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Abstract Clinical exome sequencing is a powerful approach to overcome the wide clinical and genetic heterogeneity of mucopolysaccharidosis. These data could be useful for prenatal diagnosis of MPS VII, genetic counseling, and preimplantation genetic testing.

This study evaluated the osteoconductive potential of four biomaterials used to fill bone defects. For this, 24 male Albino rabbits were submitted to the creation of a bilateral 8 mm calvarial bone defect. The animals were divided into four groups—bovine hydroxyapatite, Bio-Oss® (BIO); Lumina-Bone Porous® (LBP); Bonefill® (BFL); and an alloplastic material, Clonos® (CLN)—and were euthanized at 14 and 40 days. The samples were subjected to histological and histometric analysis for newly formed bone area. Immunohistochemical analysis for Runt-related transcription factor 2 (Runx2), vascular endothelial growth factor (VEGF), and osteocalcin (OC) was performed. After statistical analysis, the CLN group showed greater new bone formation (NB) in both periods analyzed (p

Background and Purpose: A NO-mediated desensitization of vasoconstrictor responses evoked by stimulation of α 1 -adrenoceptors has been reported in different vessels. We investigated the involvement of each α 1 -adrenoceptor subtype and constitutive NOS isoforms and the influence of ageing and hypertension on this process., Experimental Approach: Wistar and spontaneously hypertensive rats (SHR), 16, 32, 52 and 72 weeks-old, were used to evaluate the desensitization process. Expression of α 1 -adrenoceptor subtypes, endothelial NOS (eNOS) and neuronal NOS (nNOS) were determined in rat aorta and left ventricle (LV). Expression levels were also evaluated in LV of a group of heart failure patients with a wide age range., Key Results: Repeated application of phenylephrine decreased subsequent α 1 -adrenoceptor-mediated vasoconstriction by increasing nNOS protein expression in aorta, but not in tail or mesenteric resistance arteries, where mRNA levels of nNOS were undetectable. This desensitization process disappeared in the absence of endothelium or in the presence of L-NAME (100 μM), nNOS inhibitors, SMTC (1 μM) and TRIM (100 μM), and 5-methylurapidil (100 nM, α 1A -antagonist), but not BMY7378 (10 nM, α 1D -antagonist). The α 1A /nNOS-mediated desensitization was absent in aged SHR and Wistar animals, where the expression of α 1A -adrenoceptors was reduced in aorta and LV. In human LV, a negative correlation was found between age and α 1A -adrenoceptor expression., Conclusions and Implications: The α 1A -adrenoceptor subtype, through endothelial nNOS-derived NO, may act as a physiological 'brake' against the detrimental effects of excessive α 1 -adrenoceptor-mediated vasoconstriction. Reduced α 1A -adrenoceptor- and nNOS-mediated desensitization in aged patients could be involved in the age-dependent elevation of adrenergic activity., (© 2017 The British Pharmacological Society.)

1. The alpha(1)-adrenergic responses of rat aorta and tail artery have been analysed measuring the contractility and the inositol phosphate (IP) formation induced by noradrenaline. Three antagonists, prazosin, 5-methylurapidil (alpha(1A) selective) and BMY 7378 (alpha(1D) selective) have been used in different experimental procedures. 2. Noradrenaline possesses a greater potency inducing contraction and IP accumulation in aorta (pEC(50)-contraction=7.32+/-0.04; pEC(50)-IPs=6.03+/-0.08) than in the tail artery (pEC(50)-contraction=5.71+/-0.07; pEC(50)-IPs=5.51+/-0.10). Although the maximum contraction was similar in both tissues (E(max)-tail=619.1+/-55.6 mg; E(max)-aorta-698.2+/-40.8 mg), there were marked differences in the ability of these tissues to generate intracellular second messengers the tail artery being more efficient (E(max)-tail=1060+/-147%; E(max)-aorta=108.1+/-16.9%). 3. Concentration response curves of noradrenaline in presence of antagonist together with concentration inhibition curves for antagonists added before (CICb) or after (CICa) noradrenaline-induced maximal response in Ca(2+)-containing or Ca(2+)-free medium have been performed. A comparative analysis of the different procedures as well as the mathematical approaches used in each case to calculate the antagonist potencies, were completed. 4. The CICa was the simplest method to characterize the predominant alpha(1)-adrenoceptor subtype involved in the functional response of a tissue. 5. In aorta, where constitutively active alpha(1D)-adrenoeptors are present, the use of different experimental procedures evidenced a complex equilibrium between alpha(1D)- and alpha(1A)-adrenoceptor subtypes. 6. The appropriate management of LiCl in IP accumulation studies allowed us to reproduce the different experimental procedures performed in contractile experiments giving more technical possibilities to this methodology.

