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The well-known side effects of anticholinergic compounds used to treat urinary incontinence caused by detrusor overactivity have addressed the interest on other pharmacological intervention. The purpose of the present work was to investigate the possible changes in purinergic and cholinergic components of parasympathetic neurotransmission in obstructed rat bladders with detrusor overactivity, and to examine the effect of the association of suramin, atropine and indomethacin on nerve-mediated responses to electrical field stimulation (EFS). Mechanical responses to exogenous acetylcholine, ATP and substance P were also evaluated. Altered sensitivities to acetylcholine and to the sensory neurotransmitter substance P, but unchanged sensitivity to the stable ATP analogue α,β-methyleneATP were observed in bladders from obstructed rats. Suramin and atropine inhibited purinergic and cholinergic components of the neurogenic responses evoked by EFS in detrusor strips from control and obstructed rats. Interestingly, suramin enhanced the antagonistic effect of atropine on neurogenic responses of detrusor strips at all frequencies of stimulation tested. Our results suggest that the association between an antimuscarinic drug and an antagonist of P2X purinoceptors such as suramin might be helpful to reduce the therapeutic dosage of the antimuscarinic drug, along with its side effects. This approach may be of interest in the therapy of patients with bladder incontinence caused by detrusor overactivity, which do not even respond to a maximal dosage of antimuscarinic drug.

Diabetes mellitus reduces gender-related differences in the prevalence of cardiovascular disease by fading the vascular protective effects afforded by estrogen in females. However, the impact of estrogen treatment on and the contribution of androgens to vascular function in vessels from male diabetics are largely unknown. We investigated the effects of androgen deficiency and in vivo estrogen treatment by assessing the responsiveness to a number of vasoactive agents and the formation of eicosanoid mediators in aortic rings from intact and castrated streptozotocin-diabetic rats which had been implanted with 17beta-estradiol (E2) or its vehicle for 5 days. Castration was found to attenuate contractility to noradrenaline, to enhance tone-related release of NO, as shown by curves for N-methyl-L-arginine and superoxide dismutase (SOD), and to increase endothelium-dependent relaxation to carbachol and histamine, compared with intact animals. Smooth muscle sensitivity to exogenous NO and platelet thromboxane A2 production were unchanged but prostacyclin release by aortic tissue dropped by about 40% following castration. Treatment with E2 to intact animals still attenuated contractility to noradrenaline and potentiated relaxation to SOD and histamine but affected no other parameters. In contrast, when E2 was administered to castrated animals, responses to SOD, carbachol and histamine were significantly impaired. Thus, androgen deprivation appears to improve vascular function in male diabetic rats, whereas E2 treatment exerts some beneficial effects in intact, but not in castrated animals. Our findings therefore provide new insights into the role of sex hormones in the development of diabetic vascular complications.

The beneficial effects of estrogen on arterial function in women are well established, whereas studies concerning the vascular role of androgens have produced conflicting results. In the present study, we examined the effects of androgen deprivation and of estrogen treatment on vascular responses in male rats. Vascular reactivity was studied in aortic rings excised from intact and castrated rats, which had been implanted with capsules containing either 17beta-estradiol (E2) or its vehicle for 5 days. Contractile responses to noradrenaline were potentiated by castration and by E2 treatment. Concentration-response curves for N-methyl-L-arginine and superoxide dismutase indicated that the tone-related release of NO increased in tissues from castrated, compared with intact rats, but was not affected by E2 treatment. Endothelium-dependent relaxation elicited by carbachol and histamine were not altered by castration and were attenuated by E2 in preparations from intact, but not from castrated rats. Moreover, aortic prostacyclin release dropped by about 40% after E2 treatment in tissues from both intact and castrated animals. Similarly, smooth muscle sensitivity to NO significantly decreased following castration and E2 treatment, as assessed by responses to sodium nitroprusside. Finally, no differences among groups were detected in platelet thromboxane A2 production. Thus, vascular responses in male rats were not improved by androgen deprivation alone or by E2 treatment, whose effects differed in the presence or absence of androgens. These findings provide evidence for the gender specificity of the vascular effects of estrogen and may be consistent with a beneficial role of physiologic levels of male sex hormones in arterial function.

