The development and application of a novel safety-catch linker for BOC-based assembly of libraries of cyclic peptides.

Cyclic peptides are appealing targets in the drug-discovery process. Unfortunately, there currently exist no robust solid-phase strategies that allow the synthesis of large arrays of discrete cyclic peptides. Existing strategies are complicated, when synthesizing large libraries, by the extensive workup that is required to extract the cyclic product from the deprotection/cleavage mixture. To overcome this, we have developed a new safety-catch linker. The safety-catch concept described here involves the use of a protected catechol derivative in which one of the hydroxyls is masked with a benzyl group during peptide synthesis, thus making the linker deactivated to aminolysis. This masked derivative of the linker allows BOC solid-phase peptide assembly of the linear precursor. Prior to cyclization, the linker is activated and the linear peptide deprotected using conditions commonly employed (TFMSA), resulting in deprotected peptide attached to the activated form of the linker. Scavengers and deprotection adducts are removed by simple washing and filtration. Upon neutralization of the N-terminal amine, cyclization with concomitant cleavage from the resin yields the cyclic peptide in DMF solution. Workup is simple solvent removal. To exemplify this strategy, several cyclic peptides were synthesized targeted toward the somatostatin and integrin receptors. From this initial study and to show the strength of this method, we were able to synthesize a cyclic-peptide library containing over 400 members. This linker technology provides a new solid-phase avenue to access large arrays of cyclic peptides.

The extracellular Ca++ -independent component of luteinzing hormone (LH) secretion is manifest under the steroidal conditions known to induce gonadotrophine-releasing hormone (GnRH) self-potentiation

Quartered pituitaries obtained from intact males or gonadectomized males and females (72h) ± estradiol (24h) and/or testosterone (72h) implants were perifused at 0.25 ml/minute with Ca++ -free medium at 37°C, and sequential effluent fractions collected every 10 minutes, in an attempt to determine if the steroidal conditions known to induce or prevent GnRH self-potentiation would activate or inhibit the extracellular Ca++ -independent component of luteinizing hormone (LH) secretion. Continuous infusions (4h) of 1nmol GnRH or 1µmol of phorbol 12-myristate 13-acetate (PMA) did not stimulate LH secretion from the pituitaries of castrated males, intact males, estradiol-treated intact males or ovariectomized females. In contrast, estradiol induced delayed (20-30 minutes), protein synthesis-dependent components of LH secretion in response to both GnRH and PMA from pituitaries of gonadectomized males and females. Implantation of testosterone capsules immediately following gonadectomy resulted in an inhibition of the estradiol- induced GnRH- and PMA-stimulated responses from pituitaries of castrated or ovariectomized animals. These results suggest that estradiol can induce extracellular Ca++ -independent components of LH secretion from pituitaries of gonadectomized animals; responses which depend on de novo protein synthesis and which could involve protein kinase C. Additionally, the effects of estradiol are prevented by testosterone, indicating that this component of LH secretion is only apparent under the steroidal conditions known to facilitate GnRH self-potentiation.

The extracellular Ca++ -independent component of luteinzing hormone (LH) secretion is manifest under the steroidal conditions known to induce gonadotrophine-releasing hormone (GnRH) self-potentiation

Quartered pituitaries obtained from intact males or gonadectomized males and females (72h) ± estradiol (24h) and/or testosterone (72h) implants were perifused at 0.25 ml/minute with Ca++ -free medium at 37°C, and sequential effluent fractions collected every 10 minutes, in an attempt to determine if the steroidal conditions known to induce or prevent GnRH self-potentiation would activate or inhibit the extracellular Ca++ -independent component of luteinizing hormone (LH) secretion. Continuous infusions (4h) of 1nmol GnRH or 1µmol of phorbol 12-myristate 13-acetate (PMA) did not stimulate LH secretion from the pituitaries of castrated males, intact males, estradiol-treated intact males or ovariectomized females. In contrast, estradiol induced delayed (20-30 minutes), protein synthesis-dependent components of LH secretion in response to both GnRH and PMA from pituitaries of gonadectomized males and females. Implantation of testosterone capsules immediately following gonadectomy resulted in an inhibition of the estradiol- induced GnRH- and PMA-stimulated responses from pituitaries of castrated or ovariectomized animals. These results suggest that estradiol can induce extracellular Ca++ -independent components of LH secretion from pituitaries of gonadectomized animals; responses which depend on de novo protein synthesis and which could involve protein kinase C. Additionally, the effects of estradiol are prevented by testosterone, indicating that this component of LH secretion is only apparent under the steroidal conditions known to facilitate GnRH self-potentiation.

