UK-414,495, a selective inhibitor of neutral endopeptidase, potentiates pelvic nerve-stimulated increases in female genital blood flow in the anaesthetized rabbit.

BACKGROUND AND PURPOSE: Female sexual arousal consists of a number of physiological responses resulting from increased genital blood. Vasoactive intestinal peptide (VIP), neuropeptide Y and to a lesser extent nitric oxide are neurotransmitters found in the vasculature of the genitalia. Neutral endopeptidase (NEP) modulates the activity of neuropeptides including VIP. The aim of this study was to investigate the control of genital blood flow by VIP and endogenous neuropeptides using a selective NEP inhibitor [UK-414,495, ((R)-2-({1-[(5-ethyl-1,3,4-thiadiazol-2-yl) carbamoyl]cyclopentyl}methyl) valeric acid)]. EXPERIMENTAL APPROACH: Vaginal and clitoral blood flow (VBF and CBF) were monitored using laser Doppler in terminally anaesthetized New Zealand rabbits. Increases in VBF and CBF were induced by either electrical stimulation of the pelvic nerve or by i.v. infusion of VIP. KEY RESULTS: Stimulation of the pelvic nerve increased VBF and CBF, compared with basal flow. Increases were mimicked by infusion of exogenous VIP. UK-414,495 dose-dependently potentiated pelvic nerve-stimulated increases in VBF (EC(50)= 37 +/- 9 nM; 3.6 x IC(50) rabbit NEP). Nerve-stimulated increases in VBF and CBF were both enhanced after UK-414,495. UK-414,495 increased the amplitude and duration of VIP-induced increases in VBF. UK-414,495 had no effect on basal VBF or cardiovascular parameters. CONCLUSIONS AND IMPLICATIONS: Inhibition of NEP potentiates pelvic nerve-stimulated increases in genital blood flow. This suggests that the endogenous neurotransmitter mediating genital blood flow is a substrate for NEP (most likely VIP). NEP inhibitors may restore sexual arousal in women adversely affected by female sexual arousal disorder.

Effect of the selective phosphodiesterase type 5 inhibitor sildenafil on erectile dysfunction in the anesthetized dog.

PURPOSE: The effects of sildenafil, a highly selective inhibitor of cyclic guanosine monophosphate-specific phosphodiesterase type 5, on erectile function in the anesthetized dog were evaluated. MATERIALS AND METHODS: In pentobarbital-anesthetized dogs, increases in intracavernosal pressure in the corpus cavernosum and penile blood flow were induced by pelvic nerve stimulation over a frequency range of 1 to 16 hertz. The effects of increasing doses of sildenafil on electrically stimulated intracavernosal pressure, penile blood flow, blood pressure, and heart-rate were evaluated. In parallel experiments, the effects of the nitric oxide synthase inhibitor N omega-Nitro-L-Arginine (L-NOArg) on these same parameters also were assessed. RESULTS: The effects of nerve stimulation on intracavernosal pressure and blood flow to the penis were blocked by L-NOArg, 0.1-3 mg./kg., in a dose-related manner, confirming the important role of nitric oxide in producing erections. Sildenafil, 1-100 microg./kg administered intravenously, had no direct effect on intracavernosal pressure but potentiated the increase in intracavernosal pressure induced by nerve stimulation. This potentiation occurred at sildenafil plasma concentrations consistent with its relaxation effect on isolated human cavernosal tissue and its inhibition of phosphodiesterase type 5 in vitro. Sildenafil had no significant effect on blood pressure or heart rate. CONCLUSIONS: By inhibiting cyclic guanosine monophosphate-specific phosphodiesterase type 5, sildenafil augments the neuronal mechanism responsible for penile erection. This mechanism explains the significant improvements reported in the rigidity and duration of erections seen in patients with erectile dysfunction who have been treated with oral sildenafil.

Effects of sildenafil on the relaxation of human corpus cavernosum tissue in vitro and on the activities of cyclic nucleotide phosphodiesterase isozymes.