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 alpha2-macroglobulin, alpha2-HS-glycoprotein, C-reactive protein and kallikrein-binding protein.

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-β-estradiol (E 2 ) 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 E 2 replacement. Concentration-response curves to norepinephrine (NE) showed attenuation of the contractile response in E 2 -treated diabetic, with respect to non-diabetic preparations. This response was further impaired in diabetic, E 2 -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 E 2 -treated diabetic rats, no further effect being induced by E 2 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 E 2 deprivation. However, E 2 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.

1. Nitric oxide (NO) is known from previous studies to be the principle transmitter in NANC inhibitory nerves supplying the hamster urethra. However, the identity of the cotransmitter(s) responsible for the responses remaining following block with L-NG-nitroarginine methyl ester (L-NAME) is not known. 2. Electrical field stimulation (EFS) of circular strips of hamster proximal urethra precontracted with arginine vasopressin (AVP 10(-8) M), and in the presence of phentolamine (10(-6) M), propranolol (10(-6) M) and atropine (10(-6) M), caused frequency-dependent relaxation, which was attenuated by suramin (10(-4) M) and reactive blue 2 (RB2; 2 x 10(-4) M), but not by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 10(-4) M), alpha-chymotrypsin (10-50 u ml(-1)) or by the vasoactive intestinal polypeptide (VIP) antagonist, [Lys1, Pro2,5, Arg3,4, Tyr6]-VIP, (5 x 10(-7)-10(-6) M). In the presence of indomethacin (10(-6) M) frequency-dependent relaxations to EFS were enhanced, particularly at the lower frequencies of stimulation. EFS-induced relaxation was blocked by tetrodotoxin (10(-6) M), indicating its neurogenic origin. 3. Exogenous ATP (10(-7)-10(-3) M) produced concentration-related relaxations which were attenuated by the P2-purinoceptor antagonists suramin (10(-4) M) and RB2 (2 x 10(-4) M) but not by PPADS (10(-4) M). ATP-induced relaxations were also reduced significantly by indomethacin (10(-6) M). The inhibitory responses to ATP were urothelium- and NO-independent, since they were not affected by either removal of urothelium or by L-NAME (10(-4) M). 4. Exogenous VIP (10(-9)-10(-7) M) induced concentration-related relaxations which were not affected by urothelium removal, L-NAME (10(-4) M), alpha-chymotrypsin (10-50 u ml(-1)) or by [Lys1, Pro2,5, Arg3,4, Tyr6]-VIP (3 x 10(-7)-10(-6) M). Nevertheless, suramin (10(-4) M) and RB2 (2 x 10(-4) M) but not PPADS (10(-4) M) antagonized the VIP-induced relaxant responses. Calcitonin gene-related peptide (CGRP: 10(-9)-10(-7) M) was devoid of any effect or only elicited a small relaxant response in AVP-precontracted strips. 5. Exogenous prostaglandin E2 (PGE2; 10(-9)-3 x 10(-6) M) and the NO donor, sodium nitroprusside (SNP; 10(-8)-3 x 10(-5) M) elicited concentration-related relaxations on the hamster proximal urethra which were not attenuated by suramin (10(-4) M), RB2 (2 x 10(-4) M), or by PPADS (10(-4) M), indicating a specific inhibitory effect of the antagonists used. 6. In summary, these results are consistent with the view that ATP is an inhibitory transmitter released from inhibitory nerves supplying the NANC relaxation of hamster proximal urethra. The relaxant effect of ATP is NO- and urothelium-independent. The present study did not demonstrate whether VIP is released from parasympathetic nerves during EFS, since both alpha-chymotrypsin and [Lys1, Pro2,5, Arg3,4, Tyr6]-VIP were ineffective on neurogenic responses.