Insights into mechanisms of antiarrhythmic drug action from experimental models of atrial fibrillation.

Atrial fibrillation (AF) remains a challenge to medical therapy. Over the past several years, a variety of experimental models of AF have been developed. These have provided insights into mechanisms underlying AF and antiarrhythmic drug action against the arrhythmia. A variety of drugs effective against clinical AF, including flecainide, propafenone, procainamide, and sotalol, have been found to terminate experimental AF. All of these agents appear to act by prolonging the wavelength for atrial reentry at rapid rates, thereby increasing the size and decreasing the number of functional circuits maintaining the arrhythmia. While the ability to terminate AF is determined by refractoriness prolongation at rapid rates, refractoriness prolongation at slow rates (e.g., sinus rhythm) can prevent AF induction by premature beats. Thus, drugs with strong reverse use-dependence (like sotalol) may be much more effective in preventing than in terminating AF. Spacial heterogeneity in refractoriness is an important contributor to AF occurrence in some models, particularly vagal AF, and is reduced by some (but not all) drugs that terminate AF. New insights are being gained into mechanisms of electrical remodeling, which promotes AF maintenance when rapid atrial rates are maintained, such as during AF. This electrical remodeling may be an interesting novel target for therapy of AF. Insights into AF mechanisms obtained in experimental models of AF should help in the development of new and improved therapeutic approaches.

Adenosine 3',5'-cyclic monophosphate and the self-priming effect of gonadotrophin-releasing hormone.

An in vitro perifusion system was used to ascertain the role of cAMP in the genesis of the self-priming effect of gonadotrophin-releasing hormone (GnRH) in rat pituitaries. Ten-minute pulses of 20 nmol/l GnRH administered 150 min apart resulted in the manifestation of the self-priming effect, an effect which was inhibited by 5 mumol/l cycloheximide. Forskolin (1 mumol/l) which does not stimulate luteinizing hormone (LH) secretion or affect the initial LH response to GnRH significantly potentiated the second response through protein synthesis-dependent mechanisms. Additionally, an initial 10-min pulse of forskolin alone was sufficient to prime the pituitary to a subsequent pulse of GnRH 150 min later. Interestingly, similar amounts of LH were secreted in response to forskolin + GnRH or GnRH administered 150 min after forskolin. Flufenamate, an inhibitor of GnRH-stimulated increases in cAMP production prevented the manifestation of the self-priming effect of GnRH. Forskolin which bypasses the inhibitory effects of flufenamate on cAMP production reversed the flufenamate-induced inhibition of the self-priming effect of GnRH through protein synthesis-dependent processes. These results suggest that cAMP does not mediate the LH response to an initial exposure of GnRH, but does play a pivotal role in the genesis of the self-priming effect of GnRH through the stimulation of de novo protein synthesis. Once the newly synthesized proteins are available, the nucleotide is not required for the manifestation of the phenomenon.

Calcium mobilization is a prerequisite for the expression of phorbol ester-stimulated luteinizing hormone secretion from pituitaries of male and acutely ovariectomized rats.

Ionomycin, which mobilizes Ca2+, and phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), were used to compare the effects/interactions of Ca2+ and PKC on luteinizing hormone (LH) secretion from pituitaries of intact male and acutely ovariectomized (72 h) rats. Quartered pituitaries from donor animals were perifused at 0.25 ml/min and sequential effluent fractions were collected every 10 min. Continuous administration (4 h) of 1 nmol of gonadotropin-releasing hormone (GnRH) resulted in an increase in LH secretion. Cycloheximide (5 mumol) dissociated the GnRH-stimulated LH responses into protein synthesis-independent and -dependent components. While ionomycin (10 mumol) stimulated LH secretion from pituitaries of both sexes by protein synthesis-independent mechanisms, PMA (1 mumol) and the inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (PDD), were ineffective secretagogues. However, PMA (but not PDD) interacted synergistically with ionomycin and GnRH to augment LH secretion by protein synthesis-dependent mechanisms. These results suggest a similarity in the effects/interactions of Ca2+ and PKC in gonadotropes from male and ovariectomized rats. If the effects of PMA can be attributed to PKC activation, then it also appears that Ca2+ mobilization is necessary for the manifestation of PKC as a mediator of LH secretion from these gonadotropes. While PKC activity can be divorced from the protein synthesis-independent component of LH release (this component appears to be mediated by Ca2+ mobilization), the enzyme might be involved in amplifying the response to Ca2+ mobilization through synergistic protein synthesis-dependent mechanisms.

