Lina Puglisi

NOVEL LIPID-LOWERING PROPERTIES OF VACCINIUM MYRTILLUS L. LEAVES, A TRADITIONAL ANTIDIABETIC TREATMENT, IN SEVERAL MODELS OF RAT DYSLIPIDAEMIA: A COMPARISON WITH CIPROFIBRATE

Vaccinium myrtillus L. (blueberry) leaf infusions are traditionally used as a folk medicine treatment of diabetes. To further define this therapeutical action, a dried hydroalcoholic extract of the leaf was administered orally to streptozotocin-diabetic rats for 4 days. Plasma glucose levels were consistently found to drop by about 26% at two different stages of diabetes. Unexpectedly, plasma triglyceride (TG) were also decreased by 39% following treatment. Subsequent to the latter observation, possible lipid-lowering properties of the extract were investigated on other models of hyperlipidaemia and ciprofibrate, a well-established hypolipidaemic drug, was used as a reference compound. Both drug reduced TG levels of rats on hyperlipidaemic diet in a dose-dependent fashion. When administered at single doses over the same experimental period, blueberry and ciprofibrate were effective in lowering TG concentrations in ethanol-treated normolipidaemic animals and in genetically hyperlipidaemic Yoshida rats. Unlike ciprofibrate, however, blueberry failed to prevent the rise in plasma TG elicited by fructose and did not affect free fatty acid levels in any of the above experimental conditions. In rats treated with Triton WR-1339, blueberry feeding induced an hypolipidaemic activity one hour after injection but proved to be ineffective at later time points, thus suggesting that its hypolipidaemic action may reflect improved TG-rich lipoprotein catabolism. In addition, ciprofibrate and the extract were tested for antithrombotic activity using a collagen-triggered model of venous thrombosis in diabetic and Yoshida rats. Only ciprofibrate, however, significantly reduced thrombus formation in diabetics, possibly because of its effects on free fatty acid metabolism, whereas no effect was observed in Yoshida rats. In conclusion, the present findings indicate that active consituent(s) of Vaccinium myrtillus L. leaves may prove potentially useful for treatment of dyslipidaemiae associated with impaired TG-rich lipoprotein clearance.

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 (50 μM) had only a minor effect on the ATP response, whereas pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (5 μM) significantly depressed the maximum response. 4 Extracellular Ca2+ did not contribute to the observed [Ca2+]i rise: removing calcium from the extracellular medium (with 1 mM EGTA) or blocking its influx by means of either Ni2+ (1 mM) or Mn2+ (1 mM) did not modify the nucleotide responses. 5 Furthermore, after preincubation with 10 μM thapsigargin, the nucleotide‐evoked [Ca2+]i increments were completely abolished. In contrast, 10 mM 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) (1 mM) and preincubation with pertussis toxin (PTx) (350 ng ml−1) partially inhibited the nucleotide‐mediated responses. Moreover, the phospholipase C (PLC) inhibitor U‐73122, but not its inactive stereoisomer U‐73343 (5 μM), 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...

Prostaglandin-release impairment in the bladder epithelium of streptozotocin-induced diabetic rats

Isolated epithelial layer preparations were obtained from urinary bladders of 4-week streptozotocin-diabetic rats and used for endogenous prostaglandins E(2) and F(2alpha) determination. Tissues were incubated in modified Krebs solution under basal conditions, or in the presence of either indomethacin (5x10(-7) M), ATP (10(-5) and 10(-3) M) or bradykinin (10(-7) and 10(-5) M), and samples of incubation medium were collected at 15 and 30 min. In the presence of indomethacin, the release of prostaglandins in the incubation medium was under the detection limit of the enzyme immunoassay (EIA). The epithelium from diabetic rat urinary bladders was thicker and heavier and the absolute amount of endogenous prostaglandins E(2) and F(2alpha) was higher than for control animals, but when prostaglandin production was expressed as a fraction of tissue weight, it was reduced in diabetic epithelium. ATP and bradykinin has significantly increased the endogenous release of both prostaglandins from the epithelium when compared with the release under basal conditions. This increase was time-dependent and was higher in diabetic than in control tissues. ATP evoked a phasic and tonic contraction in bladder strips that was abolished by epithelium removal. Concentration-response curves for ATP did not differ among groups. Bradykinin evoked a long-lasting tonic contraction that was reduced significantly by epithelium removal in diabetic rat bladders only. Concentration-response curves for prostaglandin E(2) and F(2alpha) in diabetic rat bladder differed significantly from that in controls and epithelium removal did not alter these responses. It is suggested that bradykinin receptors and P2X nucleotide receptors already found in the smooth muscle detrusor might be present in the epithelial layer of the bladder. The prostaglandin-release impairment observed in this study might be responsible, in part, for bladder abnormalities observed in pathological conditions, such as diabetes.

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.

A pharmacological and histochemical study of hamster urethra and the role of urothelium.

1 Electrical field stimulation (EFS) of circular strips of hamster proximal urethra caused frequency‐dependent relaxations at raised tone. Phentolamine (10−6 m), propranolol (10−6 m) and atropine (10−6 m) were present throughout the experiment. Neurogenic relaxation was attenuated by L‐NG‐nitroarginine methyl ester (L‐NAME) (10−4 m), was restored by L‐arginine (3 × 10−3 m) but not by D‐arginine (3 × 10−3 m) and completely blocked by tetrodotoxin (10−6 m). Neurogenic relaxation was also reduced by suramin (10−4 m) and totally blocked by suramin together with L‐NAME. Strips of hamster urethra devoid of urothelium showed little, if any, relaxant response to EFS. 2 An immunohistochemical study showed nitric oxide synthase‐immunoreactive nerves in the smooth muscle layers and in the lamina propria, just beneath the urothelium, but no nitric oxide synthase (NOS) staining in the urothelial layer. 3 Noradrenaline elicited a significantly greater contraction in strips without urothelium than in control strips. L‐NAME (10−4 m) did not affect noradrenaline‐induced contraction in both control and urothelium‐free strips. The contractile response to acetylcholine was not dependent on the presence or absence of urothelium. Nevertheless the response induced by exogenous acetylcholine (10−3 m) was increased by L‐NAME (10−4 m), both in intact and in urothelium‐free strips. 4 Prostaglandin E2 (10−8‐5 × 10−6 m) and 2‐methyl‐thio‐ATP (10−9‐10−5 m) relaxed proximal urethra. Suramin (10−4 m) significantly inhibited the relaxation induced by 2‐methyl‐thio‐ATP. The amplitude of these responses was not significantly different between intact and urothelium‐free strips and was not blocked by L‐NAME (10−4 m). 5 These results suggest that nitric oxide (NO) is the principal transmitter involved in the non‐adrenergic, non‐cholinergic (NANC) relaxation of hamster proximal urethra possibly together with another inhibitory transmitter released from nerves. NO can be released from nerves located in the circular smooth muscle layer and in the lamina propria rather than in the urothelium. The reduced neurogenic relaxation in urothelium‐free preparations suggests that a NO‐dependent inhibitory factor is released from the urothelium. In addition, ATP and prostaglandin E2 may be involved, together with NO, in the urethra during micturition.