1. Purinergic and cholinergic components of parasympathetic neurotransmission and contractile responses to exogenous alpha,beta-methylene ATP, acetylcholine, substance K, substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide and capsaicin have been investigated in the urinary bladder of hibernating hamsters (4 weeks), cold exposed (4 weeks) and age-matched controls. 2. Electrical field stimulation (EFS) evoked increased frequency-dependent contractions in the detrusor strips from hibernating hamsters compared with those obtained from cold-exposed and age-matched animals. Tetrodotoxin (10(-6) M) completely blocked the frequency-dependent contractions in all groups. 3. The purinergic component of the parasympathetic neurotransmission was not affected in hibernating and cold-exposed animals while the cholinergic component was increased with respect to age-matched animals. The neurogenic response to EFS, still present after incubation with atropine (10(-6) M) and suramin (10(-4) M), was attenuated by indomethacin (10(-6) M) and blocked by tetrodotoxin (10(-6) M). 4. Exogenous administration of alpha,beta-methylene ATP elicited a significantly reduced contraction in strips from hibernating and cold-exposed hamsters relative to age-matched animals. The contractile response to exogenous acetylcholine was greater in the detrusors from hibernating hamsters than in cold-exposed and age-matched animals. Substance K elicited reduced contractions in preparations from hibernating animals compared with cold-exposed and control animals. Calcitonin gene-related peptide, vasoactive intestinal polypeptide, substance P and capsaicin did not elicit any relaxant or contractile response either at resting tone or in carbachol (5 x 10(-7) M)-precontracted tissues. 5. In summary, our findings indicate that 4 weeks of hibernation can significantly increase neurogenic responses in the hamster urinary bladder. This appears to be due to an increase in postjunctional responses to acetylcholine. In contrast, there was a decrease of the postjunctional responses to the parasympathetic cotransmitter ATP and also to the sensory-motor neurotransmitter substance K.

1. A brief challenge of rat astrocytes with either alpha, beta-methyleneATP (alpha, beta-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 alpha, beta-meATP- but not bFGF-induced astrocytic elongation. Similar effects on astrocytic elongation were also observed with ATP and other P2 receptor agonists (beta, gamma meATP, ADP beta S, 2meSATP and, to a lesser extent, UTP). 2. Pertussis toxin completely abolished alpha, beta-meATP- but not bFGF-induced effects. No effects were exerted by alpha, beta-meATP on cyclic AMP production; similarly, neomycin had no effects on elogation of processes induced by the purine analogue, suggesting that adenylyl cyclase and phospholipase C are probably not involved in alpha, beta-meATP-induced effects (see also the accompanying paper by Centemeri et al., 1997). The tyrosine-kinase inhibitor genistein greatly reduced bFGF- but not alpha, beta-meATP-induced astrocytic elongation. 3. Challenge of cultures with alpha, beta-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 alpha, beta-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 alpha, beta-meATP- but not bFGF-induced effects. 4. Both alpha, beta-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 alpha, beta-meATP- and bFGF-morphological differentiation of astrocytes occurs via independent transductional pathways. For the purine analogue, signalling involves a Gi/G(o) 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.

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) (ADP beta S), as well as uridine-5'-triphosphate (UTP) resembled that of ATP, while alpha, beta-methylene-ATP (alpha, beta-meATP) and beta, gamma-methylene-ATP (beta, gamma-meATP) were totally ineffective. 3. Suramin (50 microM) had only a minor effect on the ATP response, whereas pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (5 microM) 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 microM 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) (GDP beta S) (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 microM), 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.

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.

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 x 10(-3) M) but not by D-arginine (3 x 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 x 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.

The understanding of the biological effects of estrogen on the vessel wall has improved dramatically since the discovery of estrogen receptors (ERs). Most, but not all estrogen-mediated effects in blood vessels are thought to be mediated by ERs. Two major ER subclasses have been characterized so far: the ERα and the more recently described ERβ. This review will primarily focus on a new perspective that highlights ERs as essential mediators of the vascular effects of estrogen. In view of the rising research interest in this area, it can be also expected that tissue- and ER subclass-selective agonists and antagonists will be developed over the next few years, thus providing invaluable tools for pharmacological and clinical applications. © 2001 John Wiley & Sons, Inc. Med Res Rev, 21, No. 2, 171–184, 2001

