M.D. Lograno

Receptor-responses in fresh human ciliary muscle.

1 The physiological and pharmacological properties of ciliary muscle isolated from fresh human eyes were investigated. 2 The muscle exhibited no spontaneous activity. Concentration‐dependent contractions in response to carbachol were competitively antagonized by atropine (pA2 = 8.95). 3 The muscle, precontracted by carbachol (2.7 × 10−4 M), responded to the application of isoprenaline by concentration‐dependent relaxation blocked by propranolol (3.5 × 10−9 M to 3.5 × 10−8 M; pA2 = 9.15). 4 Angiotensin‐evoked contractions were antagonized by 8‐Ala‐angiotensin II (4.5 × 10−8 M) in a competitive manner, but were not inhibited by phentolamine or propranolol. 5 Contractions generated by electrical stimulation of the muscle (30 ms, 20 Hz, 60 pulses) were antagonized by atropine (10−7 M) and tetrodotoxin (6.3 × 10−7 M). Phentolamine and propranolol did not influence these responses. 6 An increase of the external potassium concentration ([K+]o) from 5.4 to 158.8 mM produced a mechanical response, antagonized by atropine, but not influenced by tetrodotoxin, phentolamine or propranolol. 7 The human ciliary muscle appears to carry muscarinic and angiotensin receptors and β2‐adrenoceptors. The estimate of Katropine for muscarinic receptors mediating carbachol‐induced contractions agrees with estimates of Katropine reported for human and rabbit iris.

Acute effects of transdermal estradiol administration on plasma levels of nitric oxide in postmenopausal women

OBJECTIVE To investigate the acute effects of transdermal E2 administration on nitric oxide (NO) plasma levels in postmenopausal women. DESIGN Randomized, placebo-controlled trial. SETTING Normal human volunteers in an academic research environment. PATIENT(S) Twenty healthy postmenopausal women. INTERVENTION(S) Transdermal administration of 100 micrograms/d E2 or placebo. MAIN OUTCOME MEASURE(S) Plasma concentrations of NO stable oxidation products and serum concentrations of E2 were assessed before and 24 hours after the administration. RESULT(S) In the group treated with E2 mean concentration of NO metabolites 24 hours after patch application (37.31 +/- 7.62 mumol/L) resulted significantly higher than baseline (21.04 +/- 5.71 mumol/L) and the control group (23.50 +/- 4.03 mumol/L). The correlation between the mean percent increase in NO metabolites and absolute E2 concentrations 24 hours after the E2 administration was statistically significant. CONCLUSION(S) Transdermal administration of E2 to healthy postmenopausal women increases the plasma levels of NO and this supports the hypothesis that a NO-related mechanisms may contribute to the cardiovascular protective effect of estrogens in postmenopause.

Growth hormone secretagogues modulate the electrical and contractile properties of rat skeletal muscle through a ghrelin-specific receptor

Growth hormone secretagogues (GHS) exhibit potent growth hormone (GH)‐releasing activity through the activation of a pituitary receptor. Here, we consider the possibility that GHS can target a specific receptor in rat skeletal muscle and have a role in the control of muscle function. By means of the intracellular microelectrode technique, we found that in vitro application of hexarelin and L‐163,255 dose dependently reduced resting chloride (gCl) and potassium (gK) conductances in rat skeletal muscle. These effects were prevented by the GHS‐receptor antagonist [D‐Lys‐3]‐GHRP‐6, and by either phospholipase C or protein kinase C (PKC) inhibitors. Ghrelin, a natural ligand of GHS receptors, also induced a reduction of muscle gCl and gK, which was antagonised by [D‐Lys‐3]‐GHRP‐6. Both GHS shifted the mechanical threshold for the contraction of muscle fibres towards more negative voltages. Accordingly, by means of FURA‐2 fluorescent measurements, we demonstrated that L‐163,255 induced a resting [Ca2+]i increase, which was reversible and not blocked by nifedipine or removal of external Ca2+. Ageing is a condition characterised by a deficit of GH secretion, which in turn modifies the electrical and contractile properties of skeletal muscle. In contrast to GH, chronic treatment of aged rats with hexarelin or L‐163,255 failed to restore the electrical and contractile muscle properties. Moreover, the two GHS applied in vitro were able to antagonise the beneficial effect on gCl and gK obtained through chronic treatment of aged animals with GH. Thus, skeletal muscle expresses a specific GHS receptor able to decrease gCl and gK through a PKC‐mediated intracellular pathway. This peripheral action may account for the lack of restoration of skeletal muscle function in long‐term GHS‐treated aged animals.

