Hypoglycemic and Hypotensive Effects of Calycotome villosa Subsp. intermedia Seeds in Meriones shawi Rats: In Vivo, Ex Vivo, and In Vitro Investigations.

Calycotome villosa subsp. intermedia is used in traditional medicine for the prevention and self-treatment of a variety of illnesses, including diabetes mellitus, obesity, and hypertension. The present study aims to investigate the in vivo, ex vivo, and in vitro hypoglycemic and hypotensive effects of the lyophilized aqueous extract of Calycotome villosa subsp. intermedia seeds (CV) on Meriones shawi submitted to hypercaloric diet and physical inactivity (HCD/PI) for 12 weeks. This diet induces a type 2 diabetes/metabolic syndrome phenotype with hypertension. Furthermore, HCD/PI decreased aorta contraction due to noradrenaline, enhanced L-arginine, and depressed insulin-evoked relaxation, while the relaxing effects of the NO donor SNAP and of diazoxide were unchanged. In vivo experiments showed that the oral administration of the CV extract (50 mg/kg b.wt) for 3 consecutive weeks significantly attenuated the development of type 2 diabetes, obesity, dyslipidemia, and hypertension. These effects may involve the improvement of lipid metabolism, insulin sensitivity, systolic arterial pressure, and urine output. Additionally, ex vivo and in vitro investigations revealed that CV treatment improved vascular contraction to noradrenaline, induced a slight aorta relaxation in response to carbachol, increased the vasorelaxation effect evoked by insulin, and depressed the L-arginine evoked relaxation. However, CV did not change the endothelium-independent vasorelaxation response evoked by SNAP or diazoxide. Hence, the present study provides useful information and supports the traditional use of CV in the prevention and self-treatment of numerous ailments. Overall, it can be concluded that Calycotome villosa subsp. intermedia seed extracts might be useful in the management of type 2 diabetes and hypertension.

Ajuga iva water extract antihypertensive effect on stroke-prone spontaneously hypertensive rats, vasorelaxant effects ex vivo and in vitro activity of fractions.

ETHNOPHARMACOLOGICAL RELEVANCE: Ajuga iva (L.) Schreb. (Labiatae) (AI) is used in folk medicine for a variety of ailments, including diabetes mellitus and hypertension. AIM OF THE STUDY: In this work, we aimed to investigate the antihypertensive and vasorelaxant effects of AI aqueous extract in stroke prone spontaneously hypertensive rats (SHR-SP). MATERIAL AND METHODS: Male SHR-SP rats were orally force-fed AI aqueous extract (500 mg/kg body weight) daily for one week. Systolic blood pressure and urine output were recorded in vivo by non-invasive methods. AI vasoactive effects on noradrenaline contractile response and acetylcholine-evoked relaxation were assessed ex vivo on aorta rings of treated and untreated SHR-SP rats. AI extract was then subjected to bio-guided fractionation using solvents of increasing polarity. For each fraction, in vitro vasorelaxation assay was performed on noradrenaline-precontracted aorta of Wistar rats, in the absence/presence of N-nitro-L-arginine (L-NNA). HPLC analysis of AI total extract, and the most in vitro active AI residual aqueous extract fraction (A1) was performed using naringin, naringenin, apigenin, apigenin 7-O-glucoside as marker compounds. RESULTS: AI aqueous extract (500 mg/kg) significantly (P < 0.05) decreased systolic blood pressure (SBP) in SHR-SP rats, while not affecting the urine output. In ex vivo experiments, the total extract decreased contractile response to noradrenaline of aortic rings isolated from AI-treated SHR-SP rats with or without addition of N-nitro-L-arginine, but endothelium dependent relaxation evoked by acetylcholine in noradrenaline-contracted aortic rings was not affected by the extract treatment. In vitro experiments on AI aqueous extract fractions showed that its polar fraction was the only one affecting in vitro noradrenaline induced contractions, but only in an endothelium dependent manner. This fraction was shown by HPLC-UV to contain flavonoid glycosides among other polar compounds whose activity and mode of action may be modified in vivo by metabolization. CONCLUSION: These results support the use of AI as antihypertensive treatment in folk medicine. The systolic blood pressure decrease may be attributed at least in part to vasorelaxant glycosylated/polar phenolic compounds as flavonoids and/or their metabolites.