PURPOSE: Sildenafil, an inhibitor of cGMP-specific phosphodiesterase 5 (PDE5), is currently undergoing evaluation as an oral therapy for penile erectile dysfunction. The aims of this study were to investigate the mechanism of action of sildenafil on the neurogenic relaxation of human corpus cavernosum (HCC) in vitro and to determine the activity of sildenafil against a full range of PDE isozymes. MATERIALS AND METHODS: Strips of HCC tissue were precontracted with phenylephrine. Relaxation responses resulting from electrical field stimulation (EFS) were then determined in the presence and absence of sildenafil. The effects of sildenafil on PDE1 to 5 prepared from human tissues and PDE6 from bovine retina were determined by measuring the conversion of [3H]-cGMP or [3H]-cAMP to their respective [3H]-5'-mononucleotides. RESULTS: Sildenafil (0.001 to 1 microM) enhanced the EFS-induced, nitric oxide (NO) dependent, relaxation of HCC in a concentration-dependent manner to a maximum of 3 times the pretreatment level at 1 microM sildenafil. Compared with zaprinast, an early PDE5 inhibitor, sildenafil was approximately 240-fold more potent, inhibiting PDE5 from HCC with a geometric mean IC50 of 3.5 nM. For sildenafil, IC50 values for inhibition of PDE1 to 4 were 80 to more than 8500 times greater than that for PDE5 and the IC50 for PDE6 (33 nM) was approximately 9-fold greater. CONCLUSIONS: The data support the proposal that enhancement of penile erection by sildenafil in patients with erectile dysfunction involves potentiation of the NO-stimulated cGMP signal mediating relaxation of cavernosal smooth muscle during sexual stimulation. Sildenafil is a potent inhibitor of PDE5 from HCC, with high selectivity for PDE5 relative to other PDE isozymes.

Effects of sildenafil, a type-5 cGMP phosphodiesterase inhibitor, and papaverine on cyclic GMP and cyclic AMP levels in the rabbit corpus cavernosum in vitro.

OBJECTIVE: To investigate further the mechanisms of action of sildenafil, a highly selective and potent inhibitor of type 5 cGMP phosphodiesterase (PDE5) that has proved effective in the treatment of erectile dysfunction, by assessing its effect on the in vitro formation of cGMP and cAMP in the corpus cavernosum of the rabbit. MATERIALS AND METHODS: Male New Zealand White rabbits (2.5 kg) were killed and their penises rapidly excised, cut into segments and pooled. Penile segments were then incubated with various concentrations of sildenafil or papaverine. The formation of cGMP was stimulated with increasing concentrations of sodium nitroprusside (SNP) and the cGMP and cAMP concentrations measured by radioimmunoassay. Responses were compared to those obtained with papaverine, which is used therapeutically as an erectogen. RESULTS: In the presence or absence of SNP, sildenafil increased cGMP concentrations in rabbit penile tissue with increasing dose; the increase was greatest (about 28-fold) when cGMP was stimulated with SNP (up to 10 mumol/L). At all stimulatory concentrations of SNP, the effective concentrations for 50% stimulation (EC50) of sildenafil were 430-520 nmol/L. Concentrations of cAMP were unaltered by sildenafil. Papaverine enhanced cGMP formation in response to SNP, but at much higher concentrations than did sildenafil (> or = 10 mumol/L). CONCLUSIONS: Sildenafil specifically increases cGMP levels in rabbit corpora cavernosa; the increase is greater in the presence of SNP indicating that, in vivo, sildenafil may enhance erection by the augmentation of nitric oxide-mediated relaxation pathways. The erectogenic effect of sildenafil is mediated by a specific enhancement of cGMP accumulation in the corpus cavernosum, consistent with the known activity of sildenafil as a potent and highly selective inhibitor of cGMP-specific PDE.

Sildenafil: an orally active type 5 cyclic GMP-specific phosphodiesterase inhibitor for the treatment of penile erectile dysfunction.

Sildenafil (Viagra, UK-92,480) is a novel oral agent under development for the treatment of penile erectile dysfunction. Erection is dependent on nitric oxide and its second messenger, cyclic guanosine monophosphate (cGMP). However, the relative importance of phosphodiesterase (PDE) isozymes is not clear. We have identified both cGMP- and cyclic adenosine monophosphate-specific phosphodiesterases (PDEs) in human corpora cavernosa in vitro. The main PDE activity in this tissue was due to PDE5, with PDE2 and 3 also identified. Sildenafil is a selective inhibitor of PDE5 with a mean IC50 of 0.0039 microM. In human volunteers, we have shown sildenafil to have suitable pharmacokinetic and pharmacodynamic properties (rapid absorption, relatively short half-life, no significant effect on heart rate and blood pressure) for an oral agent to be taken, as required, prior to sexual activity. Moreover, in a clinical study of 12 patients with erectile dysfunction without an established organic cause, we have shown sildenafil to enhance the erectile response (duration and rigidity of erection) to visual sexual stimulation, thus highlighting the important role of PDE5 in human penile erection. Sildenafil holds promise as a new effective oral treatment for penile erectile dysfunction.