Cannabinoid agonists induce relaxation in the bovine ophthalmic artery: evidences for CB1 receptors, nitric oxide and potassium channels

Glaucoma pathophysiology appears to involve vascular deficits, which may contribute to initiation and progression of the disease. Anandamide, the endogenous cannabinoid ligand, and WIN55212‐2, a synthetic cannabinoid agonist, are able to evoke concentration‐dependent relaxations in bovine ophthalmic artery rings, precontracted with 5‐hydroxytryptamine (5‐HT) (1 μM). Endothelium removal reduces cannabinoid agonist potency and efficacy. The selective cannabinoid 1 (CB1) receptor antagonists SR141716A (100 nM) and AM251 (100 nM) cause a shift to the right in the concentration–response curves to anandamide and WIN55212‐2 in arterial rings both in the presence and in the absence of endothelium. In endothelium‐intact arteries, the nitric oxide synthase inhibitor, NG‐monomethyl‐L‐arginine (L‐NMMA, 300 μM), completely blocked the anandamide‐ and WIN55212‐2‐relaxant responses; by contrast, the nitric oxide donor S‐nitroso‐N‐acetylpenicillamine (SNAP, 100 μM) induced an increase in vasorelaxant responses to cannabinoid agonists. Relaxations to anandamide and WIN55212‐2 were inhibited by iberiotoxin (IbTX, 200 nM), a blocker of large conductance, Ca2+‐activated K+ channel (BKCa), and by 4‐aminopyridine (4‐AP; 1 mM), a blocker of delayed rectifier K+ channel, whereas the blockade of KATP channels by glibenclamide (5 μM) and of small conductance Ca2+‐activated K+ channels (SKCa) by apamin (100 nM) did not produce any effects. These data suggest that anandamide and WIN55212‐2 relax the bovine ophthalmic artery by involving CB1 the cannabinoid receptor‐sensitive pathway. In endothelium‐intact arteries, relaxation occurs through activation of nitric oxide synthase cyclic GMP and Ca2+‐activated K+ channels. They also cause endothelium‐independent relaxation by involving potassium channel opening.

Epigallocatechin-3-gallate relaxes the isolated bovine ophthalmic artery: Involvement of phosphoinositide 3-kinase-Akt-nitric oxide/cGMP signalling pathway

The present study investigates the direct action and the underlying mechanism(s) of epigallocatechin-3-gallate (EGCG) vasomotor effects on the bovine isolated ophthalmic artery. Adjacent rings were cut from each artery and mounted in a wire miograph system for isometric recording. Concentration-response curves for EGCG were constructed by adding cumulative concentrations of the drug to arterial rings pre-contracted with 5-HT (1 microM). Effects of mechanical endothelial cell removal and of selective blockers of the nitric oxide (NO)/cGMP pathways were investigated on the EGCG relaxant responses. EGCG relaxed ophthalmic arteries and maximum relaxation was 78.4+/-2.64%. Mechanical removal of endothelium, blockade of soluble guanylyl cyclase by 1H-1,2,4-oxadiazolo [4,3-a]quinoxalin-1-one (ODQ, 1 and 5 microM) or inhibition of nitric oxide (NO) synthase by N(G)-nitro-L-arginine (L-NAME, 50 and 100 microM) reduced significantly the relaxant response to catechin; moreover, the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 100 microM) significantly increased the vasorelaxant responses to EGCG. Relaxation to EGCG was inhibited by iberiotoxin (200 nM), a blocker of big-conductance Ca(2+)-activated K(+) (BK(Ca)) channel, whereas the blockade of K(ATP) channel by glibenclamide (5 microM) and of small-conductance Ca(2+)-activated K(+) (SK(Ca)) channel by apamin (100 nM) elicited no effect. Interestingly, also inhibition of phosphoinositide-3-kinase (PI3K) by wortmannin (100 nM) and of Akt by SH6 (1 microM) markedly decreased the EGCG-evoked vasorelaxation. These data suggest that EGCG induced vasorelaxation in ophthalmic arteries with endothelium-intact via the activation of the NO/cGMP signalling pathway and defined an intriguing role for PI3K and Akt as upstream mediators for activation of NO-mediated relaxant responses.

Effects of bevacizumab on neuronal viability of retinal ganglion cells in rats

The aim of this study was to investigate the effects of single and repeated intravitreal injections of bevacizumab on various retinal layers focusing more on retinal ganglion cells (RGCs) in healthy rats. Male Wistar rats were treated with intravitreal injection of bevacizimab (4 μL) within right eye. Left eyes were injected with the same volume of balanced salt solution (BSS) and used as control. Ten rats received a single intravitreal injection and ten rats had three injections, with seven days time interval. Histological and immunohistochemical evaluations and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay were performed in order to find out if some degree of apoptosis could occur on RGCs. Histological and immunohistochemical analyses showed that bevacizumab induces neuronal loss compared to control eyes, after multiple injections. RGCs apoptosis after multiple treatments was demonstrated to occur by TUNEL, Annexin V and Bax assays. The loss of ganglion cells following repeated injections was confirmed and quantified by the decrease in RGC specific protein Brn3a measured by western blotting in ten additional rats. The present results need to be considered when multiple intravitreal injection of bevacizumab are performed to treat retinal diseases.

Involvement of the peroxisome proliferator-activated receptor (PPAR) alpha in vascular response of endocannabinoids in the bovine ophthalmic artery.