Evaluation of cytotoxic effects and acute and chronic toxicity of aqueous extract of the seeds of Calycotome villosa (Poiret) Link (subsp. intermedia) in rodents.

OBJECTIVE: The present investigation was carried out to evaluate the safety of an aqueous extract of the seeds of Calycotome villosa (Poiret) Link (subsp. intermedia) by determining its cytotoxicity and potential toxicity after acute and sub-chronic administration in rodents. MATERIALS AND METHODS: Cytotoxic activity was tested in cancer and non-cancer cell lines HeLa, Mel-5, HL-60 and 3T3. Acute toxicity tests were carried out in mice by a single oral administration of Calycotome seed-extract (0 - 12 g/kg) as well as intraperitoneal doses of 0 - 5 g/kg. Sub-chronic studies were conducted in Wistar rats by administration of oral daily doses for up to 90 days. Changes in body and vital organ weights, mortality, haematology, clinical biochemistry and histologic morphology were evaluated. RESULTS: The lyophilized aqueous extract of C. villosa exhibited a low cytotoxicity in all cell lines tested with an IC50 > 100 µg/ml. In the acute study in mice, intra-peritoneal administration caused dose-dependent adverse effects and mortality with an LD50 of 4.06 ± 0.01 g/kg. In the chronic tests, neither mortality nor visible signs of lethality was seen in rats. Even AST and ALT were not affected while a significant decrease in serum glucose levels, at 300 and 600 mg/kg was detected. Histopathological examination of the kidney and liver did not show any alteration or inflammation at the end of treatment. CONCLUSION: In conclusion, the aqueous extract of C. villosa seed appeared to be non-toxic and did not produce mortality or clinically significant changes in the haematological and biochemical parameters in rats.

Characterization of vascular dysregulation in meriones shawi after high-calorie diet feeding.

The present study was initiated to characterize vascular dysregulations (contraction and relaxation) associated with metabolic defects in Merions shawi, a rodent from the gerbillidae family, submitted to 12 weeks high-calorie diet. This diet induces a type 2 diabetes/metabolic syndrome phenotype with hypertension. In diabetic meriones, body weight increase was associated with hyperglycemia, increased insulinemia, and insulin resistance. Compared to lean meriones, diabetic meriones showed decreased aorta contraction to noradrenaline, which was normalized after NOS inhibition. Endothelium-dependent relaxation to carbachol was enhanced, while relaxing effects of the NO donor SNAP and of diazoxide were unchanged. Insulin-evoked relaxation was depressed in aorta from diabetic meriones, and L-arginine relaxed contracted arteries from diabetic meriones, but not from lean meriones. Urine NOX level and iNOS mRNA muscle expression were significantly higher in diabetic meriones compared to lean animals. These data strongly suggest that iNOS may have a pathogenic role in vascular dysfunction observed in diet-induced diabetic meriones.

Extracellular calcium modulates the inhibitory effect of 4-aminopyridine on Kv current in vascular smooth muscle cells.