Evaluation of the pharmacological selectivity profile of alpha 1 adrenoceptor antagonists at prostatic alpha 1 adrenoceptors: binding, functional and in vivo studies.

1. The profile of a range of alpha 1 adrenoceptor antagonists was determined in vitro against cloned human alpha 1A, alpha 1B and alpha 1D adrenoceptors and against noradrenaline-mediated contractions of rat aorta and human prostate. The in vivo profile of compounds was determined in an anaesthetized dog model which allowed the simultaneous assessment of antagonist potency against phenylephrine-mediated increases in blood pressure and prostatic pressure. 2. The quinazoline antagonists, prazosin, doxazosin and alfuzosin displayed high affinity but were non selective for the three cloned human alpha 1 adrenoceptors. Indoramin and SNAP 1069 showed selectivity for alpha 1A and alpha 1B adrenoceptors relative to the alpha 1D subtype. Rec 15/2739, WB 4101, SL 89,0591, (+)- and (-)- tamsulosin showed selectivity for alpha 1A and alpha 1D adrenoceptors relative to the alpha 1B subtype. RS 17053 showed high affinity and selectivity for alpha 1A adrenoceptors (pKi 8.6) relative to alpha 1B (pKi = 7.3) and alpha 1D (pKi = 7.1) subtypes. 3. (+)-Tamsulosin, (-)-tamsulosin, SL 89,0591, Rec 15/2739, SNAP 1069 and RS 17053 appeared to act as competitive antagonists of noradrenaline-mediated contractions of rat aorta yielding pA2 affinity estimates which were similar to binding affinities at cloned human alpha 1D adrenoceptors. The following rank order was obtained: prazosin = (-)-tamsulosin > doxazosin > SL 89,0591 = (+)-tamsulosin > Rec 15/2739 > RS 17053 = SNAP 1069. 4. (-)-Tamsulosin was a very potent, insurmountable antagonist of noradrenaline-mediated contractions of human prostate, yielding an approximate pA2 estimate of 9.8 at 1 nM. The corresponding (+)-enantiomer was 30 fold weaker. SL 89,0591, SNAP 1069 and Rec 15/2739 yielded pA2 estimates which compared well with their alpha 1A binding affinities. The affinity estimate for prazosin on human prostate was lower than the corresponding binding affinity determined at alpha 1A adrenoceptors and RS 17053 was a very weak antagonist on human prostate (pA2 = 6.0) relative to the high affinity (pKi = 8.6) determined at cloned human alpha 1A adrenoceptors. 5. In the anaesthetized dog, in vivo pseudo "pA2' values showed that doxazosin, (+)- and (-)-tamsulosin inhibited phenylephrine-induced increases in prostatic and blood pressure with similar affinity, implying that these agents show little or no selectivity for prostatic responses in this model. SL 89,0591 and SNAP 1069 were moderately selective (3 and 6 fold respectively) for prostatic pressure relative to blood pressure. Rec 15/2739 was a more potent antagonist of phenylephrine-mediated increases in prostatic pressure ("pA2' = 8.74) compared to blood pressure ("pA2' = 7.51). 6. Data in this study suggest that the alpha 1 adrenoceptor mediating noradrenaline-induced contractions of human prostate, whilst having some of the characteristics of an alpha 1A adrenoceptor, cannot be satisfactorily aligned with cloned alpha 1A, alpha 1B or alpha 1D adrenoceptors. In addition, studies in the anaesthetized dog have shown that agents having high affinity and selectivity for prostatic alpha 1 adrenoceptors, particularly over the alpha 1D subtype, appear to inhibit phenylephrine-induced increases in prostatic pressure selectively compared to blood pressure.

Activation of the micturition reflex by NK2 receptor stimulation in the anaesthetized guinea-pig.

