Chiara Bolego

Safety considerations for statins.

Purpose of review The hydroxymethyl glutaryl coenzyme A reductase inhibitors or statins offer important benefits for the large populations of individuals at high risk for coronary heart disease. These drugs have a good safety profile. Nevertheless, differences in physicochemical and pharmacokinetic properties between statins may translate into significant differences in long-term safety. This review focuses on long-term adverse effects related to statin use, namely hepatotoxicity and myopathy. Moreover, the most common drugs used in combination with statins in long-term therapies are analyzed in terms of possible drug/drug interactions affecting the safety of statins. Recent findings The withdrawal of cerivastatin from the global market in 2001, because of severe cases of rhabdomyolysis, highlighted concerns regarding the safety of the entire class. Afterwards, the role of statins and their interactions with other drugs in precipitating this condition have been carefully reviewed. In approximately 60% of the total number of cases, statin-related rhabdomyolysis was found to be related to drug/drug interactions. Recently, all cases of fatal rhabdomyolysis associated with statin use have been reported to the US Food and Drug Administration. This has shown that fatal rhabdomyolysis among statin users is a rare event, the reporting rates being much less than one death per million prescriptions in the case of all statins except cerivastatin. Summary The safety and tolerability of the available statins support their use as the first-line treatment of patients at high risk for coronary heart disease, since the clinical benefits greatly outweigh the small risk of myopathy. Nevertheless, clinicians should be aware of the adverse effects possibly related to statin therapy, particularly in patients at high risk for coronary heart disease and requiring long-term multiple-drug therapies.

Characterization of the signalling pathways involved in ATP and basic fibroblast growth factor‐induced astrogliosis

1 A brief challenge of rat astrocytes with either α,β‐methyleneATP (α,β‐meATP) or basic fibroblast growth factor (bFGF) resulted, three days later, in morphological differentiation of cells, as shown by marked elongation of astrocytic processes. The P2 receptor antagonist suramin prevented α,β‐meATP‐ but not bFGF‐induced astrocytic elongation. Similar effects on astrocytic elongation were also observed with ATP and other P2 receptor agonists (β,γmeATP, ADPβS, 2meSATP and, to a lesser extent, UTP). 2 Pertussis toxin completely abolished α,β‐meATP‐ but not bFGF‐induced effects. No effects were exerted by α,β‐meATP on cyclic AMP production; similarly, neomycin had no effects on elongation of processes induced by the purine analogue, suggesting that adenylyl cyclase and phospholipase C are probably not involved in α,β‐meATP‐induced effects (see also the accompanying paper by Centemeri et al., 1997 ). The tyrosine‐kinase inhibitor genistein greatly reduced bFGF‐ but not α,β‐meATP‐induced astrocytic elongation. 3 Challenge of cultures with α,β‐meATP rapidly and concentration‐dependently increased [3H]‐arachidonic acid (AA) release from cells, suggesting that activation of phospholipase A2 (PLA2) may be involved in the long‐term functional effects evoked by purine analogues. Consistently, exogenously added AA markedly elongated astrocytic processes. Moreover, various PLA2 inhibitors (e.g. mepacrine and dexamethasone) prevented both the early α,β‐meATP‐induced [3H]‐AA release and/or the associated long‐term morphological changes, without affecting the astrocytic elongation induced by bFGF. Finally, the protein kinase C (PKC) inhibitor H7 fully abolished α,β‐meATP‐ but not bFGF‐induced effects. 4 Both α,β‐meATP and bFGF rapidly and transiently induced the nuclear accumulation of Fos and Jun. Both c‐fos and c‐jun induction by the purine analogue could be fully prevented by pretreatment with suramin. In contrast, the effects of bFGF were unaffected by this P2 receptor antagonist. 5 It was concluded that α,β‐meATP‐ and bFGF‐morphological differentiation of astrocytes occurs via independent transductional pathways. For the purine analogue, signalling involves a Gi/Go protein‐coupled P2Y‐receptor which may be linked to activation of PLA2 (involvement of an arachidonate‐sensitive PKC is speculated); for bFGF, a tyrosine kinase receptor is involved. Both pathways merge on some common intracellular target, as suggested by induction of primary response genes, which in turn may regulate late response genes mediating long‐term phenotypic changes of astroglial cells. 6 These findings implicate P2 receptors as novel targets for the pharmacological regulation of reactive astrogliosis, which has intriguing implications in nervous system diseases characterized by degenerative events.

