Torcetrapib-induced blood pressure elevation is independent of CETP inhibition and is accompanied by increased circulating levels of aldosterone.

BACKGROUND AND PURPOSE: Inhibition of cholesteryl ester transfer protein (CETP) with torcetrapib in humans increases plasma high density lipoprotein (HDL) cholesterol levels but is associated with increased blood pressure. In a phase 3 clinical study, evaluating the effects of torcetrapib in atherosclerosis, there was an excess of deaths and adverse cardiovascular events in patients taking torcetrapib. The studies reported herein sought to evaluate off-target effects of torcetrapib. EXPERIMENTAL APPROACH: Cardiovascular effects of the CETP inhibitors torcetrapib and anacetrapib were evaluated in animal models. KEY RESULTS: Torcetrapib evoked an acute increase in blood pressure in all species evaluated whereas no increase was observed with anacetrapib. The pressor effect of torcetrapib was not diminished in the presence of adrenoceptor, angiotensin II or endothelin receptor antagonists. Torcetrapib did not have a contractile effect on vascular smooth muscle suggesting its effects in vivo are via the release of a secondary mediator. Treatment with torcetrapib was associated with an increase in plasma levels of aldosterone and corticosterone and, in vitro, was shown to release aldosterone from adrenocortical cells. Increased adrenal steroid levels were not observed with anacetrapib. Inhibition of adrenal steroid synthesis did not inhibit the pressor response to torcetrapib whereas adrenalectomy prevented the ability of torcetrapib to increase blood pressure in rats. CONCLUSIONS AND IMPLICATIONS: Torcetrapib evoked an acute increase in blood pressure and an acute increase in plasma adrenal steroids. The acute pressor response to torcetrapib was not mediated by adrenal steroids but was dependent on intact adrenal glands.

Membrane depolarization, elevated Ca(2+) entry, and gene expression in cerebral arteries of hypertensive rats.

Elevated intracellular Ca(2+) ([Ca(2+)](i)) has been implicated in contractile and phenotypic changes in arterial smooth muscle during hypertension. This study examined the role of membrane potential and [Ca(2+)](i) in altered gene expression in cerebral arteries of a rat (Dahl) genetic model of salt-sensitive hypertension. Cerebral arteries from hypertensive animals (Dahl salt-sensitive) exhibited a tonic membrane depolarization of approximately 15 mV compared with normotensive (Dahl salt-resistant) animals. Consistent with this membrane depolarization, voltage-dependent K(+) currents were decreased in cerebral artery myocytes isolated from hypertensive animals. Arterial wall Ca(2+) was elevated in cerebral arteries from hypertensive animals, an effect reversed by diltiazem, a blocker of voltage-dependent Ca(2+) channels. This depolarization-induced increase in [Ca(2+)](i) was associated with increased activation of the transcription factor, cAMP response element binding protein, and increased expression of the immediate early gene c-fos, both of which are reversed by acute exposure to the voltage-dependent Ca(2+) channel blocker nisoldipine. This study provides the first information linking altered Ca(2+) handling to changes in gene expression in cerebral arteries during hypertension.

NFAT4 movement in native smooth muscle. A role for differential Ca(2+) signaling.

The transcription factor NFAT (nuclear factor of activated T-cells) plays a central role in mediating Ca(2+)-dependent gene transcription in a variety of cell types. Sustained increases in intracellular calcium concentration ([Ca(2+)]i) are presumed to be required for NFAT dephosphorylation by the Ca(2+)/calmodulin-dependent protein calcineurin and its subsequent nuclear translocation. Here, we provide the first identification and characterization of NFAT in native smooth muscle, showing that NFAT4 is the predominant isoform detected by reverse transcriptase-polymerase chain reaction and Western blot analysis. PDGF induces NFAT4 translocation in smooth muscle, leading to an increase in NFAT transcriptional activity. NFAT4 activation by PDGF depends on Ca(2+) entry through voltage-dependent Ca(2+) channels, because its nuclear accumulation is prevented by the Ca(2+) channel blocker nisoldipine and the K(+) channel opener pinacidil. Interestingly, elevation of [Ca(2+)]i by membrane depolarization or ionomycin treatment are not effective stimuli for NFAT4 nuclear accumulation, indicating that Ca(2+) influx is necessary but not sufficient for NFAT4 activation. In contrast, membrane depolarization readily activates the Ca(2+)-dependent transcription factor CREB (cAMP-responsive element-binding protein). The calcineurin blockers CsA and FK506 also prevented the PDGF-induced NFAT4 nuclear localization. These results indicate that both the nature of the calcium signal and PDGF-induced modulation of nuclear import-export of NFAT are critical for NFAT4 activation in this tissue.

