Christian Pinna

Development of the First Ultra-Potent “Capsaicinoid” Agonist at Transient Receptor Potential Vanilloid Type 1 (TRPV1) Channels and Its Therapeutic Potential

Olvanil (N-9-Z-octadecenoyl-vanillamide) is an agonist of transient receptor potential vanilloid type 1 (TRPV1) channels that lack the pungency of capsaicin and was developed as an oral analgesic. Vanillamides are unmatched in terms of structural simplicity, straightforward synthesis, and safety compared with the more powerful TRPV1 agonists, like the structurally complex phorboid compound resiniferatoxin. We have modified the fatty acyl chain of olvanil to obtain ultra-potent analogs. The insertion of a hydroxyl group at C-12 yielded a compound named rinvanil, after ricinoleic acid, significantly less potent than olvanil (EC50 = 6 versus 0.7 nM), but more versatile in terms of structural modifications because of the presence of an additional functional group. Acetylation and phenylacetylation of rinvanil re-established and dramatically enhanced, respectively, its potency at hTRPV1. With a two-digit picomolar EC50 (90 pM), phenylacetylrinvanil (PhAR, IDN5890) is the most potent vanillamide ever described with potency comparable with that of resiniferatoxin (EC50, 11 pM). Benzoyl- and phenylpropionylrinvanil were as potent and less potent than PhAR, respectively, whereas configurational inversion to ent-PhAR and cyclopropanation (but not hydrogenation or epoxidation) of the double bond were tolerated. Finally, iodination of the aromatic hydroxyl caused a dramatic switch in functional activity, generating compounds that behaved as TRPV1 antagonists rather than agonists. Since the potency of PhAR was maintained in rat dorsal root ganglion neurons and, particularly, in the rat urinary bladder, this compound was investigated in an in vivo rat model of urinary incontinence and proved as effective as resiniferatoxin at reducing bladder detrusor overactivity.

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.

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.

Prolonged Ovarian Hormone Deprivation Impairs the Protective Vascular Actions of Estrogen Receptor αAgonists

The vascular consequences of estrogen treatment may be driven by its initiation timing. We tested the hypothesis that the duration of ovarian hormone deprivation before estrogen reintroduction affects the role of estrogen as mediator of endothelial function and vascular relaxation in nondiseased vessels. Rats were ovariectomized and implanted with 17&bgr;-estradiol (E2) or oil capsules 1, 4, and 8 months after surgery. After the longest hypoestrogenicity period, acetylcholine-mediated aortic relaxation was attenuated and insensitive to E2 administration despite endothelial integrity. Whereas no rapid vasorelaxant responses were elicited by an estrogen receptor (ER) &bgr;–selective agonist, responses to E2 and an ER&agr; selective agonist waned postovariectomy at any given time and were restored by E2 treatment after 1 and 4 months but not 8 months postovariectomy. Accordingly, endothelial ER&agr; mRNA and protein expression declined ≈6-fold after prolonged hypoestrogenicity and was restored by estrogen replacement starting 1 month but not 8 months postovariectomy. Furthermore, the amount of active phosphorylated endothelial NO synthase rose significantly after E2 replacement after 1 and 4 months but not 8 months postovariectomy. The present findings document that the functional impairment of the ER&agr;/endothelial NO synthase signaling network after an extended period of hypoestrogenicity was not restored by E2 administration, providing experimental support to early initiation of estrogen replacement with preferential ER&agr; targeting to improve cardiovascular outcomes.

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.

Selective Agonists of Estrogen Receptor Isoforms. New Perspectives for Cardiovascular Disease

The cloning of estrogen receptors (ERs) and generation of ER-deficient mice have increased our understanding of the molecular mechanisms underlying the cardiovascular effects of estrogen. It is conceivable that clinical trials of estrogens so far failed to improve cardiovascular health because of the poor ER isoform selectivity and tissue specificity of endogenous hormones as well as incorrect treatment timing and regimens. Tissue-selective ER modulators (SERMs) may be safer agents than endogenous estrogens for cardiovascular disease. Yet, designing isoform-selective ER ligands (I-SERMs) with agonist or antagonist activity is required to pursue improved pharmacological control of ERs, especially taking into account emerging evidence for the beneficial role of vascular ER&agr; activation. Ideally, the quest for unique ER ligands targeted to the vascular wall should lead to compounds that merge the pharmacological profiles of SERM and I-SERM agents. This review highlights the current bases for and approaches to selective ER modulation in the cardiovascular system.

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.

ATP and vasoactive intestinal polypeptide relaxant responses in hamster isolated proximal urethra

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; 2×10−4 m), but not by pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS; 10−4 m), α‐chymotrypsin (10–50 u ml−1) or by the vasoactive intestinal polypeptide (VIP) antagonist, [Lys1, Pro2,5, Arg3,4, Tyr6]‐VIP, (5×10−7–10−6 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 (2×10−4 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), α‐chymotrypsin (10–50 u ml−1) or by [Lys1, Pro2,5, Arg3,4, Tyr6]‐VIP (3×10−7–10−6 m). Nevertheless, suramin (10−4 m) and RB2 (2×10−4 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; 10−9–3×10−6 m) and the NO donor, sodium nitroprusside (SNP; 10−8–3×10−5 m) elicited concentration‐related relaxations on the hamster proximal urethra which were not attenuated by suramin (10−4 m), RB2 (2×10−4 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 α‐chymotrypsin and [Lys1, Pro2,5, Arg3,4, Tyr6]‐VIP were ineffective on neurogenic responses.

A possible role for urinary bladder epithelium in bradykinin-induced contraction in diabetic rats

Diabetes provokes a greater responsiveness of rat urinary bladder preparations to bradykinin and a greater formation and release of prostaglandin F2 alpha, without affecting prostaglandin E2 release significantly. Inhibition of cyclooxygenase by indomethacin (1 microM) inhibits the contraction elicited by bradykinin and leads to identical contractile responses of control and diabetic urinary bladder strips. Removal of the urinary bladder epithelium does not modify the contractile response evoked by bradykinin in control preparations but significantly decreases the contraction of preparations of diabetic tissues. Quantitatively, the activity of control urinary bladder strips with epithelium and the activity of diabetic preparations without epithelium are the same. More prostaglandin F2 alpha is released into the medium by urinary bladder strips devoid of epithelium in both control and in diabetic rats. These results indicate a role for epithelial cells in the smooth muscle contraction evoked by bradykinin in diabetic rats.