinetic properties of the Na+-H+ antiporter was characterized. MATERIALS AND METHODS: pHi was determined in cell suspensions using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein tetraacetoxymethyl ester, a fluorescent pH indicator. The kinetics properties of the Na+-H+ antiporter activity were determined using platelets acidified with nigericin and challenged with varying extracellular concentrations of Na+. RESULTS: AFC (20 micromol/l) decreased basal pHi significantly (7.047 +/- 0.011 versus 7.133 +/- 0.012 for control, P< 0.001). The acidification was dose-dependent and reached steady state 3 min after AFC addition. In the absence of extracellular Na+, the platelets were acidified to the same extent with AFC or with ethanol (control): 6.530 +/- 0.031 versus 6.532 +/- 0.031 (P= 0.97). However, upon addition of Na+, the platelets treated with AFC showed a significant decrease in the maximal value for initial pHi recovery compared with controls: 0.788 +/- 0.041 versus 0.983 +/- 0.047 pH/min (P< 0.02). AFC also increased the Hill coefficient (2.89 +/- 0.22 versus 2.14 +/- 0.16, P < 0.03), and tended to decrease K0.5, the [Na+] corresponding to half-maximal activation (51.3 +/- 1.8 versus 60.5 +/- 3.9 mmol/l, P = 0.06) of the antiporter. CONCLUSION: Our data indicate that inhibition of carboxyl methylation reduces basal pHi and alters the kinetic properties of the Na+-H+ antiporter in human platelets, suggesting that carboxyl methylation is implicated in the regulation of intracellular pH homeostasis.

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Earlier experiments with animal and human arteries have shown that farnesol, a natural 15-carbon (C15) isoprenoid, is an inhibitor of vasoconstriction (Roullet, J.-B., Xue, H., Chapman, J., McDougal, P., Roullet, C. M., and McCarron, D. A. (1996) J. Clin. Invest. 97, 2384-2390). We report here that farnesol reduced KCl- and norepinephrine-dependent cytosolic Ca2+ transients in fura-2-loaded intact arteries. An effect on Ca2+ signaling was also observed in cultured aortic smooth muscle cells (A10 cells). In these cells, farnesol reduced KCl-induced [Ca2+]i transients and mimicked the inhibitory effect of Ca2+-free medium on the [Ca2+]i response to both 12,13-phorbol myristate acetate, a protein kinase C activator, and thapsigargin, a specific endoplasmic reticulum ATPase inhibitor. Perforated patch-clamp experiments further showed in two vascular smooth muscle cell lines (A10 and A7r5), a reversible, dose-dependent inhibitory effect of farnesol on L-type Ca2+ currents (IC50 = 2.2 microM). Shorter (C10, geraniol) and longer (C20, geranylgeraniol) isoprenols were inactive. L-type Ca2+ channel blockade also occurred under tight (gigaohm) seal configuration using cell-attached, single-channel analysis, thus suggesting a possible action of farnesol from within the intracellular space. We finally demonstrated that farnesol did not affect Ca2+-sensitive pathways implicated in smooth muscle contraction, as tested with alpha-toxin permeabilized arteries. Altogether, our results indicate that farnesol is an inhibitor of vascular smooth muscle Ca2+ signaling with plasma membrane Ca2+ channel blocker properties. The data have implications for the endogenous and pharmacological regulation of vascular tone by farnesol or farnesol analogues.

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BACKGROUND: Previous studies have shown that farnesol, a 15-carbon nonsterol derivative of mevalonic acid, inhibits vasoconstriction. Because of its lipophilic properties, we hypothesized that farnesol increased membrane dynamics, thus reducing uptake of Ca2+ and contraction. OBJECTIVE: To characterize the effect of farnesol on cell membrane fluidity. DESIGN: The study was conducted using A7r5 cells, a rat aortic vascular smooth muscle cell line. Inhibition of Ca2+ uptake by farnesol was first established in these cells. Then, the effect of farnesol on membrane dynamics was determined. Finally, to ascertain that activation of Ca2+ extrusion and reuptake processes by farnesol did not occur, Ca2+-ATPase activity was examined. METHODS: Membrane fluidity in cell homogenates was estimated using two fluorescent dyes (1,6-diphenyl-1,3,5-hexatriene) and (1-[-(trimethylamino)-phenyl]-6-phenyl-1,3,5-hexatriene). Ca2+ uptake was determined by monitoring the changes in cytosolic Ca2+ concentration ([Ca2+]i) in fura-2-loaded cells after addition of KCI. Ca2+-ATPase activity was measured in 100000 x g cell fractions. RESULTS: Farnesol reduced KCI-induced (Ca2+]i transients significantly (P < 0.001), but did not modify membrane dynamic properties [0.214+/-0.007 versus 0.218+/-0.007 (n = 10) and 0.142+/-0.002 versus 0.146+/-0.003 (n = 5) for 1 -[-(trimethylamino)-phenyl]-6-phenyl-1,3,5-hexatriene and 1,6-diphenyl-1,3,5-hexatriene anisotropies, respectively; NS]. Administration of up to 30 micromol/l farnesol did not affect Ca2+-ATPase activity. CONCLUSION: Farnesol inhibits KCI-dependent rise of [Ca2+]i in A7r5 cells. This effect of farnesol is not related to a global change in plasma membrane lipid organization or to activation of Ca2+ pumps. Other mechanisms such as direct inhibition of voltage-dependent Ca2+ channels could therefore explain the biologic action of farnesol in the vascular tissue.