Membrane depolarization mediates phosphorylation and nuclear translocation of CREB in vascular smooth muscle cells.

Diverse signals have the potential to modulate gene transcription through the Ca2+ and cAMP response element binding protein (CREB) in vascular smooth muscle cells (VSMCs). A key step in the transmission of these signals is import into the nucleus. Here, we provide evidence that the Ran GTPase, which regulates nuclear import, exerts different regulation over PDGF-BB, Ca2+, and cAMP signaling to CREB in VSMCs. PDGF-BB, membrane depolarization, and forskolin increased levels of activated CREB (P-CREB) and c-fos in VSMCs and intact aorta. The calcium channel antagonist nimodipine reduced the level of P-CREB stimulated by membrane depolarization, but not by PDGF-BB or forskolin. Block of Ran-mediated nuclear import, by wheat germ agglutinin or an inactivating Ran mutant (T24N Ran), significantly reduced nuclear P-CREB in response to PDGF-BB or membrane depolarization, but enhanced levels of P-CREB in response to forskolin. Contrary to expectation, block of nuclear import led to the appearance of P-CREB in the cytoplasm after depolarization. Furthermore, blocking nuclear export with leptomycin B reduced P-CREB stimulation by both depolarization and PDGF-BB. These results suggest that translocation of CREB between the nucleus and the cytoplasm provides an important role in CREB activating pathways in VSMCs.

Voltage dependence of Ca2+ sparks in intact cerebral arteries.

Ca2+ sparks have been previously described in isolated smooth muscle cells. Here we present the first measurements of local Ca2+ transients ("Ca2+ sparks") in an intact smooth muscle preparation. Ca2+ sparks appear to result from the opening of ryanodine-sensitive Ca2+ release (RyR) channels in the sarcoplasmic reticulum (SR). Intracellular Ca2+ concentration ([Ca2+]i) was measured in intact cerebral arteries (40-150 micron in diameter) from rats, using the fluorescent Ca2+ indicator fluo 3 and a laser scanning confocal microscope. Membrane potential depolarization by elevation of external K+ from 6 to 30 mM increased Ca2+ spark frequency (4. 3-fold) and amplitude (approximately 2-fold) as well as global arterial wall [Ca2+]i (approximately 1.7-fold). The half time of decay ( approximately 50 ms) was not affected by membrane potential depolarization. Ryanodine (10 microM), which inhibits RyR channels and Ca2+ sparks in isolated cells, and thapsigargin (100 nM), which indirectly inhibits RyR channels by blocking the SR Ca2+-ATPase, completely inhibited Ca2+ sparks in intact cerebral arteries. Diltiazem, an inhibitor of voltage-dependent Ca2+ channels, lowered global [Ca2+]i and Ca2+ spark frequency and amplitude in intact cerebral arteries in a concentration-dependent manner. The frequency of Ca2+ sparks (<1 s-1 . cell-1), even under conditions of steady depolarization, was too low to contribute significant amounts of Ca2+ to global Ca2+ in intact arteries. These results provide direct evidence that Ca2+ sparks exist in quiescent smooth muscle cells in intact arteries and that changes of membrane potential that would simulate physiological changes modulate both Ca2+ spark frequency and amplitude in arterial smooth muscle.

Frequency modulation of Ca2+ sparks is involved in regulation of arterial diameter by cyclic nucleotides.

Forskolin, which elevates cAMP levels, and sodium nitroprusside (SNP) and nicorandil, which elevate cGMP levels, increased, by two- to threefold, the frequency of subcellular Ca2+ release ("Ca2+ sparks") through ryanodine-sensitive Ca2+ release (RyR) channels in the sarcoplasmic reticulum (SR) of myocytes isolated from cerebral and coronary arteries of rats. Forskolin, SNP, nicorandil, dibutyryl-cAMP, and adenosine increased the frequency of Ca(2+)-sensitive K+ (KCa) currents ["spontaneous transient outward currents" (STOCs)] by two- to threefold, consistent with Ca2+ sparks activating STOCs. These agents also increased the mean amplitude of STOCs by 1.3-fold, an effect that could be explained by activation of KCa channels, independent of effects on Ca2+ sparks. To test the hypothesis that cAMP could act to dilate arteries through activation of the Ca2+ spark-->KCa channel pathway, the effects of blockers of KCa channels (iberiotoxin) and of Ca2+ sparks (ryanodine) on forskolin-induced dilations of pressurized cerebral arteries were examined. Forskolin-induced dilations were partially inhibited by iberiotoxin and ryanodine (with no additive effects) and were entirely prevented by elevating external K+. Forskolin lowered average Ca2+ in pressurized arteries while increasing ryanodine-sensitive, caffeine-induced Ca2+ transients. These experiments suggest a new mechanism for cyclic nucleotide-mediated dilations through an increase in Ca2+ spark frequency, caused by effects on SR Ca2+ load and possibly on the RyR channel, which leads to increased STOC frequency, membrane potential hyperpolarization, closure of voltage-dependent Ca2+ channels, decrease in arterial wall Ca2+, and, ultimately, vasodilation.