Secretion of matrix metalloproteinases (MMPs) by macrophages and smooth muscle cells (SMC) may impair atherosclerotic cap integrity leading to atherosclerosis complications. Selective estrogen receptor modulators (SERMs) have favourable impact on plasma lipid levels, but their role in the prevention of atherosclerosis still remains unclear. We investigated the effects of raloxifene, a second generation SERM, on MMP expression and activity in cultured macrophages and SMC, and in rabbit carotid lesions. Human monocyte-derived macrophages were isolated from blood of healthy donors. SMC were isolated from the intima-media layers of collared rabbit carotid arteries. Cells were incubated for 24 h with increasing concentrations of raloxifene. Ovariectomized rabbits fed a 1% cholesterol-rich diet were subjected to pericarotid collar placement and treated with or without 10 mg kg −1 d −1 raloxifene for 2 weeks. In macrophages, raloxifene treatment (0.1–10 μM) significantly reduced MMP-9 gelatinolytic potential in a concentration-dependent manner, without affecting MMP-9 activation. This effect was estrogen receptor (ER)-dependent and due to the inhibition of MMP-9 promoter-driven transcription following an interaction with NF-kB pathway. Similarly, in cultured SMC, raloxifene inhibited up to 40% MMP-2 gelatinolytic activity. In vivo , raloxifene decreased the expression of MMP-2, MMP-3, and MMP-9 by intimal cells and the total gelatinolytic activity of collared carotids. These effects were accompanied by reduction of lesion size and inhibition of macrophage accumulation. Overall, results indicate that raloxifene may reduce MMPs expression and activity in macrophages and smooth muscle cells and favourably affect lesion formation.

Background and aim Based on the reported cardioprotective effects of resveratrol, a polyphenolic antioxidant abundant in grapes that binds to estrogen receptors, and the well-characterized anti-inflammatory properties of 17β-estradiol, the effects of resveratrol on the functional expression of inflammatory enzymes were assessed in vascular smooth muscle cells (SMC) from normoglycaemic and streptozotocin-diabetic rats. Methods and results SMC were isolated from the aorta four weeks after treating rats with streptozotocin or its vehicle. In SMC exposed to a cytokine mixture for 24 h, unexpectedly, treatment with resveratrol (0.1–100 μM) as well as the structurally related isoflavone genistein (1 nM–1 μM) enhanced expression of inducible NO synthase (iNOS). Genistein failed to mimic the elevated iNOS activity induced by resveratrol. Inhibition of estrogen receptors by the pure antiestrogen ICI 182,780 reversed the action of resveratrol on iNOS. In addition, resveratrol failed to alter cyclooxygenase-2 protein levels but reduced the accumulation of prostaglandin E2 in the culture medium of SMC from normoglycaemic, but not diabetic rats. Conclusions These results indicate that resveratrol, at concentrations approaching putative peak plasma levels in vivo, exhibited no anti-inflammatory properties in vascular SMC from normal and diabetic rats. By contrast, resveratrol displayed a potential pro-inflammatory activity in settings of vascular inflammation.

The vascular endothelium is a well-recognized target of damage for factors leading to increased cardiovascular risk. Among the agents playing an important role in cardiovascular homeostasis, nitric oxide and prostacyclin represent key markers of endothelial integrity. In the present work, we report for the first time the reduced expression of both endothelial nitric oxide synthase and cyclooxygenase-2 (COX-2) proteins, as well as decreased prostacyclin production, in unstimulated human endothelial cells from insulin-dependent diabetic mothers when compared to cells from non-diabetic, control subjects. According to a major role of COX-2 as a source of prostacyclin production even in unstimulated endothelial cells, prostacyclin production was concentration-dependently inhibited by the selective COX-2 inhibitor SC236. Overall, our results suggest a possible link between reduced endothelial COX-2 and NO-synthase expression and the increased risk of cardiovascular diseases affecting diabetic patients, and point to the use of endothelial cells from diabetic patients as a tool for investigating early dysfunction in pathological endothelium.

The activity of a recent K ATP channel opener, the N -(4-Phenylsulfonylphenyl)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide (ZM226600) was investigated on a female rat model of overactive bladder with outlet obstruction. Both ZM226600 and pinacidil instilled into the bladder (10 − 7 M, 30 min) or following systemic administration (10, 100 nmol/kg e.v.) almost completely abolished bladder overactivity and improved residual volume and frequency of micturition. However, pinacidil affected arterial pressure. Oxybutynin instilled into the bladder (10 − 7 , 10 − 6 , 10 − 5 M, 30 min) decreased detrusor overactivity by about 16%, 25% and 46% respectively, but also blocked micturition reflexes at highest doses tested. Oxybutynin reduced detrusor overactivity by about 50% and 80%, after systemic administration (10, 100 nmol/kg e.v.), but also blocked micturition reflexes at the highest dose tested. In conclusion, ZM226600 is more active than oxybutynin in reducing bladder overactivity, and it is devoid of vascular side effects observed with pinacidil. Its short duration of action (about 1 h) is probably the main problem to solve, in order to consider this compound a valid alternative to antimuscarinics in the therapy of bladder overactivity.