1. The mechanisms underlying stimulation of bladder contractions and bronchoconstriction by the selective NK2 receptor agonist, [beta-Ala8]NKA(4-10), were examined in the anaesthetized guinea-pig. 2. Atropine, alpha,beta-methylene-ATP and ganglion blocking agents were used to examine the contribution of reflex arc activation and/or potentiation of efferent mechanisms to the NK2 receptor-mediated responses seen in these two tissues. 3. [beta-Ala8]NKA(4-10)-induced bronchoconstriction was immediate, dose-dependent and was unaffected by pretreatment with ganglion blockers (hexamethonium or chlorisondamine), blockade of muscarinic receptors by atropine, or desensitization of P2 purinoceptors by alpha,beta-methylene-ATP. 4. At does of 5 micrograms kg-1 and above, [beta-Ala8]NKA(4-10) induced bladder contractions that appeared to be of an 'all-or-nothing' nature. These contractions occurred after a delay of 10 to 30 s and were often biphasic, comprised of an initial rapid component followed by a slower tonic component. 5. Pretreatment of the animals with either atropine or the desensitizing purinoceptor agonist alpha,beta-methylene-ATP, resulted in partial inhibition of bladder contractile responses to [beta-Ala8]NKA(4-10). The combination of atropine and alpha,beta-methylene-ATP pretreatment resulted in additive inhibition leading to complete blockade of the response. 6. The bladder responses to [beta-Ala8]NKA(4-10) (5 micrograms kg-1) were inhibited by pretreatment with the ganglion blockers, hexamethonium and chlorisondamine, indicating a preganglionic mechanism of action. 7. These findings demonstrate the indirect nature of the bladder contractions induced by activation of NK2 receptors in the anaesthetized guinea-pig. Contractions occur secondary to the release of endogenous cholinergic and NANC transmitters by activation of neuronal NK2 receptors located at apreganglionic site, possibly on capsaicin-sensitive sensory afferent nerves, where NK2 sites have been demonstrated autoradiographically. In contrast, [beta-Ala8]NKA(4- 10)-induced bronchoconstriction in the anaesthetized guinea-pig is a direct smooth muscle contractile response that is unaffected by ganglionblockade or blockade of muscarinic receptors.

Characterization of an alpha 1D-adrenoceptor mediating the contractile response of rat aorta to noradrenaline.

1. The affinities of a number of alpha 1-adrenoceptor antagonists were determined by displacement of [3H]-prazosin binding from cloned human alpha 1A-adrenoceptors (previously designated cloned alpha 1c subtype), alpha 1B alpha 1D and rat alpha 1D-adrenoceptors, stably expressed in rat-1 fibroblasts. Functional affinity estimates for these compounds were also determined from noradrenaline-mediated contractions of rat aorta. 2. BMY 7378 displayed high affinity for cloned human alpha 1D-adrenoceptors (pKi = 8.2 +/- 0.10) and was selective over alpha 1A (pKi = 6.2 +/- 0.10) and alpha 1B subtypes (6.7 +/- 0.11). WB 4101, benoxathian and phentolamine displayed high affinity for alpha 1A and alpha 1D adrenoceptors compared to the alpha 1B subtype. Spiperone displayed high affinity and selectivity for alpha 1B adrenoceptors (pKi 8.8 +/- 0.16). 5-Methyl-urapidil was selective for cloned alpha 1A adrenoceptors. 3. Comparative binding affinities (pKi) for compounds at cloned human and rat1D adrenoceptors were almost identical (r = 0.99, slope = 1.08). 4. Prazosin, doxazosin and 5-methyl-urapidil were potent, competitive antagonists of noradrenaline-mediated contractions of rat aorta (pA2 values of 9.8, 8.8 and 7.8 respectively). The selective alpha 1D antagonist BMY 7378 was also a potent antagonist on rat aorta (pKB = 8.3 +/- 0.1) but the interaction of this compound was not consistent with competitive antagonism at a single population of receptors. 5. Functional affinities for compounds determined against noradrenaline-mediated contractions of rat aorta correlated well with binding affinities at cloned alpha 1D-adrenoceptors (r = 0.96), but not with alpha 1A (r = 0.61) or alpha 1B (r = 0.46) subtypes. 6. Noradrenaline-mediated contractions of rat aorta were sensitive to the alkylating effects of chlorethylclonidine (CEC). CEC (10 microM) caused a small rightward shift in the noradrenaline concentration-response curve. CEC at 100 microM caused a further shift and suppression of the maximum response to noradrenaline.7. The results of this study suggest that noradrenaline predominantly, but not exclusively, mediates contraction of rat aorta through the activation of an alphalD-adrenoceptor.