4-Aminopyridine is widely used as a Kv channel blocker. However, its mechanism of action is still a matter of debate. Extracellular calcium as well as 4-aminopyridine have been reported to interact with the activation kinetics of particular Kv channels. The objective of the present study was to investigate whether extracellular calcium could modulate the inhibition of Kv current by 4-aminopyridine in vascular myocytes. Kv current was recorded by using whole-cell patch-clamp in freshly isolated smooth muscle cells from rat mesenteric artery. Macroscopic properties of Kv current were not affected by change in extracellular calcium from 0 to 2mM. During a 10s depolarizing pulse, 4-aminopyridine inhibited the peak current without affecting the end-pulse current. The concentration-effect curve of 4-aminopyridine was shifted to the left in the presence of 2mM calcium compared to 0 calcium. After 4-aminopyridine washout, current recovery from block was slower in the presence than in the absence of calcium. Inhibition of Kv current by 4-aminopyridine (0.5mM) and the Kv2 blocker stromatoxin (50nM) was additive and stromatoxin did not alter the potentiation of 4-aminopyridine effect by extracellular calcium. These results showed that extracellular calcium modulated the inhibitory potency of 4-aminopyridine on Kv current in vascular myocytes. The component of Kv current that was inhibited by 4-aminopyridine in a calcium-sensitive manner was distinct from Kv2 current.

Redundant control of migration and adhesion by ERM proteins in vascular smooth muscle cells.

Ezrin, radixin, and moesin possess a very similar structure with a C-terminal actin-binding domain and a N-terminal FERM interacting domain. They are known to be involved in cytoskeleton organization in several cell types but their function in vascular smooth muscle cells (VSMC) is still unknown. The aim of this study was to investigate the role of ERM proteins in cell migration induced by PDGF, a growth factor involved in pathophysiological processes like angiogenesis or atherosclerosis. We used primary cultured VSMC obtained from rat aorta, which express the three ERM proteins. Simultaneous depletion of the three ERM proteins with specific siRNAs abolished the effects of PDGF on cell architecture and migration and markedly increased cell adhesion and focal adhesion size, while these parameters were only slightly affected by depletion of ezrin, radixin or moesin alone. Rac1 activation, cell proliferation, and Ca(2+) signal in response to PDGF were unaffected by ERM depletion. These results indicate that ERM proteins exert a redundant control on PDGF-induced VSMC migration by regulating focal adhesion turn-over and cell adhesion to substrate.

Different effect of Rho kinase inhibition on calcium signaling in rat isolated large and small arteries.

In addition to its role in the regulation of artery contraction, Rho kinase (ROCK) was reported to be involved in the cytosolic calcium response to vasoconstrictor agonists in rat aorta and superior mesenteric artery (SMA). However, it remains to be determined whether ROCK also contributes to calcium signaling in resistance arteries, which play a major role in blood pressure regulation. The investigation of the effect of ROCK inhibition on the calcium and contractile responses of rat resistance mesenteric artery (RMA), in comparison with aorta and SMA, indicated that the calcium response to noradrenaline was inhibited by the ROCK inhibitor Y-27632 in aorta and SMA but not in RMA. The effect of Y-27632 on the calcium signal was unaffected by cytochalasin-D. ROCK activation in noradrenaline-stimulated arteries was confirmed by the inhibition of myosin light chain phosphorylation by Y-27632. Moreover, noradrenaline-induced calcium signaling was similarly inhibited by nimodipine in aorta, SMA and RMA, but nimodipine sensitivity of the contraction increased from the aorta to the RMA, suggesting that the contraction was controlled by different sources of calcium. In pressurized RMA, Y-27632 and H-1152 depressed pressure-induced calcium responses and abolished myogenic contraction. These results stress the important differences in calcium signaling between conductance and resistance arteries.

Rho kinase regulation of vasopressin-induced calcium entry in vascular smooth muscle cell: comparison between rat isolated aorta and cultured aortic cells.