1. The role of a constitutively active population of alpha(1D)-adrenoceptors was analysed in arteries obtained from spontaneously hypertensive rats (SHR) and controls (WKY) divided into three groups: young prehypertensive, adult hypertensive, and adult animals chronically treated with captopril (50 mg kg(-1) per day orally) in order to prevent the hypertensive state. 2. In adult SHR, a significant increase in BMY 7378 potency (not in prazosin potency) was observed in aorta, mesenteric artery, and the first and second branches of the small mesenteric arteries with respect to WKY rats. This difference was not observed in iliac and tail arteries, which suggests an increased functional role of alpha(1D)-adrenoceptors only in some vessels of SHR. 3. The increase in the resting tone (IRT) observed in absence of agonist, inhibited by BMY 7378, that represents the constitutively active population of alpha(1D)-adrenoceptors, was also significantly greater in aorta and mesenteric artery from adult SHR. 4. In young and captopril treated adult animals, no differences between strains with respect to BMY 7378 potency, or IRT were observed. 5. The increase in the functional role of alpha(1D)-adrenoceptors and their constitutive activity observed in hypertension is prevented by captopril treatment. The pathological consequence of this change is the slower rate of recovery of the basal tone after removal of an adrenergic stimulus, observed in vessels from hypertensive animals that had shown an increase in the functionality of constitutively active alpha(1D)-adrenoceptors. This change was not observed in prehypertensive or captopril treated animals.

1. In the present work, the properties of 3-methyl isoquinoline, 3,4-dihydropapaverine, tetrahydropapaverine and tetrahydropapaveroline were compared with those of papaverine and laudanosine. The work includes functional studies on rat isolated aorta contracted with noradrenaline, caffeine or KCl, and a determination of the affinity of the compounds for alpha1-adrenoceptors and calcium channel binding sites, with [3H]-prazosin, [3H]-nitrendipine and [3H]-(+)-cis-diltiazem binding to rat cerebral cortical membranes. The effects of papaverine derivatives on the different molecular forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aorta were also determined. 2. The three papaverine derivatives show greater affinity than papaverine for the [3H]-prazosin binding site. They are therefore more selective as inhibitors of [3H]-prazosin binding as opposed to [3H]-(+)-cis-diltiazem, while papaverine appears to have approximately equal affinity for both. [3H]-nitrendipine binding was not affected by either papaverine or papaverine derivatives in concentrations up to 100 microM. 3-Methylisoquinoline had no effect on any of the binding sites assayed. 3. Contractions evoked by noradrenaline (1 microM) in rat aorta were inhibited in a concentration-dependent manner by 3,4-dihydropapaverine, tetrahydropapaverine and with a lower potency, by tetrahydropapaveroline. In Ca2+-free solution, tetrahydropapaverine and to a lesser extent, tetrahydropapaveroline, inhibited the noradrenaline (1 microM) evoked contraction in a concentration-dependent manner and did not modify the phasic contractile response evoked by caffeine (10 mM). This suggests that these alkaloids do not act at the intracellular level, unlike papaverine which inhibits the contractile response to caffeine and noradrenaline. 4. Inositol phosphates formation induced by noradrenaline (1 microM) in rat aorta was inhibited by tetrahydropapaverine (100 microM) and tetrahydropapaveroline (300 microM), thus suggesting that alpha1D-adrenoceptors are coupled to phosphoinositide metabolism in rat aorta. 5. Unlike papaverine, which has a significant effect on all the PDE isoforms, the three alkaloids assayed did not have an inhibitory effect on the different forms of PDE isolated from bovine aorta. 6. These results provide evidence that papaverine derivatives with a partially or totally reduced isoquinoline ring have a greater affinity for alpha1-adrenoceptors and a lower affinity for benzothiazepine sites in the Ca2+-channel than papaverine. This structural feature also implies a loss of the inhibitory activity on PDE isoforms. The planarity of the isoquinoline ring (papaverine) impairs the interaction with the alpha1-adrenoceptor site and facilitates it with the Ca2+-channels and PDEs, whereas the more flexible tetrahydroisoquinoline ring increases the binding to alpha1-adrenoceptors.