Comparative mechanisms of antiarrhythmic drug action in experimental atrial fibrillation. Importance of use-dependent effects on refractoriness.

BACKGROUND: Antiarrhythmic drugs are considered to terminate atrial fibrillation by prolonging refractoriness, but direct experimental evaluation of this concept has been limited. The atria are activated rapidly during atrial fibrillation, and antiarrhythmic drugs are known to have important rate-dependent actions. The potential role of such properties in determining drug effects during atrial fibrillation has not been evaluated. METHODS AND RESULTS: We evaluated the effects of representative class Ia (procainamide), Ic (propafenone), and III (sotalol) antiarrhythmic drugs on sustained cholinergic atrial fibrillation and atrial electrophysiological properties in anesthetized, open-chest dogs. Loading and maintenance doses were used to produce stable plasma concentrations, and computer-based 112-electrode epicardial mapping was used to study atrial conduction and activation during atrial fibrillation. Clinically used doses of procainamide and propafenone terminated atrial fibrillation in 13 of 13 (100%) and 7 of 10 (70%) dogs, respectively, but a dose of sotalol (2 mg/kg IV) in the clinical range terminated atrial fibrillation in only 2 of 8 (25%) dogs (P = .0005 vs procainamide, P = .08 vs propafenone). Procainamide and propafenone prevented atrial fibrillation induction in 13 of 13 (100%) and 7 of 10 (70%) dogs, respectively, compared with none of 8 dogs for 2 mg/kg sotalol (P < .0001 vs procainamide, P = .004 vs propafenone). A larger dose of sotalol (cumulative dose, 8 mg/kg) was uniformly effective in terminating atrial fibrillation and preventing its induction. All drugs significantly increased atrial refractory period, with effects that were use dependent for propafenone but reverse use dependent for sotalol. Effective doses of all drugs significantly increased the wavelength for reentry at rapid atrial rates in the presence of vagal stimulation into the range observed under drug-free conditions in the absence of vagal input. The inefficacy of clinical doses of sotalol was explained by the reverse use dependence of its effects on refractoriness, which resulted in reduced effects on wavelength at rapid rates. The effects of propafenone on refractoriness were significantly increased at rapid rates, contributing to its ability to increase wavelength and terminate atrial fibrillation. Activation mapping showed that drugs terminated atrial fibrillation by reducing the number and increasing the size of reentry circuits, leading to termination by mechanisms related to block in the remaining circuit(s). CONCLUSIONS: We conclude that antiarrhythmic drugs terminate experimental atrial fibrillation by increasing the wavelength for reentry at rapid rates, leading to a reduction in the number of functional reentry circuits and, eventually, failure of reentrant excitation. Use-dependent effects on refractoriness can limit (in the case of the reverse use dependence of sotalol) or contribute (in the case of propafenone) to antiarrhythmic drug efficacy against atrial fibrillation by determining drug-induced changes in wavelength at rapid atrial rates.

Estradiol modulation of PMA--and ionomycin-stimulated LH secretion from pituitaries of castrated rats.

Quartered pituitaries from castrated (72 h) +/- estradiol (E2)-treated (24 h) rats were used in a perifusion system to investigate estradiol modulation of ionomycin and ionomycin + PMA stimulated LH secretion. Estradiol enhanced the LH responses to GnRH (1 nM) and ionomycin (10 microM), and was necessary for the manifestation of PMA-stimulated (1 microM) LH secretion. Cycloheximide (5 microM) inhibited the E2-enhanced responses to GnRH, ionomycin and PMA. The protein synthesis inhibitor also partially suppressed the GnRH response from pituitaries of castrates, but was totally ineffective against the ionomycin-induced LH secretion. Protein synthesis-dependent, synergistic interactions between PMA and ionomycin were evident from pituitaries of castrates (even though PMA alone was an ineffective secretagogue). Synergistic interactions were not apparent from pituitaries of castrated + E2-treated rats. These results indicate that: (i) estradiol enhances the responsiveness of male gonadotropes to ionomycin and PMA by protein synthesis-dependent mechanisms which appear to mask their synergistic interactions; and (ii) increases in cytoplasmic Ca2+ might be a prerequisite for an expression of the involvement of PKC as a mediator of LH secretion in the absence of high concentrations of estradiol.