In addition to its role in artery contraction, Rho kinase (ROCK) is reported to be involved in the Ca(2+) response to vasoconstrictor agonist in rat aorta. However the signaling pathway mediated by ROCK had not been investigated so far and it was not known whether ROCK also contributed to Ca(2+) signaling in cultured vascular smooth muscle cells (VSMC), which undergo profound phenotypic changes. Our results showed that in VSMC, ROCK inhibition by Y-27632 or H-1152 had no effect on the Ca(2+) response to vasopressin, while in aorta the vasopressin-induced Ca(2+) entry was significantly decreased. The inhibition of myosin light chain kinase (MLCK) by ML-7 depressed the vasopressin-induced Ca(2+) signal in aorta but not in VSMC. The difference in ROCK sensitivity of vasopressin-induced Ca(2+) entry between aorta and VSMC was not related to an alteration of the RhoA/ROCK pathway. However, MLCK expression and activity were depressed in cultured cells compared to aorta. We concluded that the regulation of vasopressin-induced Ca(2+) entry by ROCK in aorta could involve the myosin cytoskeleton and could be prevented by the downregulation of MLCK in VSMC. These results underline the important differences in Ca(2+) regulation between whole tissue and cultured cells.

The liver toxicity biomarker study phase I: markers for the effects of tolcapone or entacapone.

The Liver Toxicity Biomarker Study is a systems toxicology approach to discover biomarkers that are indicative of a drug's potential to cause human idiosyncratic drug-induced liver injury. In phase I, the molecular effects in rat liver and blood plasma induced by tolcapone (a "toxic" drug) were compared with the molecular effects in the same tissues by dosing with entacapone (a "clean" drug, similar to tolcapone in chemical structure and primary pharmacological mechanism). Two durations of drug exposure, 3 and 28 days, were employed. Comprehensive molecular analysis of rat liver and plasma samples yielded marker analytes for various drug-vehicle or drug-drug comparisons. An important finding was that the marker analytes associated with tolcapone only partially overlapped with marker analytes associated with entacapone, despite the fact that both drugs have similar chemical structures and the same primary pharmacological mechanism of action. This result indicates that the molecular analyses employed in the study are detecting substantial "off-target" markers for the two drugs. An additional interesting finding was the modest overlap of the marker data sets for 3-day exposure and 28-day exposure, indicating that the molecular changes in liver and plasma caused by short- and long-term drug treatments do not share common characteristics.

EBP50 is involved in the regulation of vascular smooth muscle cell migration and cytokinesis.

Ezrin, Radixin, Moesin binding phosphoprotein 50 (EBP50) is a scaffold protein that possesses two PDZ interacting domains. We have shown that, in isolated artery stimulated with noradrenaline, EBP50 interacts with several elements of the cytoskeleton. However, the contribution of EBP50 to the organization of the cytoskeleton is unknown. We have used primary cultured vascular smooth muscle cells to investigate the involvement of EBP50 in the regulation of cell architecture, motility and cell cycle, and to identify its target proteins and subsequent action mechanism. The results showed that depletion of EBP50 by siRNA transfection induced changes in cell architecture and increased cell migration. The same phenotype was induced by inhibition of myosin IIa and this effect was not additive in cells depleted for EBP50. Moreover, a larger proportion of binucleated cells was observed after EBP50 depletion, indicating a defect in cytokinesis. The identification, after co-immunoprecipitation, of a direct interaction of EBP50 with both tubulin and myosin IIa suggested that EBP50 could regulate cell migration and cytokinesis by linking myosin IIa fibers and microtubule network. Indeed, depletion of EBP50 also dismantled myosin IIa fibers and induced the formation of stable microtubules in lamellae expansions and Rac1 activation. This signaling cascade leads to the formation of lamellipodia, trailing tails and decrease of focal adhesion formation, triggering cell migration.

Angiotensin I-converting enzyme inhibitory activity of gelatin hydrolysates and identification of bioactive peptides.

In this project we report on the angiotensin I-converting enzyme (ACE)-inhibitory activity of a bovine gelatin hydrolysate (Bh2) that was submitted to further hydrolysis by different enzymes. The thermolysin hydrolysate (Bh2t) showed the highest in vitro ACE inhibitory activity, and interestingly a marked in vivo blood pressure-lowering effect was demonstrated in spontaneously hypertensive rats (SHR). In contrast, Bh2 showed no effect in SHR, confirming the need for the extra thermolysin hydrolysis. Hence, an angiotensin I-evoked contractile response in isolated rat aortic rings was inhibited by Bh2t, but not by Bh2, suggesting ACE inhibition as the underlying antihypertensive mechanism for Bh2t. Using mass spectrometry, seven small peptides, AG, AGP, VGP, PY, QY, DY and IY or LY or HO-PY were identified in Bh2t. As these peptides showed ACE inhibitory activity and were more prominent in Bh2t than in Bh2, the current data provide evidence that these contribute to the antihypertensive effect of Bh2t.