1. The selectivity of action of boldine and the related aporphine alkaloids, predicentrine (9-O-methylboldine) and glaucine (2,9-O-dimethylboldine) and alpha 1-adrenoceptor subtypes was studied by examining [3H]-prazosin competition binding in rat cerebral cortex. WB 4101 and benoxathian were used as selective alpha 1A-adrenoceptor antagonists. 2. In the competition experiments [3H]-prazosin (0.2 nM) binding was inhibited by WB 4101 and benoxathian. The inhibition curves displayed shallow slopes which could be subdivided into high and low affinity components (pKi = 9.92 and 8.29 for WB 4101, 9.35 and 7.94 for benoxathian). The two antagonists recognized approximately 37% of the sites with high affinity from among the total [3H]-prazosin specific binding sites. 3. Boldine, predicentrine and glaucine also competed for [3H]-prazosin (0.2 nM) binding with shallow and biphasic curves recognizing 30-40% of the sites with high affinity. Drug affinities (pKi) at the high and low affinity sites were, 8.31 and 6.50, respectively, for boldine, 8.13 and 6.39 for predicentrine, and 7.12 and 5.92 for glaucine. The relative order of selectivity for alpha 1A-adrenoceptors was boldine (70 fold alpha 1A-selective) = predicentrine (60 fold, alpha 1A-selective) > glaucine (15 fold, alpha 1A-selective). 4. Pretreatment of rat cerebral cortex membranes with chloroethylclonidine (CEC, 10 microM) for 30 min at 37 degrees C followed by thorough washing out reduced specific [3H]-prazosin binding by approximately 70%. The CEC-insensitive [3H]-prazosin binding was inhibited by boldine monophasically (Hill slope = 0.93) with a single pKi value (7.76). 5. These results suggest that whereas the aporphine structure shared by these alkaloids is responsible for their selectively of action for the alpha 1A-adrenoceptor subtype in rat cerebral cortex, defined functional groups, namely the 2-hydroxy function, induces a significant increase in alpha 1A-subtype selectivity and affinity.

1. In the present study, depletion of internal Ca2+ stores sensitive to noradrenaline (1 microM) in rat aorta, is the signal for the entry of extracellular Ca2+, not only to refill the stores but also, in our experimental conditions, to activate the contractile proteins. This induces an increase in the resting tone that constitutes, the first functional evidence of this Ca2+ entry. 2. The fact that methoxamine (100 microM) reproduces the same processes as noradrenaline but clonidine (1 microM) does not, indicates that alpha(1)-adrenoceptor activation is related to the increase in the resting tone observed after depletion of adrenoceptor-sensitive internal Ca2+-stores. 3. Benoxathian and WB 4101 (alpha(1A)- and alpha(1D)-adrenoceptor antagonists) selectively inhibit, in a concentration-dependent manner, this mechanical response observed in absence of the agonist, which suggests that these agents can act as inverse agonists and provide a functional model for studying this phenomenon. Since chloroethylclonidine (100 microM) has no effect on this response, the participation of alpha(1B)-adrenoceptors can be ruled out. 4. Contractile responses to noradrenaline (1 microM) in Ca2+-free medium were selectively blocked by chloroethylclonidine. This suggests that the response to noradrenaline in Ca2+-free medium mainly depends on the activation of the alpha(1B)-adrenoceptor subtype.

