Multiple effects of sibutramine on ejaculation and on vas deferens and seminal vesicle contractility.

Sibutramine is an inhibitor of norepinephrine and 5-HT reuptake largely used in the management of obesity. Although a fairly safe drug, postmarketing adverse effects of sibutramine were reported including abnormal ejaculation in men. This study investigates the effects of sibutramine on ejaculation and vas deferens and seminal vesicle contractility. Adult male rats received sibutramine (5; 20; or 50 mg kg(-1), ip) and after 60 min were exposed to receptive females for determination of ejaculation parameters. The vasa deferentia and seminal vesicles of untreated rats were mounted in isolated organ baths for recording of isometric contractions and HEK293 cells loaded with fluorescent calcium indicator were used to measure intracellular Ca(2+) transients. Sibutramine 5 and 20 mg kg(-1) reduced ejaculation latency whereas 50 mg kg(-1) increased ejaculation latency. Sibutramine 3 to 30 microM greatly increased the sensitivity of the seminal vesicle and vas deferens to norepinephrine, but at concentrations higher than 10 microM there were striking depressions of maximal contractions induced by norepinephrine, carbachol and CaCl(2). In HEK293 cells, sibutramine 10 to 100 microM inhibited intracellular Ca(2+) transients induced by carbachol. Depending on the doses, sibutramine either facilitates or inhibits ejaculation. Apart from its actions in the central nervous system, facilitation of ejaculation may result from augmented sensitivity of smooth muscles to norepinephrine while reductions of intracellular Ca(2+) may be involved in the delayed ejaculation observed with high doses of sibutramine.

L-arginine signalling potential in the brain: the peripheral gets central.

L-arginine is a basic amino acid that has versatile metabolic roles, being involved in the generation of a wide range of biologically active intermediates such as nitric oxide (NO), polyamines, creatine and L-amino acids [1]. Because the levels of L-arginine reflect a metabolic crossroads, the mechanisms of its synthesis and degradation in peripheral tissues are very well described. However, there is an increasing amount of data also implicating this amino acid as a mediator of central nervous system activities and those are not yet fully understood. Here we shall summarize the tissue-specific pathways controlling L-arginine intracellular and blood levels and also the emerging evidence pointing to a role of this metabolite in the central regulation of diverse physiological processes, as blood pressure control and inflammatory response. As a conclusion we shall discuss the advantages of targeting L-arginine metabolism over other NO donors, as a general strategy to correct NO deficiency related diseases and discuss a few new patents recently deposited which take into account the rational perspective outlined in the present review.

Identification of novel bradykinin-potentiating peptides (BPPs) in the venom gland of a rattlesnake allowed the evaluation of the structure-function relationship of BPPs.

Aiming to extend the knowledge about the diversity of bradykinin-potentiating peptides (BPPs) and their precursor proteins, a venom gland cDNA library from the South American rattlesnake (Crotalus dursissus terrificus, Cdt) was screened. Two novel homologous cDNAs encoding the BPPs precursor protein were cloned. Their sequence contain only one single longer BPP sequence with the typical IPP-tripeptide, and two short potential BPP-like molecules, revealing a unique structural organization. Several peptide sequences structurally similar to the BPPs identified in the precursor protein from Cdt and also from others snakes, were chemically synthesized and were bioassayed both in vitro and in vivo, by means of isolated smooth muscle preparations and by measurements of blood pressure in anaesthetized rats, respectively. We demonstrate here that a pyroglutamyl residue at the N-terminus with a high content of proline residues, even with the presence of a IPP moiety characteristic of typical BPPs, are not enough to determine a bradykinin-potentiating activity to these peptides. Taken together, our results indicate that the characterization of the BPPs precursor proteins and identification of characteristic glutamine residues followed by proline-rich peptide sequences are not enough to predict if these peptides, even with a pyroglutamyl residue at the N-terminus, will present the typical pharmacological activities described for the BPPs.

Novel pharmaceutical composition of bradykinin potentiating penta peptide with beta-cyclodextrin: physical-chemical characterization and anti-hypertensive evaluation.

This work describes chemical properties and anti-hypertensive activity of an oral pharmaceutical formulation obtained from the complexation of beta-cyclodextrin (beta-CD) with bradykinin potentiating penta peptide (BPP-5a) founded in the Bothrops jararaca poison. Physical chemistry characterizations were recorded in order to investigate the intermolecular interactions between species in complex. Circular dichroism data indicated conformational changes of BPP-5a upon complexation with beta-CD. ROESY and theoretical calculations showed a selective approximation of triptophan moiety into cavity of beta-CD. Isothermal titration calorimetry data indicated an exothermic formation of the complex, which is accomplished by reduction of entropy. The anti-hypertensive activity of the BPP-5a/beta-CD complex has been evaluated in spontaneous hypertensive rats, showing better results than pure BPP-5a.