Ca2+ channels, ryanodine receptors and Ca(2+)-activated K+ channels: a functional unit for regulating arterial tone.

Local calcium transients ('Ca2+ sparks') are thought to be elementary Ca2+ signals in heart, skeletal and smooth muscle cells. Ca2+ sparks result from the opening of a single, or the coordinated opening of many, tightly clustered ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In arterial smooth muscle, Ca2+ sparks appear to be involved in opposing the tonic contraction of the blood vessel. Intravascular pressure causes a graded membrane potential depolarization to approximately -40 mV, an elevation of arterial wall [Ca2+]i and contraction ('myogenic tone') of arteries. Ca2+ sparks activate calcium-sensitive K+ (KCa) channels in the sarcolemmal membrane to cause membrane hyperpolarization, which opposes the pressure induced depolarization. Thus, inhibition of Ca2+ sparks by ryanodine, or of KCa channels by iberiotoxin, leads to membrane depolarization, activation of L-type voltage-gated Ca2+ channels, and vasoconstriction. Conversely, activation of Ca2+ sparks can lead to vasodilation through activation of KCa channels. Our recent work is aimed at studying the properties and roles of Ca2+ sparks in the regulation of arterial smooth muscle function. The modulation of Ca2+ spark frequency and amplitude by membrane potential, cyclic nucleotides and protein kinase C will be explored. The role of local Ca2+ entry through voltage-dependent Ca2+ channels in the regulation of Ca2+ spark properties will also be examined. Finally, using functional evidence from cardiac myocytes, and histological evidence from smooth muscle, we shall explore whether Ca2+ channels, RyR channels, and KCa channels function as a coupled unit, through Ca2+ and voltage, to regulate arterial smooth muscle membrane potential and vascular tone.

Increased myogenic tone and diminished responsiveness to ATP-sensitive K+ channel openers in cerebral arteries from diabetic rats.

Diabetes mellitus has profound adverse effects on vascular and, in particular, endothelial function. Although pressure-induced constriction ("myogenic tone") is a major contributor to the regulation of blood flow, little is known about the effects of diabetes on this response. Diabetes has been shown to diminish the dilation of cerebral arteries to synthetic ATP-sensitive K+ (KATP) channel openers. In this study, we explored the effects of diabetes induced in rats by streptozotocin on cerebral artery (250 to 300 microns) myogenic tone and on vasodilations to the synthetic KATP channel openers pinacidil and levcromakalim. Elevation of intravascular pressure caused a graded membrane potential depolarization and constriction, which was greater in arteries from diabetic rats compared with normal rats (at 60 mm Hg, 5 mV more depolarized and 22 microns more constricted). Pressurized arteries (at 60 mm Hg) from diabetic rats were 5- to 15-fold less sensitive to pinacidil and levcromakalim than were control arteries (EC50 values for pinacidil and levcromakalim were 1.4 and 0.6 mumol/L, respectively, in diabetic animals and 0.3 and 0.04, respectively, in control animals; P < .05). Removal of the endothelium or addition of a NO synthase inhibitor, NG-nitro-L-arginine (LNNA), in control arteries decreased the sensitivity to KATP channel openers and depolarized and constricted control arteries to levels similar to those observed in arteries from diabetic animals. Sodium nitroprusside caused a membrane potential hyperpolarization and enhanced the response to pinacidil in arteries from diabetic animals. Removal of the endothelium or LNNA had little effect on the apparent KATP channel opener sensitivity, the membrane potential, and pressure-induced constrictions of arteries from diabetic animals. The results are consistent with the hypothesis that this type of diabetes leads to a decrease in tonic NO release from the endothelium, which in turn causes membrane potential depolarization and vasoconstriction, resulting in a diminished response to KATP channel openers.