1. The activity of (+/-)-laudanosine, a benzyltetrahydroisoquinoline alkaloid, was investigated in pithed rats and rat isolated aorta. Its effects on [3H]-(+)-cis-diltiazem and [3H]-nitrendipine binding to rat cerebral cortical membranes, and on the different molecular forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aorta were investigated. 2. The dose-response curve to methoxamine (3-300 micrograms kg-1, i.v.) in normotensive pithed rats was shifted to the right by (+/-)-laudanosine, 3 and 6 mg kg-1. 3. (+/-)-Laudanosine inhibited in a concentration-dependent manner the contractile responses evoked by noradrenaline (NA 1 microM), depolarizing solution (KCl 80 mM) or depolarizing solution plus phentolamine (10 microM) in rat isolated aorta. The alkaloid appeared to be more potent against NA-induced contractions. 4. In Ca(2+)-free solution, (+/-)-laudanosine (100 microM) inhibited the contraction evoked by NA and did not modify the phasic contractile response evoked by caffeine. The alkaloid did not modify the refilling of the intracellular Ca(2+)-sotres sensitive to NA or caffeine. 5. (+/-)-Laudanosine inhibited [3H]-prazosin binding to cortical membranes and also inhibited [3H]-(+)-cis-diltiazem but with a lower potency. [3H]-nitrendipine binding was not affected by laudanosine. 6. (+/-)-Laudanosine does not have a significant effect on the different forms of PDEs isolated from bovine aorta. In contrast, compounds structurally related to this alkaloid such as papaverine and its derivatives, had a non-selective or more specific inhibitory effect on these PDE forms. These differences can be explained on the basis of their structural features: the planarity of the isoquinoline ring(papaverine) facilitates the interaction with receptor sites, and the different position of the benzyl group does not modify the activity unless this position leads to the presence of a chiral centre (laudanosine).7. These results suggest that (+/-)-laudanosine has a selective activity as an alpha1-adrenoceptor blocker. Its lack of action on different PDE forms provides us with information about a group of benzylisoquinolines that with small structural changes show a different effect on PDE-forms isolated from vascular smooth muscle.

1. In the present study we assessed the activity of antioquine, a bisbenzyltetrahydroisoquinoline alkaloid isolated from Pseudoxandra sclerocarpa, by examining its effects on the contractile activity of rat isolated aorta, specific binding of [3H]-(+)-cis-diltiazem, [3H]-nitrendipine and [3H]-prazosin to cerebral cortical membranes and the different molecular forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aorta. 2. Contractions in rat aorta induced by high concentrations of KCl (80 mM) and noradrenaline (1 microM) were inhibited by antioquine in a concentration-dependent manner (0.1 microM- 300 microM). The alkaloid appeared more potent against KCl-induced contractions. This inhibitory effect was observed at both 37 degrees C and 25 degrees C. 3. Paradoxically, at the highest concentration tested (300 microM) antioquine induced a contractile response of similar magnitude in the presence and absence of extracellular calcium, at 37 degrees C. This activity was greatly attenuated at 25 degrees C. Antioquine-induced contractions were not inhibited by prazosin (0.1 microM), nifedipine (1 microM) or diltiazem (100 microM). On the contrary, prazosin and nifedipine slightly increased the contractions in the presence of extracellular calcium. Papaverine (100 microM) partially inhibited the contractile response to antioquine both in the presence and absence of extracellular calcium. 4. At 25 degrees C, in Ca(2+)-free solution, antioquine (300 microM) did not modify the contractile response (phasic and tonic) evoked by noradrenaline, but increased the phasic contraction induced by caffeine. At 37 degrees C, the contraction elicited by antioquine made it impossible to observe the noradrenaline-induced one. 5. Antioquine showed affinity for the [3H]-prazosin binding site and for the [3H]-(+)-cis-diltiazembinding site of the Ca2+-channel receptor complex, but had no effect at the dihydropyridine binding site in rat cerebral cortex.6. Antioquine weakly inhibited some PDE forms isolated from bovine aorta: a CaM-PDE (PDE I)which preferentially hydrolyzes cyclic GMP and is activated by calmodulin, and a rolipram-sensitive cyclic AMP-PDE (PDE IV) which hydrolyzed cyclic AMP. Antioquine did not exert any inhibitory effect on the other forms of PDE, a cyclic GMP selective form (PDE V) and a low Km cyclic AMP-PDEthat is inhibited by cyclic GMP (CGI-PDE, PDE III).7. The present work provides evidence that antioquine has properties both as a calcium entry blocker(possibly through the benzothiazepine recognition site in the calcium channel) and as a contractile agent.Its mechanism of action as a contractile agent is not related to Ca2+-entry and is hypothetically similar to that of calyculin-A or okadaic acid. The possible involvement of a-adrenoceptors in this paradoxical effect cannot be excluded. The rigidity of the molecule provides an interesting model for analyzing this contractile mechanism and the intracellular processes involved.