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Recent studies have suggested that nonsterol, mevalonate-derived metabolites are implicated in the control of vascular tone and blood pressure. Because of the metabolic importance of farnesyl pyrophosphate, a 15-carbon (C15) intermediate of the cholesterol pathway, the vasoactive properties of the farnesyl motif were investigated. Two farnesyl analogues were used: farnesol, the natural dephosphorylated form of farnesyl pyrophosphate, and N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), a synthetic mimic of the carboxyl terminus of farnesylated proteins. Both compounds inhibited NE-induced vasoconstriction in rat aortic rings at micromolar concentration. Their action was rapid, dose dependent, and reversible. Shorter (C10) and longer (C20) isoprenols as well as N-acetyl-S-geranyl-L-cysteine (C10) did not inhibit the response to NE. In contrast, N-acetyl-S-geranylgeranyl-L-cysteine (C20), exhibited vasoactive properties similar to AFC. It was further demonstrated that AFC and farnesol inhibited KCl and NaF-induced contractions, suggesting a complex action on Ca2+ channels and G protein-dependent pathways. Finally, the effect of farnesol and AFC on the NE response was reproduced in human resistance arteries. In conclusion, mevalonate-derived farnesyl analogues are potent inhibitors of vasoconstriction. The study suggests that farnesyl cellular availability is an important determinant of vascular tone in animals and humans, and provides a basis for exploring farnesyl metabolism in humans with compromised vascular function as well as for using farnesyl analogues as regulators of arterial tone in vivo.

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Previous data in rat conductance vessels indicated that cellular mevalonate contributes to vascular tone and systemic blood pressure control. Using exogenous mevalonate (M) or lovastatin, a 3-hydroxy-3-methyl-glutaryl CoA (HMG-CoA) reductase inhibitor (L), we characterized the role of mevalonate availability in resistance artery function, both in experimental animals and humans. Rat mesenteric artery resistance vessels (MARV, n = 9) were incubated for 48 h with either L, M, L + M, or vehicle (V) and tested for reactivity to NE, serotonin, acetylcholine, atrial natriuretic peptide, and sodium nitroprusside (SNP). Lovastatin increased sensitivity to NE (P < 0.03) and serotonin (P < 0.003), and significantly impaired the response to all three vasodilators. These effects were reversed by co-incubation with mevalonate. Mevalonate alone had no effect. In separate experiments, intravascular free Ca2+ concentration (ivfCa2+) was determined in fura-2AM loaded MARV. Basal ivfCa2+ was increased after a 48-h exposure to L (52.7 +/- 4.6 nM, L, vs. 29.7 +/- 2.4 nM, V, n = 12, P < 0.003), as were ivfCa2+ levels following stimulation with low (100 nM) NE concentrations. Similar ivfCa2+ concentrations were achieved during maximum contraction with NE (10 mM) in both groups. Human resistance arteries of human adipose tissue were also studied. Lovastatin increased the sensitivity to NE (ED50 = 372 +/- 56 nM, V, and 99 +/- 33 nM, L, P < 0.001) and significantly decreased the relaxation to acetylcholine and SNP of human vessels. We conclude that mevalonate availability directly contribute to resistance vessel function and vascular signal transduction systems in both experimental animals and humans. The study calls for the identification of non-sterol, mevalonate-derived vasoactive metabolites, and suggests that disorders of the mevalonate pathway can alter vascular tone and cause hypertension.

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The abnormal intestinal Ca2+ transport reported in spontaneously hypertensive rats (SHR) has been attributed to decreased responsiveness to calcitriol. We reexamined this hypothesis by studying the calcitriol regulation of SHR duodenal calbindin-D9K and calmodulin and the relation of calcitriol to Ca2+ uptake by isolated enterocytes. SHR and normotensive Wistar-Kyoto (WKY) rats were injected with either 50 ng/d calcitriol (vit-D) or vehicle alone (control) for 3 days. Decreased calbindin-D9K (P < .001) and cellular Ca2+ flux (P < .001) were observed in control SHR. Calcitriol increased total cell and brush border calbindin-D9K (P < .0001); this variation paralleled plasma calcitriol levels in both strains. In contrast, Ca2+ flux, which increased in vit-D animals, remained lower in SHR for plasma calcitriol levels similar to those in WKY rats. Immunoreactive calmodulin was similar in both strains whether assayed in total cell or brush border membranes. In contrast, when measured by ligand blotting (45Ca), calmodulin was lower in SHR than in WKY rats (P < .01), suggesting the existence of a calmodulin pool with reduced Ca2+ binding capacity in the hypertensive strain. Calcitriol had no effect on calmodulin in either strain. In conclusion, Ca2+ binding protein regulation by calcitriol is normal in the SHR, and decreased hormone responsiveness cannot account for the defective duodenal calcium transport of this experimental model of hypertension.

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