Effect of alpha 1 adrenoceptor antagonists on prostatic pressure and blood pressure in the anesthetized dog.

OBJECTIVES: In the current study we have profiled a range of compounds at alpha 1 adrenoceptor subtypes in vitro and have assessed their effects in vivo using the anesthetized dog in an attempt to elucidate the predominant alpha 1 adrenoceptor subtype mediating contractile responses of the canine prostate. METHODS: The affinity of compounds for alpha 1 adrenoceptor subtypes was determined by displacement of [3H] prazosin binding from stably transfected rat 1 fibroblasts expressing alpha 1A, alpha 1B, and alpha 1C, adrenoceptor subtypes. The potency of these agents was then assessed in vivo using an anesthetized dog model allowing simultaneous measurement of prostatic pressure and blood pressure following intravenous (i.v.) administration of phenylephrine (1 to 128 micrograms/kg). RESULTS: All compounds examined in this study showed high and similar affinity for alpha 1 adrenoceptor subtypes, with the exception of 5-Methyl-urapidil, which was selective for alpha 1C (pKi = 9.3) over alpha 1B (pKi = 7.2) and alpha 1A (pKi = 8.1). Doxazosin, terazosin, alfuzosin, and tamsulosin were potent antagonists of phenylephrine responses and in vivo derived "pseudo pA2" determinations showed that the drugs did not discriminate between prostatic and vascular receptors. 5-Methyl-urapidil was also a potent antagonist of phenylephrine-induced responses but was selective for prostatic pressure ("pseudo pA2" = 8.7) over blood pressure ("pseudo pA2" = 7.2). CONCLUSIONS: Data in the present study suggest a predominant role of the alpha 1C adrenoceptor subtype in the contractile response of the canine prostate to phenylephrine in vivo. This model therefore provides a suitable means of assessing putative prostate-selective antagonists for the treatment of benign prostatic hyperplasia.

Three-dimensional structure of echistatin and dynamics of the active site.

The snake venom protein echistatin contains the cell recognition sequence Arg-Gly-Asp and is a potent inhibitor of platelet aggregation. The three-dimensional structure of echistatin and the dynamics of the active RGD site are presented. A set of structures was determined using the Distance Geometry method and subsequently refined by Molecular Dynamics and energy minimization. Disulfide pairings are suggested, based on violations of experimental constraints. The structures satisfy 230 interresidue distance constraints, derived from nuclear Overhauser effect measurements, five hydrogen-bonding constraints, and 21 torsional constraints from vicinal spin-spin coupling constants. The segment from Gly5 to Cys20 and from Asp30 to Asn42 has a well-defined conformation and the Arg-Gly-Asp sequence, which adopts a turn-like structure, is located at the apex of a nine-residue loop connecting the two strands of a distorted beta-sheet. The mobility of the Arg-Gly-Asp site has been quantitatively characterized by 15N relaxation measurements. The overall correlation time of echistatin was determined from fluorescence measurements, and was used in a model-free analysis to determine internal motional parameters. The active site has order parameters of 0.3-0.5, i.e., among the smallest values ever observed at the active site of a protein. Correlation of the flexible region of the protein as characterized by relaxation experiments and the NMR solution structures was made by calculating generalized order parameters from the ensemble of three-dimensional structures. The motion of the RGD site detected experimentally is more extensive than a simple RGD loop 'wagging' motional model, suggested by an examination of superposed solution structures.

Pharmacological properties of the cloned alpha 1A/D-adrenoceptor subtype are consistent with the alpha 1A-adrenoceptor characterized in rat cerebral cortex and vas deferens.