Characterization of the Ca2+ responses evoked by ATP and other nucleotides in mammalian brain astrocytes

1 This study was aimed at characterizing ATP‐induced rises in cytosolic free calcium ion, [Ca2+]i, in a population of rat striatal astrocytes loaded with the fluorescent Ca2+ probe Fura2, by means of fluorescence spectrometry. 2 ATP triggered a fast and transient elevation of [Ca2+]i in a concentration‐dependent manner. The responses of the purine analogues 2‐methylthio‐ATP (2‐meSATP), adenosine‐5′‐O‐(2‐thiodiphosphate) (ADPβS), as well as uridine‐5′‐triphosphate (UTP) resembled that of ATP, while α,β‐methylene‐ATP (α,β‐meATP) and β,γ‐methylene‐ATP (β,γ‐meATP) were totally ineffective. 3 Suramin (50u2003μM) had only a minor effect on the ATP response, whereas pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (5u2003μM) significantly depressed the maximum response. 4 Extracellular Ca2+ did not contribute to the observed [Ca2+]i rise: removing calcium from the extracellular medium (with 1u2003mM EGTA) or blocking its influx by means of either Ni2+ (1u2003mM) or Mn2+ (1u2003mM) did not modify the nucleotide responses. 5 Furthermore, after preincubation with 10u2003μM thapsigargin, the nucleotide‐evoked [Ca2+]i increments were completely abolished. In contrast, 10u2003mM caffeine did not affect the responses, suggesting that thapsigargin‐, but not caffeine/ryanodine‐sensitive stores are involved. 6 Both application of the G‐protein blocker guanosine‐5′‐O‐(2‐thiodiphosphate) (GDPβS) (1u2003mM) and preincubation with pertussis toxin (PTx) (350u2003ngu2003ml−1) partially inhibited the nucleotide‐mediated responses. Moreover, the phospholipase C (PLC) inhibitor U‐73122, but not its inactive stereoisomer U‐73343 (5u2003μM), significantly reduced the ATP‐evoked [Ca2+]i rise. 7 In conclusion, our results suggest that, in rat striatal astrocytes, ATP‐elicited elevation of [Ca2+]i is due solely to release from intracellular stores and is mediated by a G‐protein‐linked P2Y receptor, partially sensitive to PTx and coupled to PLC.

17β-Estradiol Decreases Nitric Oxide Synthase II Synthesis in Vascular Smooth Muscle Cells1

Several studies have provided evidence for a direct effect of 17β-estradiol on vessel wall via interaction with the constitutively expressed nitric oxide synthase (NOS) by endothelium. The aim of the present study was to investigate the effect of 17β-estradiol on inducible NOS (NOS II) in primary culture of smooth muscle cells (SMC) from rat aorta. We here prove that 17β-estradiol decreases the content and activity of NOS II in SMC. This effect appears to be the consequence of ER activation, because: 1) ERα and ERβ are expressed in rat aorta SMC grown in culture; 2) low concentrations of hormone modulate NOS II activity; 3) the specific ERα antagonist ICI182,780 completely blocks 17β-estradiol effect. On the other hand, progesterone is deprived of any effect on NOS II content or activity, proving the specificity of 17β-estradiol effect. In addition, we show that 17β-estradiol can counteract the increase in NOS II activity following cytokine treatment. The observation could indicate a novel mechanism for t...

Differential effects of low- and high-dose estrogen treatments on vascular responses in female rats.

In an attempt to study the mechanisms by which estrogens affect vascular responses, we utilized aortic preparations from intact and ovariectomized female rats receiving low- and high-dose subcutaneous estrogen treatments. Oil-treated, as well as male rats, served as controls. In ovariectomized females, low-dose 17-beta-estradiol injections (5 microg/kg daily for two days) affected the basal release of nitric oxide, as evaluated by concentration-related curves to superoxide dismutase and N(G)-Methyl-L-arginine acetate, which was found to be greater in 17-beta-estradiol-treated females compared to oil-treated females or males. Conversely, the nitric oxide-related vascular relaxation evoked by acetylcholine and sodium nitroprusside was unchanged. Prostacyclin production was also evaluated. Aortic rings from ovariectomized 17-beta-estradiol-treated females released significantly more prostacyclin than those from oil-treated females. These results point out a possible role for nitric oxide and prostacyclin in the vascular protection brought about by physiological levels of estrogens. When intact females were treated with high doses of ethynilestradiol (100 microg/Kg daily for one month), a component of contraceptive pills, either the basal release of nitric oxide, or acetylcholine-induced relaxation underwent a significant decrease. Likewise, the relaxant responses to sodium nitroprusside were impaired in the aortic rings obtained from ethynilestradiol-treated animals when compared to controls. Similarly, the amount of prostacyclin released from aortic tissues obtained from ethynilestradiol-treated animals was significantly reduced. These results may provide a possible explanation for the higher incidence of cardiovascular disease in women who take contraceptive preparations containing high doses of estrogens.