Identification and functional implication of a Rho kinase-dependent moesin-EBP50 interaction in noradrenaline-stimulated artery.

Ezrin, radixin, and moesin (ERM) proteins are known to be substrates of Rho kinase (ROCK), a key player in vascular smooth muscle regulation. Their function in arteries remains to be elucidated. The objective of the present study was to investigate ERM phosphorylation and function in rat aorta and mesenteric artery and the influence of ERM-binding phosphoprotein 50 (EBP50), a scaffold partner of ERM proteins in several cell types. In isolated arteries, ERM proteins are phosphorylated by PKC and ROCK with different kinetics after either agonist stimulation or KCl-induced depolarization. Immunoprecipitation of EBP50 in noradrenaline-stimulated arteries allowed identification of its interaction with moesin and several other proteins involved in cytoskeleton regulation. This interaction was inhibited by Y27632, a ROCK inhibitor. Moesin or EBP50 depletion after small interfering RNA transfection by reverse permeabilization in intact mesenteric arteries both potentiated the contractility in response to agonist stimulation without any effect on contractile response induced by high KCl. This effect was preserved in ionomycin-permeabilized arteries. These results indicate that, in agonist-stimulated arteries, the activation of ROCK leads to the binding of moesin to EBP50, which interacts with several components of the cytoskeleton, resulting in a decrease in the contractile response.

Vasoconstrictor and inotropic effects induced by the root bark extracts of Anthocleista schweinfurthii.

The present study was undertaken to investigate the cardiovascular effect of three extracts from the root bark of Anthocleista schweinfurthii Gilg.: an aqueous extract (AE), a dichloromethane extract (DCMR) and a fraction enriched in cardiac glycoside type compounds (CARDAN). In isolated perfused frog heart, bolus injection of the extracts produced a positive inotropic effect. The responses to AE and DCMR, but not to CARDAN, were depressed by propranolol. In isolated rat aorta, DCMR produced a transient increase in contractile tension while AE and CARDAN induced a sustained constriction. AE vasoconstrictor effect was abolished by phentolamine, while contraction evoked by CARDAN was antagonized by verapamil. In aortic rings contracted in low K+ media, the addition of K+ evoked a relaxation, which was abolished by ouabain, depressed by DCMR but not affected by either A(E) or CARDAN. These observations indicate that Anthocleista schweinfurthii contains substances that promote vasoconstriction and increase cardiac contraction. The effect of DCMR was only partially mediated by inhibition of the Na+ pump while the mechanism of action of A(E) and CARDAN was distinct from the inhibition of the Na+, K+ - ATPase pump, but could involve adrenergic receptors, or either direct or indirect activation of L-type calcium channels.

Vasorelaxant activity of essential oils from Croton zambesicus and some of their constituents.

In this study, we determined the vasorelaxant activity of essential oils of different samples of CROTON ZAMBESICUS collected in the same area in Benin at different periods and analysed their compositions by GC-FID and GC-MS. 68 compounds were identified among which 20 have not been described previously in this plant's essential oils. We observed quantitative differences among essential oils but all possess significant vasorelaxant activity on intact rat aortae contracted by KCl (IC (50) 5.6-11.8 µg/mL). This activity may, at least in part, be explained by the presence of vasorelaxant diterpenes such as ENT-18-hydroxy-trachyloban-3-one, isopimara-7,15-dien-3ß-ol, and ENT-18-hydroxy-isopimar-7,15-dien-3ß-ol, previously isolated from the dichloromethane extract of the leaves, but also to linalool (IC (50) 43.4 µg/mL) and particularly to caryophyllene oxide (IC (50) 2.5 µg/mL).