1. In the present study, the properties of glaucine (an aporphine structurally related to papaverine) were compared with those of papaverine, diltiazem, nifedipine and prazosin. The work includes functional studies on rat isolated aorta contracted with noradrenaline, caffeine or KCl, and a determination of the affinity of glaucine at calcium channel binding sites of alpha-adrenoceptors, by use of [3H]-(+)-cis-diltiazem, [3H]-nitrendipine and [3H]-prazosin binding to cerebral cortical membranes. The effects of glaucine on the different molecular forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aorta were also determined. 2. Contraction evoked by noradrenaline (1 microM) or depolarizing solution (60 mM KCl) were inhibited in a concentration-dependent manner by all the compounds tested. As expected, prazosin showed a greater selectivity of action on NA-induced contraction, whereas nifedipine and diltiazem appeared more potent on KCl-induced contraction. Glaucine had a greater potency on the contraction elicited by noradrenaline whereas papaverine acted non specifically. 3. In Ca(2+)-free solution, prazosin (0.1 microM) and glaucine (0.1 mM) inhibited the contraction evoked by NA; diltiazem (0.1 mM) diminished this contraction whereas nifedipine (1 microM) had no effect. Preincubation of tissues with glaucine, diltiazem, nifedipine and prazosin did not modify the contractile response induced by caffeine. In contrast, papaverine (0.1 mM) significantly inhibited the contractions evoked by NA or caffeine in Ca(2+)-free medium. 4. Glaucine and papaverine show affinity at the [3H]-prazosin binding site and at the benzothiazepine binding site of the Ca(2+)-channel receptor complex, but have no effect at the dihydropyridine binding site in rat cerebral cortex. Glaucine exerts some selectivity as an inhibitor of [3H]-prazosin binding as opposed to [3H]-(+ )-cis-diltiazem binding while papaverine appears to have approximately equal affinity in this respect.5. This study confirms the presence of four phosphodiesterase (PDE) activities in bovine aorta: a calmodulin-activated PDE (CaM-PDE type I) which hydrolyzed preferentially guanosine 3':5'-cyclic monophosphate (cyclic GMP); a cyclic GMP selective form (cGMP-PDE type V); and two low Km adenosine 3':5'-cyclic monophosphate (cyclic AMP) PDEs that are insensitive to the stimulatory effect of CaM, one of which was inhibited by cyclic GMP (CGI-PDE, type III) and the other by rolipram (cAMP-PDE, type IV). Glaucine selectively inhibits one of the two forms of Ca2+-independent low Km cAMP-PDE, the type IV. In contrast, papaverine exerts a non-selective inhibitory effect upon all PDE forms.6. The present work provides evidence that glaucine, a benzyltetrahydroisoquinoline alkaloid, has interesting properties as an alpha l-adrenoceptor antagonist, calcium entry blocker (through the benzothiazepine recognition site in the calcium channel) and as a selective inhibitor of the rolipram-sensitive cAMP-PDE, type IV PDE.