Background— Previous reports from our group have shown that 17β-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 17β-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, 17β-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 ERα and, to a greater extent, of ERβ, were increased in diabetic compared with nondiabetic SMC. Cytokines decreased ERα and ERβ expression in both groups. However, 17β-estradiol dose-dependently restored the expression of ERα but further downregulated that of ERβ, 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 ERβ than of ERα protein, whereas 17β-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.

ATP and 2-methyl-thio-ATP (2-Me-SATP) increase cytosolic calcium concentrations ([Ca 2+ ] i ) in rat striatal astrocytes (Centemeri et al. [1997] Br J Pharmacol 121:1700-1706). The aim of the present study was to: (1) characterize pyrimidine-induced [Ca 2+ ] i increases in the same experimental system, and (2) try to identify the multiple P2Y receptor subtypes mediating Ca 2+ mobilization. UDP and UTP triggered a concentration-dependent [Ca 2+ ] i elevation (EC 50 s = 0.58 μM ± 0.4 and 31 μM ± 6, respectively). Pyrimidine-evoked [Ca 2+ ] i elevation was solely due to mobilization from intracellular stores, because: (1) removing calcium from extracellular medium or (2) blocking its influx with Ni 2+ did not modify UTP responses; (3) the store-depleting agent thapsigargin completely abolished UTP-evoked [Ca 2+ ] i increments. Guanosine-5'-O-(2-thiodiphosphate) partially inhibited the UTP response, whereas pertussis toxin (PTx) had no effect. The phospholipase C inhibitor U-73122 significantly reduced the UTP-evoked [Ca 2+ ] i rise. Computer-assisted analysis indicated that the UTP and UDP responses are mediated by a single receptor, while ATP and 2-Me-SATP interact with two distinct receptors. The selective P2Y 1 receptor antagonist MRS2179 abolished the ATP higher potency component. Sequential challenges with the same nucleotides resulted in almost complete homologous desensitization. Pre-exposure to UTP lowered the subsequent responses to either ATP or 2-Me-SATP Maximally active concentrations of UTP and ATP were not additive. In conclusion, [Ca 2+ ] elevation in astrocytes by purines and pyrimidines is mediated by two distinct P2Y receptors, likely the P2Y 1 and P2Y 6 subtypes.

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.

Electrical field stimulation caused frequency-dependent relaxations in precontracted strips of hamster proximal urethra, which were attenuated by l -NG-nitroarginine methyl ester (10−4 M) and completely blocked by tetrodotoxin (10−6 M). Strips of hamster urethra devoid of urothelium showed reduced relaxant responses to electrical field stimulation which were abolished by l -NG-nitroarginine methyl ester (10−4 M). Western blot analysis showed the presence of a constitutive endothelial nitric oxide synthase in the urothelial layer, suggesting that urothelium may release nitric oxide in response to electrical field stimulation and that this release is blocked by tetrodotoxin. It is suggested that the urothelium may contribute to relaxations of the smooth muscle of hamster urethra produced by nerve stimulation.

This investigation sought to determine how different components of the hemostatic system affect the development of venous thrombosis in rats displaying hyperlipidemia, either on a genetic basis or secondary to metabolic disorders. On employing an experimental model of collagen-triggered venous thrombosis, both spontaneously hyperlipidemic (Yoshida strain) and streptozotocin-induced diabetic rats generated about 2.3-fold greater thrombi than normolipidemic controls. This was associated with significant platelet activation, as revealed by increased levels of serum thromboxane B2 in diabetics (1.5-fold) as well as in Yoshida (8-fold) rats, in comparison with controls. In contrast, ex vivo total fibrinolytic activity, as measured by euglobulin lysis time, did not differ between normo- and hyperlipidemic or diabetic animals. Plasminogen activator inhibitor activity was lower in both Yoshida and diabetic rats than in controls. However, tissue-type plasminogen activator activity was differently affected by the genetic or the diabetes-related hyperlipidemia, showing significantly lower values in Yoshida (-26%), but significantly higher values in diabetic rats (+29%) than in normolipidemic controls. We conclude that platelet activation, rather than consistent modifications of the fibrinolytic system, is likely to influence the enhanced thrombus development associated with primary or secondary forms of hyperlipidemia.