The biphasic response of rat vesical smooth muscle to ATP

1 Adenosine‐5′‐triphosphate (ATP) is known to exert a variety of biological effects via the activation of either ionotropic P2X‐ or G‐protein coupled P2Y‐purinoceptor subtypes. In this study the effects induced by ATP and ATP analogues on rat bladder strips were characterized at resting tone and in carbachol‐prestimulated tissues. 2 ATP exerted a clear concentration‐dependent biphasic response, which was maximal at 1 mM concentration and was characterized by an immediate and transient contraction, followed by a slower sustained relaxation. The receptor mediating contraction was susceptible to desensitization by ATP and by the ATP analogue, α,β‐methyleneATP (α,β‐meATP) showing the typical features of the P2X‐purinoceptor; conversely, ATP‐evoked relaxation did not undergo tachyphylaxis following either ATP or α,β‐meATP. 3 The slower and sustained relaxant phase seemed to be due to activation of P2Y‐purinoceptors, based on responses obtained with the P2Y agonist, 2‐methyl‐thioATP (2‐meSATP) and, more importantly, based on the clear involvement of the G‐proteins. In fact, the G‐protein activator, guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) significantly potentiated and the G‐protein blocking agent, guanosine 5′‐O‐(2‐thio‐diphosphate) (GDPβS) completely abolished the ATP‐induced relaxation. No effects were exerted by these two G‐protein modulators on the ATP‐induced contraction. 4 The relaxant component of the ATP response of bladder tissue was not significantly influenced by nitro‐benzyl‐thioinosine (NBTI) or by 8‐phenyltheophylline (8‐PT), suggesting that the contribution of the ATP metabolite adenosine to this response was negligible. Moreover, relaxation evoked by ATP and by the adenosine analogue, 5′‐N‐ethylcarboxamidoadenosine (NECA) was additive. 5 Suramin was unable to modify either the relaxant or the contractile responses of bladder strips to ATP. However, when tested on the concentration‐response curve to the slowly hydrolysable P2x‐agonist α,β‐meATP, a rightward shift was detected, suggesting that ATP contractile responses are mediated by suramine‐sensitive P2x‐purinoceptors. 6 Uridine‐5′‐triphosphate (UTP) only induced a rapid and concentration‐dependent contraction of the rat bladder preparation, which was not desensitized by pre‐exposure to α,β‐meATP, suggesting that UTP responses were not mediated by the ‘classical’ P2X‐purinoceptor. 7 It is therefore concluded that both P2X‐ and P2Y‐purinoceptors, which mediate ATP‐induced contraction and relaxation, respectively, are present in rat bladder. Moreover, removal of epithelium did not affect ATP‐elicited contraction, whereas ATP‐induced relaxation was significantly augmented. These data suggest that P2x‐ and P2y‐ purinoceptors are localized in smooth muscle cells and that the relaxant response is probably modulated by excitatory factor(s) released by epithelial cells.

Diabetes Undermines Estrogen Control of Inducible Nitric Oxide Synthase Function in Rat Aortic Smooth Muscle Cells Through Overexpression of Estrogen Receptor-β

Background—Previous reports from our group have shown that 17&bgr;-estradiol reduces the synthesis and activity of inducible nitric oxide synthase (iNOS) in rat aortic smooth muscle cells (SMC) in response to inflammatory mediators. In this study, we investigated the effect of 17&bgr;-estradiol on iNOS function in aortic SMC from streptozotocin-diabetic rats. Methods and Results—Comparative analysis of NO release and of iNOS mRNA and protein content after 24-hour stimulation with a cytokine mixture revealed milder iNOS activation in diabetic than in control SMC. Furthermore, 17&bgr;-estradiol dose-dependently blocked iNOS synthesis and activity in control but not in diabetic SMC. The defective estrogen response in diabetic SMC at 24 hours could not be attributed to reduced expression of estrogen receptors (ER). In fact, mRNA and protein levels of ER&agr; and, to a greater extent, of ER&bgr;, were increased in diabetic compared with nondiabetic SMC. Cytokines decreased ER&agr; and ER&bgr; expression in both groups. However, 17&bgr;-estradiol dose-dependently restored the expression of ER&agr; but further downregulated that of ER&bgr;, indicating a differential regulation of ER isoforms. Conclusions—Estrogenic control of iNOS was impaired in diabetic SMC. This was associated with a larger increase of ER&bgr; than of ER&agr; protein, whereas 17&bgr;-estradiol regulated the two isoforms in an opposite fashion. Thus, modifications in the estrogen modulation of iNOS and in the expression pattern of ER may be involved in diabetic vascular dysfunction.