1. The pharmacological characteristics of cloned mammalian alpha 1A/D-, alpha 1B- and alpha 1C-adrenoceptor subtypes expressed in rat 1 fibroblasts were determined in comparison to the binding and functional properties of these subtypes in rat tissues. 2. Analysis of [3H]-prazosin binding to membrane homogenates from rat 1 fibroblast cells expressing each of the alpha 1-subtypes indicated high affinity binding to a single population of binding sites. Binding affinities were similar for alpha 1A/D-, alpha 1B- and alpha 1C-subtypes (Kds: 0.13, 0.10 and 0.15 nM respectively) although a higher density of alpha 1B- and alpha 1C-receptors (Bmax: 4068 and 10,323 fmol mg-1 protein respectively) were expressed in comparison to alpha 1A/D (838 fmol mg-1). 3. Displacement of [3H]-prazosin from membranes expressing cloned alpha 1-adrenoceptor subtypes revealed that 5-methyl-urapidil, WB 4101, benoxathian and phentolamine displayed high affinity and selectivity for alpha 1A/D- over alpha 1B-subtypes. These compounds also had high affinity and selectivity for alpha 1C- over alpha 1B-subtypes. 5-Methyl-urapidil showed selectivity for alpha 1C (Ki 0.60 +/- 0.16 nM) over both alpha 1A/D (Ki, 9.8 +/- 2.8 nM) and alpha 1B (Ki 57.2 +/- 12 nM) subtypes. Prazosin and doxazosin were not subtype selective. 4. In comparison to [3H]-prazosin a similar pharmacological profile was obtained with [125I]-HEAT using cloned alpha 1A/D-, alpha 1B- and alpha 1C-adrenoceptors expressed in rat 1 fibroblasts. 5. The affinities of prazosin, WB 4101, 5-methyl-urapidil, phentolamine and benoxathian at cloned alpha 1A/D-receptors were consistent with alpha 1A affinities determined with chlorethylclonidine-treated rat cortical membranes. Affinities at cloned XIB-receptors were consistent with alpha 1B affinities determined with rat liver membranes.6. Using the epididymal rat vas deferens as a functional measure of alpha 1A affinity, prazosin (pA29.23 +/- 0.28), WB 4101 (pA2 9.58 +/- 0.12), phentolamine (pKB 7.90 +/- 0.16), benoxathian (pKB 9.21 +/- 0.21)and 5-methyl-urapadil (pKB 8.51 +/-0.16) were potent antagonists of noradrenaline-induced contractions.7. At present, evidence from cloning studies suggests the existence of at least three alpha 1-adrenoceptor subtypes. In contrast to the recent proposal for alpha l-adrenoceptor classification, the pharmacology of the cloned alpha 1A/D (or alpha lD)-adrenoceptor is more consistent with that of an alpha 1A-adrenoceptor characterized in rat cerebral cortex and vas deferens.

Neuropeptide-Y stimulates pituitary-adrenal activity in fetal and adult sheep.

Corticotrophin-releasing factor (CRF) and arginine vasopressin (AVP) are the primary neuropeptides regulating the secretion of ACTH from the anterior pituitary gland during fetal and adult life. However, a number of other neuropeptides including neuropeptide-Y (NPY) appear to modulate the activity of this system. The potential role of NPY in the regulation of pituitary-adrenal function was examined in fetal and adult sheep. Administration of NPY (6.5 micrograms) as a bolus injection into the third cerebral ventricle of adult ewes elicited a significant (P < 0.05) increase in plasma concentrations of ACTH. In fetal sheep at day 125 gestation (term = 145 days) a five-fold higher dose (30 micrograms) of NPY injected into the lateral cerebral ventricles also caused a significant increase in plasma concentrations of ACTH. The potential of NPY to influence ACTH secretion directly from the pituitary gland was investigated using primary cultures of fetal (day 130 gestation) and adult pituitary cells. CRF (10(-10)-10(-7) M) caused a significant (P < 0.01) dose-related increase in ACTH secretion from both fetal and adult pituitary cells. Furthermore, the secretion of ACTH from adult cells was significantly greater (P < 0.05) than that from fetal cells. NPY (10(-10)-10(-7) M) had no effect on basal or CRF-stimulated ACTH secretion from fetal or adult pituitary cells. Pre-incubation of pituitary cells with cortisol (10(-9) and 10(-7) M) for three days significantly inhibited CRF-stimulated ACTH secretion but had no effect on basal ACTH release. The simultaneous addition of NPY did not alter the ability of cortisol to inhibit CRF-stimulated ACTH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

The solution conformation of Ac-Pen-Arg-Gly-Asp-Cys-OH, a potent fibrinogen receptor antagonist.