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.

The in vitro responses of longitudinal preparations of rat stomach fundus and ileum to capsaicin at 1, 8, 4, 16 and 26 weeks and to substance P at 1 and 8 weeks from diabetes induction were studied. The results were compared with those obtained in age-matched control rats. The contractile responses to exogenous substance P and capsaicin were not affected in the stomach fundus from diabetic rats. Atropine (1 microM) did not antagonize the substance P-induced response whereas it inhibited about 90% of the capsaicin-induced response in controls and about 60% of the response in diabetic rats. At the resting tone, capsaicin induced a relaxation followed by a contraction in stomach fundus of control rats. Only a contraction was evoked in diabetic rats. In carbachol (0.05-0.1 microM) pre-stimulated strips, a complete restoration of the biphasic response was obtained in the diabetic state. The contractile response elicited by exogenous substance P was not significantly increased in the ileum preparations from diabetic rats; nevertheless the EC50 value for substance P was reduced 8 weeks after the onset of diabetes. The response elicited by capsaicin in the ileum of control rats was also biphasic. The capsaicin-induced contraction was greater in tissue from diabetic rats as compared with controls and relaxation was not evident. An age-related decrease of the contraction was also evident in both groups. Atropine (1 microM) partially antagonized the responses to substance P and capsaicin. The inhibition of the responses with atropine was more evident in control than in diabetic rats. These results suggest that the myogenic actions of several agonists in these two tissues are differently modified in experimental diabetes.

The in vitro responses of rat urinary bladder, to substance P and capsaicin were studied at 1, 4, 16, and 26 weeks of diapetes 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.

Calcium metabolism appears to be altered in human and experimental hypertension, which represents an important risk factor for thrombotic events. We investigated the possible effect of the calcium antagonist nicardipine on a model of experimental venous thrombosis in spontaneously hypertensive rats (SHR). Thrombus formation was highly enhanced in SHR with respect to normotensive Wistar Kyoto rats (WKY). Nicardipine, when administered orally (10 mg kg −1 ) at a single dose or once a day for three days, completely counteracted the increase in thrombus size caused by hypertension. Furthermore, a significant rise in prostacyclin production from aortic tissue [19.2±1.5 vs 13.2±2.4 ng (mg dry tissue) −1 ], associated with a fall in thromboxane A2 release from activated platelets (328.3±74.6 vs 705.0±88.1 ng ml −1 ), was observed in nicardipine-treated SHR. Plasma triglyceride and free fatty acid levels were also lowered by drug administration. Our results suggest that the actions of nicardipine on calcium metabolism result in antithrombotic effect through an increased availability of vasodilating eicosanoids in vessel walls and through a reduced amount of prothrombotic agents (thromboxane, free fatty acids).

We describe how marking nitrocellulose membranes with horseradish peroxidase can be a useful and cheap method to obtain specific badges on zymograms, developed via enhanced chemiluminescence (ECL).

Summary A polymer bound nicotinic acid preparation (P61/9) was administered orally to rats at various doses. Plasma levels of free nicotinic acid were determined by a newly developed analytical procedure which utilizes the multiple selected ion monitoring technique. The pharmacological activity of P61/9 was evaluated as the decrease of plasma free fatty acid (FFA) and triglyceride (TG) levels. With plasma levels of nicotinic acid as low as 170 ng/ml an active effect on FFA and TG has been observed. When compared with free nicotinic acid, P61/9 is shown to produce a more prolonged biological effect.