Forskolin but not cholera toxin bypasses flufenamate-induced inhibition of cAMP production in anterior pituitaries.

The pharmacological activators of adenylyl cyclase (cholera toxin and forskolin) were utilized in the present study to determine whether they could bypass the inhibitory effects of flufenamate on cAMP production in rat hemipituitaries. During 2 hr incubations, 10 microM flufenamate inhibited gonadotropin-releasing hormone (GnRH)-stimulated (25 nM) cAMP production. Flufenamate did not affect GnRH-receptor interactions as evidenced by its inability to significantly affect either the binding affinity or the binding capacity for GnRH. Additionally, flufenamate inhibited the cholera toxin-stimulated cAMP production, but was ineffective against forskolin-induced activation of adenylyl cyclase. These results indicate that forskolin can be used to restore cAMP production in the presence of flufenamate. Since GnRH and cholera toxin stimulate cAMP production via the GnRH receptor and the Gs protein respectively, and forskolin exerts its stimulatory effects via the catalytic component, the data are consistent with the possibility that flufenamate exerts its inhibitory effect at the level of the Gs protein.

The use of flufenamate and forskolin to evaluate the role of cAMP in gonadotropin-releasing hormone-stimulated luteinizing hormone secretion from pituitaries of ovariectomized rats.

Flufenamate which inhibited gonadotropin-releasing hormone (GnRH)-stimulated cAMP production in pituitaries from ovariectomized (72 hr) rats, was used to determine whether ovariectomy induces a change in the role of cAMP as a mediator of luteinizing hormone (LH) secretion. Additionally, the study evaluated the practicability of utilizing forskolin to restore intracellular cAMP concentrations in the presence of flufenamate. Infusions of flufenamate to perifused pituitary tissue blocks did not affect the protein synthesis-independent component of GnRH-stimulated LH secretion, but completely inhibited the protein synthesis-dependent component. Dibutyryl cAMP (dbcAMP) and forskolin potentiated the GnRH-stimulated responses, and restored the LH secretion inhibited by flufenamate, even though these agents were ineffective secretagogues when administered singly. The LH responses affected by forskolin were dependent on protein synthesis, while dbcAMP affected both the protein synthesis-dependent and -independent components of GnRH-stimulated LH secretion. Since the effects of dbcAMP on the protein synthesis-independent component of LH secretion might be due to interactions with GnRH receptors, the results suggest that forskolin might be a better choice for restoring intracellular cAMP levels in the presence of flufenamate when assessing the role of cAMP in gonadotropes. The study also indicates that ovariectomy does not result in a change in the role of cAMP, which appears to be a pivotal, but indirect mediator of the protein synthesis-dependent component of GnRH-stimulated LH secretion.

Sitting up post angioplasty: a new sheath technology.

A new angioplasty sheath has been developed which permits a patient to sit up to a 60 degree Semi-Fowler position post angioplasty with the sheath in place. To determine the safety of this device, we studied eleven (11) patients undergoing Percutaneous Transluminal Coronary Angioplasty (PTCA) compared with 11 PTCA patients treated in a standard fashion. The patients were evaluated for the length of time the sheath remained in place (study 17 +/- 6 hours versus control 19 +/- 5 hours), the time required to achieve hemostasis upon sheath removal (66 +/- 52 minutes versus 81 +/- 89 minutes), and the need for analgesics (frequency of 1.9 times versus frequency of 6.4 times per patient). Complications in the study group were 0 episodes of severe bleeding at the insertion site versus 1 episode in the control group, slight oozing in 8 study patients versus 7 control patients, small hematoma in 2 study patients versus 1 control patient, and large hematoma formation in 1 study patient versus 0 control patients. In summary, the use of a soft, flexible sheath allows the patient to safely site up in the Semi-Fowler position post PTCA with significant improvement of discomfort.

Calmodulin and protein kinase C activation duplicates the biphasic secretion of luteinizing hormone.