Ala-Val-Phe and Val-Phe: ACE inhibitory peptides derived from insect protein with antihypertensive activity in spontaneously hypertensive rats.

In this study, we evaluated the stability/bioavailability and in vivo antihypertensive activity of the tripeptide, Ala-Val-Phe, that was recently purified from insect protein (Spodoptera littoralis; Lepidoptera) and that showed in vitro angiotensin converting enzyme (ACE) inhibitory activity. This tripeptide is partly hydrolyzed by mucosal peptidases to Val-Phe, a more potent in vitro ACE inhibitor. In organ bath experiments using rat aorta, Val-Phe showed ACE inhibition, while Ala-Val-Phe did not. Single oral administration (5mg/kg body weight) to spontaneously hypertensive rats led to a significant decrease in blood pressure for both peptides. Docking experiments indicated an active character for Val-Phe and an inactive character for Ala-Val-Phe as potential inhibitors of human ACE. From our results, it can be suggested that after oral administration of Ala-Val-Phe, Val-Phe is released by in vivo peptidases and is responsible for in vivo activity of Ala-Val-Phe. To the best of our knowledge this is the first report of in vivo antihypertensive activity of peptides derived from insect protein.

Vascular activity of a natural diterpene isolated from Croton zambesicus and of a structurally similar synthetic trachylobane.

The aim of this study was to determine the vasorelaxant activity of a natural diterpene extracted from Croton zambesicus, ent-18-hydroxy-trachyloban-3-one (DT6), and a synthetic diterpene of similar structure, ent-trachyloban-14,15-dione (DT10) in rat aorta. DT6 and DT10 inhibited aorta contraction in a concentration-dependent manner. Both were more potent inhibitors of KCl-evoked contraction than noradrenaline-evoked contraction. Nitric oxide (NO) synthase inhibition did not significantly affect DT6 effect whereas it significantly decreased DT10 inhibitory potency. In fura-2 loaded aorta rings, DT10 simultaneously inhibited KCl-evoked contraction and cytosolic calcium increase in a concentration-dependent manner. Furthermore, DT10 significantly inhibited calcium channel current recorded by the patch-clamp technique in human neuroblastoma cells SH-SY5Y. However, despite potentiation of 8-bromo-cGMP-response, DT6 and DT10 as verapamil depressed acetylcholine-evoked relaxation, DT6 being the most potent, while only DT6 and DT10 depressed SNAP-evoked relaxation. In conclusion, these data suggest that vasorelaxant activity of diterpenes (DT) is associated with the blockade of L-type voltage-operated calcium channels. Inhibition of NO-dependent relaxation by DT could be related to a decrease in NO availability.

Expression of human amyloid precursor protein in rat cortical neurons inhibits calcium oscillations.

Synchronous calcium oscillations are observed in primary cultures of rat cortical neurons when mature networks are formed. This spontaneous neuronal activity needs an accurate control of calcium homeostasis. Alteration of intraneuronal calcium concentration is described in many neurodegenerative disorders, including Alzheimer disease (AD). Although processing of amyloid precursor protein (APP) that generates Abeta peptide has critical implications for AD pathogenesis, the neuronal function of APP remains unclear. Here, we report that expression of human APP (hAPP) in rat cortical neurons increases L-type calcium currents, which stimulate SK channels, calcium-dependent K(+) channels responsible for medium afterhyperpolarization (mAHP). In a neuronal network, increased mAHP in some neurons expressing hAPP leads to inhibition of calcium oscillations in all the cells of the network. This inhibition is independent of production and secretion of Abeta and other APP metabolites. In a neuronal network, reduction of endogenous APP expression using shRNA increases the frequency and reduces the amplitude of calcium oscillations. Altogether, these data support a key role for APP in the control of neuronal excitability.