A polymer binding nicotinic acid by ester bonds has been prepared. This polymer had a polysaccharide backbones and contained about 25% nicotinic acid residues. This macromolecular compound, given by oral route, shows a prolonged (12–13 hrs) antilipolytic activity in the rat in contrast to equal doses of free nicotinic acid (active for 2 hrs only). The plasma levels of nicotinic acid are lower but more constant after the administration of the macromolecular form. No rebound in FFA release follows treatment in this form.

Summary An anaphylactic reaction was induced with the specific antigen (1 mg Ovalbumin) in perfused lungs from actively sensitized guinea-pigs in order to evaluate the ability of the ginkgolide BN-52021 (0.4, 4, 40 ug/ml) to modulate the mediator release. The ginkgolide reduces in a dose dependent manner the release of histamine (22%, 45% and 75% of inhibition), TXB 2 (77% of inhibition at the maximal dose used) and of SRS-A to a lower extent (only 33% at the higher dose). BN-52021 was still powerful in reducing histamine release induced by immunological reaction in indomethacin treated animals. In conclusion, the present “in-vitro” results confirm the beneficial activity of this ginkgolide, already demonstrated by the same Authors in “in-vivo” experiment, in anaphylactic reactions, and further substantiate the wider spectrum of action of the ginkgolide beside its specific PAF receptor antagonistic activity.

Pirinixil (BR 931), a new hypolipidemic agent, which has been reported to increase high density lipoprotein cholesterol levels in experimental animals was fed for 21 days (0.05% in the diet) to Wistar rats on a semi-sy n thetic diet. Pirinixil increased plasma cholesterol levels significantly without affecting plasma triglycerides. Liver cholesterol and triglycerides were markedly reduced by treatment, while combined plasma and liver lipid levels decreased approximately 20%. Liver HMG-CoA reductase activity was not affected, but cholesterol 7α-hydroxylase was significantly reduced, thus confirming data with other hypolipidemic agents in rats. The experimental findings do not support the recent hypothesis that hypolipidemic agents may increase total body lipid levels.

Summary The polymer-bound nicotinic acid (P 61 ) designed as a long-acting form of this well known hypolipidemic drug is devoid of the undesirable side effects which are due to the very high peak level of free nicotinic acid (NA). The effect of this compound has been pharmacologically evaluated both in the plasma lipids and in the liver lipid accumulation in hypercholesterolemic rats. This polymer seems to maintain the activity of the free form of NA on plasma lipids whereas it shows a protective action on TG content in the liver.

The effects of prostaglandin E1 (PGE1) on the dose-response curves of glycerol release from adipose tissue by norepinephrine and theophylline have been investigated in vitro. PGE1 antagonizes the lipid mobilizing action of both drugs; this antagonism is competitive with theophylline but is non-competitive with norepinephrine. The possible interference of PGE1 with the rate of synthesis and breakdown of 3′5′-adenosine-monophosphate (3′5′-AMP) is discussed.

Experiments in vitro , carried out on paired rat epididymal fat pads, show that theophylline increases free fatty acid release and decreases triglyceride synthesis from glucose and palmitic acid. Prostaglandin E 1 is able to antagonize the former without influencing the latter effect of theophylline. Prostaglandin E 1 alone has no effect on basal free fatty acids release and on palmitic acid incorporation, slightly increases, however, glucose incorporation into lipids. It may be concluded that both theophylline and prostaglandin E 1 are affecting free fatty acid release by interfering rather with the breakdown than with the resynthesis of adipose tissue triglycerides.

Summary An experimental model of hypertriglyceridemia induced by subacute ethanol administration was developed in rats. A four day schedule of administration induced a 50–100% increase of serum triglycerides, without changes in cholesterol levels. Drugs effective on lipid or glucose metabolism were tested in this model. Hypoglycemic sulphonylureas, nicotinic acid, clofibrate, and the new hypolipidemic agent : diethylamide of beta-benzalbutyric acid (C11) proved to be effective agents in controlling ethanol induced hypertriglyceridemia. The activity of C11 was also confirmed in an ethanol induced hypertriglyceridemia in human volunteers.