Cloning and characterization of the human and rabbit NUDEL-oligopeptidase promoters and their negative regulation.

NUDEL-oligopeptidase is a cytosolic cysteine peptidase, active towards oligopeptides and involved in the conversion and inactivation of a number of bioactive peptides. This protein interacts with neuronal proteins and is essential for brain development and cortical organization during embryogenesis. In this study, 5'-flanking sequences of the human and rabbit NUDEL-oligopeptidase gene were cloned into the pGL3 reporter gene vector and the promoter activity of the full-length fragment and deletions series was measured in transient transfection assays using two different cell lines, namely, C6 rat glioma and NH15 human neuroblastoma. Overall, a very similar pattern of promoter activity was obtained for both rabbit and human NUDEL-oligopeptidase promoter sequences, and their respective serial deletion constructs upon transient transfection into these cell lines. The only exception was for the longest rabbit upstream sequence that displayed about 1.8-fold higher luciferase expression upon transfection into NH15 neuronal cells than that observed upon transfection into C6 glioma cells. On the other hand, no significant difference was observed for the human longest sequence. These results are in good agreement with the expression pattern of NUDEL-oligopeptidase in human and rabbit tissues.

A structure-based site-directed mutagenesis study on the neurolysin (EC 3.4.24.16) and thimet oligopeptidase (EC 3.4.24.15) catalysis.

Neurolysin (EP24.16) and thimet oligopeptidase (EP24.15) are closely related metalloendopeptidases. Site-directed mutagenesis of Tyr(613) (EP24.16) or Tyr(612) (EP24.15) to either Phe or Ala promoted a strong reduction of k(cat)/K(M) for both enzymes. These data suggest the importance of both hydroxyl group and aromatic ring at this specific position during substrate hydrolysis by these peptidases. Furthermore, the EP24.15 A607G mutant showed a k(cat)/K(M) of 2x10(5) M(-1) s(-1) for the Abz-GFSIFRQ-EDDnp substrate, similar to that of EP24.16 (k(cat)/K(M)=3x10(5) M(-1) s(-1)) which contains Gly at the corresponding position; the wild type EP24.15 has a k(cat)/K(M) of 2.5x10(4) M(-1) s(-1) for this substrate.

The Mycobacterium leprae hsp65 displays proteolytic activity. Mutagenesis studies indicate that the M. leprae hsp65 proteolytic activity is catalytically related to the HslVU protease.

The present study reports, for the first time, that the recombinant hsp65 from Mycobacterium leprae (chaperonin 2) displays a proteolytic activity toward oligopeptides. The M. leprae hsp65 proteolytic activity revealed a trypsin-like specificity toward quenched fluorescence peptides derived from dynorphins. When other peptide substrates were used (beta-endorphin, neurotensin, and angiotensin I), the predominant peptide bond cleavages also involved basic amino acids in P(1), although, to a minor extent, the hydrolysis involving hydrophobic and neutral amino acids (G and F) was also observed. The amino acid sequence alignment of the M. leprae hsp65 with Escherichia coli HslVU protease suggested two putative threonine catalytic groups, one in the N-domain (T(136), K(168), and Y(264)) and the other in the C-domain (T(375), K(409), and S(502)). Mutagenesis studies showed that the replacement of K(409) by A caused a complete loss of the proteolytic activity, whereas the mutation of K(168) to A resulted in a 25% loss. These results strongly suggest that the amino acid residues T(375), K(409), and S(502) at the C-domain form the catalytic group that carries out the main proteolytic activity of the M. leprae hsp65. The possible pathophysiological implications of the proteolytic activity of the M. leprae hsp65 are now under investigation in our laboratory.

Selective inhibition of the C-domain of angiotensin I converting enzyme by bradykinin potentiating peptides.

Somatic angiotensin I converting enzyme (ACE) contains two functional active sites. Up to now, most of the studies aimed at characterizing the selectivity of inhibitors toward the two ACE active sites relied on the use of ACE mutants containing a single functional active site. By developing new fluorogenic synthetic substrates of ACE, we demonstrated that inhibitor selectivity can be assessed directly by using somatic ACE. This useful screening approach led us to discover that some bradykinin potentiating peptides turned out to be selective inhibitors of the C-domain of ACE. The peptide pGlu-Gly-Leu-Pro-Pro-Arg-Pro-Lys-Ile-Pro-Pro, with K(i)(app) values of 30 nM and 8 microM, respectively, for the C- and N-domain of ACE, is to our knowledge the most highly selective C-domain inhibitor of ACE so far reported. Inhibitors able to block selectively either the N- or C-domain of ACE will represent unique tools to probe the function of each domain in the regulation of blood pressure or other physiopathological events involving ACE activity.