Diabetes abolishes the vascular protective effects of estrogen in female rats

Estrogen is known to exert a protective effect against cardiovascular disease. However, women with diabetes have three times the risk as compared with age-matched non-diabetic women. Our previous study on aortic rings of ovariectomized (OVX) female rats treated with 17-beta-estradiol (E2) demonstrated that the beneficial effect of estrogen is related to the basal release of NO from endothelial cells. In the present study, in order to understand why estrogen protection is abolished in diabetes, we tested vascular responses in OVX, streptozotocin-diabetic female rats and their non-diabetic controls receiving or not E2 replacement. Concentration-response curves to norepinephrine (NE) showed attenuation of the contractile response in E2-treated diabetic, with respect to non-diabetic preparations. This response was further impaired in diabetic, E2-deprived rats. The basal release of NO, as evaluated by concentration-related responses to N(G)-methyl-L-arginine acetate in NE-precontracted aortic rings, was found to be impaired in E2-treated diabetic rats, no further effect being induced by E2 deprivation. The endothelium-dependent relaxation produced by carbachol did not change between groups, whereas the relaxation produced by histamine was enhanced by both diabetes and E2 deprivation. However, E2 treatment counteracted the response to histamine only in preparations from non-diabetic animals. Finally, the relaxation induced by sodium nitroprusside, an endothelium-independent relaxant agent, was comparable between groups. These findings suggest that the lack of protective effects of estrogen in diabetes may be mainly ascribed to the failure of estrogen to reverse the impaired basal release of NO and the abnormal relaxation to histamine, which are observed in the aorta of diabetic rats.

Proteins of rat serum IV. Time-course of acute-phase protein expression and its modulation by indomethacine.

Changes in the concentration of major serum proteins were monitored from day 0 to day 4 in three experimental groups: rats injected with turpentine, rats receiving the turpentine shot and daily doses of indomethacine, and rats given indomethacine alone. In inflamed animals, peak changes for acute‐phase reactants, evaluated by two‐dimensional electrophoresis (2‐DE), were usually observed between 48 and 72 h after the phlogistic stimulus. By itself, indomethacine was found to affect the synthesis of most proteins (except one of the thiostatin variants and ceruloplasmin); the changes in serum levels, whether positive or negative, were the same as upon inflammation (except for kallikrein‐binding protein), but their extent and/or timing usually differed. When inflamed animals were given indomethacine, a clear‐cut difference in the concentration of some proteins was observed versus inflamed rats not given medication, at 24 h after the start of the treatments. Proteins mainly affected were α2‐macroglobulin, α2‐HS‐glycoprotein, C‐reactive protein and kallikrein‐binding protein.

Effect of substance P and capsaicin on urinary bladder of diabetic rats and the role of the epithelium

The in vitro responses of rat urinary bladder, to substance P and capsaicin were studied at 1, 4, 16, and 26 weeks of diabetes induction by streptozotocin. We also studied the role of epithelium in these responses. The results were compared with those obtained in age-matched control rats. The bladder contractile response to exogenous substance P was similar in both groups at all stages (1-26 weeks) studied, whereas the bladder response to capsaicin gradually decreased with the progression of diabetes. Atropine did not inhibit these responses whereas indomethacin slightly reduced substance P- but not capsaicin-induced responses in control and diabetic rats. The removal of epithelium slightly increased the substance P- and capsaicin-induced responses in control tissue; these responses were significantly reduced in tissue excised from diabetic rats. Our results indicate that, in rat urinary bladder, diabetes (1) provokes an impairment of capsaicin-sensitive sensory fibers but not of the cholinergic system even at an early stage (4 weeks) of the disease, (2) has no effect on the sensitivity of smooth muscle cells to substance P, (3) stimulates the release of epithelial contracting factors, partially non-prostanoic. Furthermore epithelium removal impairs acetylcholine-induced contraction in bladder excised from diabetic rats but not in controls.