Effect of organ culture on noradrenaline- evoked contraction, calcium signalling and TRPC expression in rat mesenteric artery.

The aim of this study was to explore the changes evoked by organ culture in the signalling pathways activated by noradrenaline in rat resistance mesenteric artery. Contractile responses and calcium signalling were significantly more sensitive to noradrenaline in arteries cultured for 48-72 h in the absence of growth factors compared to fresh arteries. Both calcium release activated by noradrenaline in calcium-free solution and calcium entry measured after the addition of external calcium were higher in cultured arteries than in fresh tissue. Blockers of non-selective cation channels (SKF-96365, flufenamic acid, Gd(3+)) more potently inhibited noradrenaline contraction in cultured arteries than in fresh ones. The src kinase inhibitors genistein or PP2 normalised the increased contraction and the increased calcium release evoked by noradrenaline in cultured arteries. In cultured arteries, trpc1 (transient receptor potential canonical 1) mRNA expression was decreased by 47 +/- 8% (n = 5, p < 0.05), while trpc6 mRNA expression was increased by 92 +/- 24% (n = 5, p < 0.05) in comparison with non-cultured arteries. Immunofluorescence analysis of protein expression confirmed the up-regulation of TRPC6 protein after culture. These results indicate that mesenteric artery culture results in src kinase-dependent increase in the responses to noradrenaline and in a change in cation channel activity, which could contribute to the increased contraction.

The liver toxicity biomarker study: phase I design and preliminary results.

Drug-induced liver injury (DILI) is the primary adverse event that results in withdrawal of drugs from the market and a frequent reason for the failure of drug candidates in development. The Liver Toxicity Biomarker Study (LTBS) is an innovative approach to investigate DILI because it compares molecular events produced in vivo by compound pairs that (a) are similar in structure and mechanism of action, (b) are associated with few or no signs of liver toxicity in preclinical studies, and (c) show marked differences in hepatotoxic potential. The LTBS is a collaborative preclinical research effort in molecular systems toxicology between the National Center for Toxicological Research and BG Medicine, Inc., and is supported by seven pharmaceutical companies and three technology providers. In phase I of the LTBS, entacapone and tolcapone were studied in rats to provide results and information that will form the foundation for the design and implementation of phase II. Molecular analysis of the rat liver and plasma samples combined with statistical analyses of the resulting datasets yielded marker analytes, illustrating the value of the broad-spectrum, molecular systems analysis approach to studying pharmacological or toxicological effects.

PKD1 haploinsufficiency is associated with altered vascular reactivity and abnormal calcium signaling in the mouse aorta.

Mutations in PKD1 are associated with autosomal dominant polycystic kidney disease (ADPKD), which leads to major cardiovascular complications. We used mice with a heterozygous deletion of Pkd1 (Pkd1+/-) and wild-type (Pkd1+/+) littermates to test whether Pkd1 haploinsufficiency is associated with a vascular phenotype in different age groups. Systolic blood pressure measured by the tail-cuff method was similar up to 20 weeks of age, but significantly higher in 30-week-old Pkd1+/- compared to Pkd1+/+. By contrast, similar telemetric recordings were obtained in unrestrained Pkd1+/- and Pkd1+/+ mice. The contractile responses evoked by KCl or phenylephrine were similar in young animals but increased in abdominal aortas of 30-week-old Pkd1+/- mice, and acetylcholine-evoked relaxation was depressed. Basal cytosolic calcium, KCl, and phenylephrine-evoked calcium signals were significantly lower in the Pkd1+/- aortas, whereas calcium release evoked by caffeine or thapsigargin was significantly larger. These changes were paralleled with a significant change in the mRNA expression of Pkd2, Trpc1, Orai1, and Serca2a in the aortas from Pkd1+/- vs. Pkd1+/+. These results are the first to indicate that haploinsufficiency in Pkd1 is associated with altered intracellular calcium homeostasis and increased vascular reactivity in the aorta with compensatory changes in transport proteins involved in the calcium signaling network.