Endocannabinoids regulate vascular tone in a variety of vascular tissues. This study aimed to investigate the role of peroxisome proliferators-activated receptors (PPARs) in anandamide- and palmitoylethanolamide-induced relaxant responses on the bovine ophthalmic artery and to evaluate the mechanisms involved. The effects of anandamide and palmitoylethanolamide were examined under myographic conditions on arterial rings pharmacologically pre-contracted with 5-HT. Anandamide and palmitoylethanolamide relaxed the ophthalmic artery rings in time- and concentration-dependent manner stimulating the PPAR alpha (PPARα). The vasorelaxation to endocannabinoids was inhibited by PPARα antagonist GW6471 (1μM), but not the PPAR gamma (PPARγ) antagonist GW9662 (1 μM). Anandamide-induced relaxation was attenuate during the first 60 min by AM251, a selective antagonist of cannabinoid CB(1) receptors, and Pertussis toxin, an inhibitor of G(i/o) protein; by the contrast, the palmitoylethanolamide-induced vasorelaxation was unaffected by cannabinoid antagonists and Pertussis toxin. Endothelium removal decreases slightly the potency and efficacy to endocannabinoids. The relaxant effect to anandamide and palmitoylethanolamide was inhibited by L-NMMA (300 μM), an inhibitor of nitric oxide synthase, and iberiotoxin (200 nM), a selective blocker of large conductance Ca²⁺-activated K⁺ (BK(Ca)). These data support the view that anandamide and palmitoylethanolamide relax the ophthalmic artery in a time-dependent manner via the transcription factors PPARα suggesting a function for them in the physiological mechanisms of vascular regulation.

Changes of vascular smooth muscle reactivity in hypertensive rats

Mechanical responses produced by high potassium solution (high-K), norepinephrine (noradrenaline; NA) and phenylephrine (Phe) were examined in the thoracic aorta isolated from control (WKY) and spontaneously hypertensive rats (SHR). In the SHRs the tissues showed increased sensitivity to high potassium compared with those from the control rats. Mechanical removal of endothelium in tissues from the controls did not change the response. The effects produced by NA and Phe were also increased in tissues from SHRs. The amplitudes of contractions were enhanced after removal of the endothelium in tissues from the controls. The relaxation in response to endothelium-dependent vasodilators (acetylcholine and histamine) was significantly depressed in aortic rings from SHRs. Experiments using modified sandwich preparations suggest that the defect in the relaxant ability of vascular smooth muscle is coupled to a reduced functionality of the endothelium.

Synthesis and Biological Evaluation of Direct Thrombin Inhibitors Bearing 4‑(Piperidin-1-yl)pyridine at the P1 Position with Potent Anticoagulant Activity

The design and synthesis of a new class of nonpeptide direct thrombin inhibitors, built on the structure of 1-(pyridin-4-yl)piperidine-4-carboxamide, are described. Starting from a strongly basic 1-amidinopiperidine derivative (6) showing poor thrombin (fIIa) and factor Xa (fXa) inhibition activities, anti-fIIa activity and artificial membrane permeability were considerably improved by optimizing the basic P1 and the X-substituted phenyl P4 binding moieties. Structure-activity relationship studies, usefully complemented with molecular modeling results, led us to identify compound 13b, which showed excellent fIIa inhibition (Ki = 6 nM), weak anti-Xa activity (Ki = 5.64 μM), and remarkable selectivity over other serine proteases (e.g., trypsin). Compound 13b showed in vitro anticoagulant activity in the low micromolar range and significant membrane permeability. In mice (ex vivo), 13b demonstrated anticoagulant effects at 2 h after oral dosing (100 mg·kg(-1)), with a significant 43% prolongation of the activated partial thromboplastin time (aPTT), over controls (P < 0.05).

Biochemical and functional evidence for the presence of dopamine D1 receptors in the bovine ciliary body

The present paper reports both functional and biochemical evidence for the presence of dopamine D1 receptors in the bovine ciliary body. Dopamine (DA) and dopamine D1 agonists (such as SKF 38,393) but not D2 agonists (such as LY 141,865) produced a concentration-related decrease in the tone induced by a maximally active concentration of carbachol (1 x 10(-4)-5 x 10(-4) M). The maximal relaxation obtained was 100% of the carbachol response using 10(-5) M dopamine or 5 x 10(-6) M SKF 38,393. SCH 23,390, a D1 antagonist, but not (-)-sulpiride, antagonized the effect of DA and SKF 38,393. In accordance with the functional data, radioreceptor binding experiments revealed the existence of a high affinity saturable [3H]SCH 23,390 binding to membranes prepared from ciliary body (Bmax: 344 fmol mg protein-1; Kd: 0.87 nM). The binding was specifically displaced by SCH 23,390, dopamine and dopamine D1 agonists, but not by norepinephrine, D2 agonists, or antagonists such as LY 141,865 and sulpiride. No specific binding was found when using dopamine D2 ligands, such as tritiated spiroperidol.