The solution conformation of Ac-Pen-Arg-Gly-Asp-Cys-OH, a potent fibrinogen receptor antagonist, was characterized in DMSO-d6 by the combination of nmr and molecular modeling. The conformational space available to the peptide was explored using a distance geometry algorithm with distance constraints derived from 1H-nmr spectra. The dynamics of the peptide were examined by relaxation time measurements and low temperature studies. The results from the low temperature studies suggest that the peptide backbone does not exist in a single, well-defined conformation but undergoes exchange between multiple conformers. This result is consistent with the inability to find a single structure that satisfies all the nmr-derived constraints. The constraints could only be satisfied by considering pairs of conformers to represent the experimental data. The low energy conformers comprise type II' or type V beta-turns with distinct side-chain directionality. The Arg-Gly-Asp portion of the ring is flexible and can be described by amide-plane rotations of the Arg-Gly and Gly-Asp peptide bonds. Although some backbone flexibility is evident, the incorporation of beta,beta-dimethyl cysteine imparted greater conformational rigidity as compared to the previously studied cyclic pentapeptide, Ac-Cys-Arg-Gly-Asp-Cys-OH.

Structure of synthetic peptide analogues of an eggshell protein of Schistosoma mansoni.

The peptide (Gly-L-Tyr-L-Asp-L-Lys-L-Tyr)6, referred to as F4-6, was synthesized as a model for a schistosome eggshell protein in which the Gly-Tyr-Asp-Lys-Tyr consensus sequence is repeated over 40 times. Analysis by CD, Fourier transform infrared spectroscopy, potentiometric and spectrophotomertric titrations, NMR, and molecular modeling suggests that F4-6 forms some type of left-handed structure. A likely possibility appears to be a left-handed alpha-helix stabilized by Lysi-Aspi +4 salt bridges and possibly Aspi-Tyri +4 hydrogen bonding and Tyr-Tyr interactions. Spectroscopic studies of a number of F4-6 analogues support this conclusion. For example, substitution of D-Ala for Gly produces a peptide with enhanced left-handed helical spectral characteristics, whereas an L-Ala substitution results in a peptide with minimal structure. These studies suggest that the F4 protein from Schistosoma mansoni may be the first example of a naturally occurring protein devoid of proline and carbohydrate that forms a left-handed helix composed of L-amino acids, although alternative forms of other left-handed structures have yet to be rigorously excluded.

Inhibition of thrombin and other trypsin-like serine proteinases by cyclotheonamide A.

Cyclotheonamide A (CA), a cyclic peptide isolated from the marine sponge of the genus Theonella was shown to be a slow-binding inhibitor of several trypsin-like serine proteinases. Values of 4.6 x 10(4), 4.8 x 10(4), 9.3 x 10(3), 2.1 x 10(3) and 2.7 x 10(2) M-1 s-1 were determined for the second-order rate constants for formation of CA complexes with thrombin, trypsin, plasmin, 2-chain t-PA and factor Xa, respectively. The equilibrium constant (Ki) was measured for dissociation of CA from the CA complex with human thrombin (Ki = 1.0 nM), bovine trypsin (Ki = 0.2 nM), human plasmin (Ki = 12 nM), human factor Xa (Ki = 50 nM) and human 2-chain tissue plasminogen activator (t-PA) (Ki = 40 nM). CA produces dose dependent increases in clotting time assays. The clotting time in the thrombin time, activated partial thromboplastin time and prothrombin time assays, were doubled by 1.5, 0.9 and 48 microM CA, respectively. A model for the binding of CA to the active site of thrombin is proposed.

Endocrine actions of central neuropeptide Y in the ewe: activation of the hypothalamo-pituitary-adrenal axis by exogenous neuropeptide Y and role of endogenous neuropeptide Y in the secretion of luteinizing hormone during the oestrous cycle.