Myocardial ischemia was induced in perfused paced isovolumic left heart preparation of the rabbit by reducing, for a period of 40 min, the flow rate from 20 ml/min to 0.2 ml/min (severe model) and to 1 ml/min (moderate model). The relationship between prostaglandin biosynthesis and cardiac ischemic damage was evaluated in the two experimental models. The results obtained indicate that the total amount of 6-keto-PGF1 alpha generated increases with the severity of the ischemia, particularly during the 20 min of reperfusion (moderate model 81.8 +/- 13.7 ng: severe model 375 +/- 102 ng). The inhibition of the prostaglandin synthesis, prostaglandin-E2, and 6-keto-prostaglandin-F1 alpha (PGE2 and 6-keto-PGF1 alpha levels below the detection limits) by Aspirin (20 micrograms/ml) and Indomethacin (1 microgram/ml) in moderate myocardial ischemia was correlated with greater increments in resting diastolic tension (nearly 100% and 40%, respectively). This phenomenon was also associated to a further decrease on cardiac contractility and increase on coronary perfusion pressure upon reperfusion. On the contrary drugs which stimulated prostaglandin generation in myocardial tissue, such as Defibrotide (400 micrograms/ml), completely protected the organ from ischemia. U-60257 (3 micrograms/ml) and FPL-55712 (2 micrograms/ml), compounds, which respectively inhibits biosynthesis and the effects of leukotrienes, displayed a beneficial activity on this moderate model of ischemia. The present data suggests that the deleterious effect of nonsteroidal antiinflammatory drugs in low flow myocardial ischemia and reperfusion damage may be associated with removal of PGI2 and PGE2 from ischemic myocardium.

Summary The presence of β 1 and β 2 adrenoceptors is demonstrated in the guinea-pig tracheal smooth muscle and both receptors subserve the same function. The functional relationship between the two β-receptors in the quinea-pig trachea, which is difficult to explain, is discussed in relation to the capacity of β 1 receptors to take over their function, if the β 2 receptors are completely blocked.

Summary Rat epididymal adipose tissue artery releases, during incubation, a PGI 2 -like substance which is active on rat blood pressure as well on aggregation induced by collagen or ADP. The radioimmunoassay of 6-keto PGF 1α , the stable metabolite of PGI 2 , confirms that this eicosanoid is sinthesized also within the blood vessel cells supplying the adipocytes.

The simultaneous incorporation of labelled glucose and palmitic acid into lipids has been investigated in vitro in epididymal fat pads of rats fed essential fatty acid-deficient diet, in order to obtain information on possible metabolic differences brought about by essential fatty acid-deficiency in adipose tissue. It has been found that the incorporation of both precursors, but especially of glucose, is greater into adipose tissue lipids (mostly triglycerides) in rats fed essential fatty acid-deficient diet than in controls, indicating a greater triglyceride synthesis in these animals. Lipid synthesis in the adipose tissue of essential fatty acid-deficient animals, however, does not seem to differ qualitatively from that of controls. The distribution of radioactivity among the lipid components suggests that no change in the metabolic utilization of essential fatty acids for the synthesis of neutral lipids occurs. Also the incorporation of palmitic acid and glucose into glycerides is similarly affected in control and essential fatty acid-deficient animals during fasting or nicotinic acid or theophylline treatment. It may be concluded that the increased free fatty acid mobilization in essential fatty acid-deficient rats is not a consequence of decreased triglyceride synthesis. On the other hand, the enhanced lipolysis observed is related to the increased synthesis of triglycerides in adipose tissue. The relationship between these observations and the role of prostaglandins, which are synthesized from essential fatty acids in adipose tissue, is discussed.

Experiments in vitro, carried out on paired rat epididymal fat pads, show that nicotinic acid increases the glucose incorporation into lipids only when great amounts of tissue FFA are available, as after fasting or norepinephrine treatment. The data indicate that nicotinic acid has no effect on glucose uptake and on lipid synthesis after phosphatidic acid formation. It is suggested that under the influence of nicotinic acid in vitro an increased re-esterification from α-GP and tissue FFA takes place.

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, alpha,beta-methyleneATP (alpha,beta-meATP) showing the typical features of the P2x-purinoceptor; conversely, ATP-evoked relaxation did not undergo tachyphylaxis following either ATP or alpha,beta-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) (GTP gamma S) significantly potentiated and the G-protein blocking agent, guanosine 5'-O-(2-thio-diphosphate) (GDP beta S) 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 alpha,beta-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 alpha,beta-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.