Neurons immunoreactive for neuropeptide Y (NPY) are abundant in the hypophysiotrophic areas of the brain. In particular, there is considerable anatomical evidence for the influence of this neuropeptide on the reproductive and hypothalamo-pituitary-adrenal axes. We therefore investigated whether central administration of NPY can alter the activities of the reproductive and hypothalamopituitary-adrenal axes in the ewe, and whether ovarian steroids are involved in the modulation of these events. We also attempted to investigate whether endogenous NPY is important in the control of luteinizing hormone-releasing hormone/luteinizing hormone (LHRH/LH) secretion in the sheep oestrous cycle. Central injection of NPY (0.15 and 1.5 nmol in 50 microliters saline), delivered by gravity flow into the third cerebral ventricle, had no effect on LH levels in ovariectomized (OVX) ewes (n = 6) or OVX ewes implanted with oestradiol (OVX/E2) (n = 7), nor was LH secretion altered by central NPY (1.5 nmol) in intact cycling animals in either the follicular or the luteal phase (n = 5). However, central administration of 1.5 nmol NPY to intact ewes during both the follicular (P < 0.05) and the luteal phase (P < 0.01), and in OVX/E2 ewes (P < 0.05) caused a large and significant increase in plasma cortisol levels. High titre antibodies were raised to NPY in sheep and the effects of peripheral and central (intracerebroventricular) administration of anti-NPY antibodies on the timing and/or characteristics of the E2-induced LH surge in anoestrous ewes and of the preovulatory surge of LH in cycling ewes were determined. Intravenous administration of anti-NPY antibodies (n = 6) had no effect on the oestradiol benzoate-induced LH surge, compared with the control injection of non-immune plasma (n = 6). Likewise, passive systemic immunization against NPY (n = 10) was without effect on the characteristics of the preovulatory LH surge, compared with the control group (n = 10). However, central (intracerebroventricular) administration of anti-NPY antibodies (n = 4) delayed or abolished the preovulatory LH surge when compared with non-immune plasma treatment in the same animals. In summary, tonic LHRH/LH secretion is unaffected by centrally administered NPY at the doses used in this study. However, the same doses of NPY activate the hypothalamo-pituitary-adrenal axis, thus lending clear support to the hypothesis that NPY is involved in the multifactorial regulation of adrenocorticotrophin and cortisol secretion in this species, probably by stimulating corticotrophin-releasing hormone and/or arginine vasopressin secretion within the hypothalamus.(ABSTRACT TRUNCATED AT 400 WORDS)

NMR and molecular modeling characterization of RGD containing peptides.

The tripeptide sequence arginine-glycine-aspartic acid (RGD) has been shown to be the key recognition segment in numerous cell adhesion proteins. The solution conformation and dynamics in DMSO-d6 of the cyclic pentapeptides, [formula: see text], a potent fibrinogen receptor antagonist, and [formula: see text], a weak fibrinogen receptor antagonist, have been characterized by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. 1H-1H distance constraints derived from two-dimensional NOE spectroscopy and torsional angle constraints obtained from 3JNH-H alpha coupling constants, combined with computer-assisted modeling using conformational searching algorithms and energy minimization have allowed several low energy conformations of the peptides to be determined. Low temperature studies in combination with molecular dynamics simulations suggest that each peptide does not exist in a single, well-defined conformation, but as an equilibrating mixture of conformers in fast exchange on the NMR timescale. The experimental results can be fit by considering pairs of low energy conformers. Despite this inherent flexibility, distinct conformational preferences were found which may be related to the biological activity of the peptides.

Characterization of the herpes simplex virus origin binding protein interaction with OriS.

The origin binding protein (OBP) of herpes simplex virus (HSV), which is essential for viral DNA replication, binds specifically to sequences within the viral replication origin(s) (for a review, see Challberg, M.D., and Kelly, T. J. (1989) Annu. Rev. Biochem. 58, 671-717). Using either a COOH-terminal OBP protein A fusion or the full-length protein, each expressed in Escherichia coli, we investigated the interaction of OBP with one HSV origin, OriS. Binding of OBP to a set of binding site variant sequences demonstrates that the 10-base pair sequence, 5' CGTTCGCACT 3', comprises the OBP-binding site. This sequence must be presented in the context of at least 15 total base pairs for high affinity binding, Ka = approximately 0.3 nM. Single base pair mutations in the central CGC sequence lower the affinity by several orders of magnitude, whereas a substitution at any of the other seven positions reduces the affinity by 10-fold or less. OBP binds with high affinity to duplex DNA containing mismatched base pairs. This property is exploited to analyze OBP binding to DNA heteroduplexes containing singly substituted mutant and wild-type DNA strands. For positions 2, 3, 5, 6, 7, 8, and 9, substitutions are tolerated on one or the other DNA strand, indicating that base-mediated interactions are limited to one base of each pair. For both Boxes I and II, these interactions are localized to one face of the DNA helix, forming a recognition surface in the major groove. In OriS, the 31 base pairs which separate Boxes I and II orient the two interaction surfaces to the same side of the DNA.