Ionomycin, which activates the Ca2+-calmodulin system, and phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), were used to investigate potential roles of these systems as mediators of the biphasic secretion of luteinizing hormone. Quartered pituitaries from diestrous II female rats were perifused at 37 degrees C, and sequential effluent fractions collected every 10 min. Gonadotropin-releasing hormone administration resulted in a biphasic response: an initial, protein synthesis-independent secretion, followed 60 min later by a secondary, augmented, protein synthesis-dependent component. Ionomycin-stimulated gonadotropin secretion was immediate and partially independent of protein synthesis, whereas the PMA-induced secretion was delayed (approximately 70 min), and was completely dependent on protein synthesis. Simultaneous infusions of ionomycin and PMA resulted in an initial, protein synthesis-independent response followed by the secondary, augmented, protein synthesis-dependent component, which exhibited synergistic interactions between calmodulin and PKC. These results suggest that calmodulin mediates the initial, protein synthesis-independent secretion, PKC mediates part of the secondary, augmented response, while calmodulin and PKC synergize to mediate the remaining component of the secondary response.

Dependency of phorbol ester-induced gonadotropin secretion on estradiol.

Phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC) was used to investigate the estradiol dependency of PKC-stimulated luteinizing hormone (LH) secretion from perifused anterior pituitaries. Infusions of PMA stimulated LH secretion from diestrous II, ovariectomized + estradiol-treated, and orchidectomized + estradiol-treated quartered pituitaries, by protein synthesis-dependent mechanisms. In contrast, pituitaries from intact, orchidectomized males, or ovariectomized females were unresponsive to PMA. Interestingly, dispersed male pituitary cells differed from male pituitary tissue blocks, in that the dispersed cells responded to PMA with increased LH secretion. These results indicate that PKC's ability to directly stimulate LH secretion is dependent on de novo protein synthesis and estradiol. Moreover, the effects of estradiol on PKC-stimulated secretion form at least one basis for the estradiol-induced increased responsiveness of gonadotrophs to GnRH. Additionally, it appears that dispersed pituitary cells may not respond to activators of PKC in a physiological manner.

The phorbol ester-induced extracellular Ca2+-independent release of LH is dependent on estradiol and de novo protein synthesis.

Phorbol 12-myristate 13-acetate (PMA) was used to determine whether the PMA-induced extracellular Ca2+-independent release of LH was dependent on sex, estradiol and de novo protein synthesis. Infusions of gonadotropin-releasing hormone (GnRH) or PMA in a perifusion system stimulated a partial secretion of LH from diestrous II and ovariectomized + estradiol-treated female pituitaries (responses inhibited by cycloheximide). In contrast, PMA was ineffective in stimulating PRL secretion from these pituitaries, as well as LH secretion from male or ovariectomized female pituitaries. These results indicate that the PMA-stimulated extracellular Ca2+-independent secretion of LH is a specific process which is dependent on sex, estradiol and de novo protein synthesis, and mimics the characteristics of the GnRH-stimulated responses.

Phencyclidine suppresses hippocampal long-term potentiation through stereospecific activation of phencyclidine receptors.

The effects of phencyclidine and the dioxolane enantiomers, dexoxadrol and levoxadrol, on long-term potentiation in the hippocampus were compared. Field potentials were evoked by stimulation of Schaffer collaterals and recorded from the CA1 region. Long-term potentiation was induced by stimulation with a single train of 25 pulses at 50 Hz. The drugs were delivered by pressure, 1 min before tetanization. Phencyclidine and its receptors ligand, dexoxadrol, abolished the induction of long-term potentiation. Levoxadrol which has very low affinity for the phencyclidine receptor was devoid of this action although it reduced the magnitude of long-term potentiation. These results indicate that phencyclidine blocks long-term potentiation by stereospecific activation of phencyclidine receptors.

Sex differences in the extracellular Ca2+-independent release of LH and FSH.

The present study was undertaken to investigate the effects of sex and estrous cycle on the manifestation of the extracellular Ca2+-independent component of gonadotropin secretion. Quartered pituitaries from male, ovariectomized (OVX) females +/- estradiol (E2) implants, and mature females at each stage of the estrous cycle were perifused with Ca2+-free medium. Gonadotropin releasing hormone (GnRH)-stimulated luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion from male and OVX pituitaries was inhibited in Ca2+-free medium. In contrast, only a partial inhibition was obtained from OVX + E2 or regularly cycling female pituitaries. This extracellular Ca2+-independent component of gonadotropin secretion was lowest at estrus and increased progressively during the estrous cycle. Estradiol replacement in OVX animals resulted in a response similar to that obtained on proestrus. These results indicate that the extracellular Ca2+-independent component of LH and FSH release is only manifest from intact female and not male pituitaries, and is dependent on estradiol.

Cyclic AMP indirectly mediates the extracellular Ca2+-independent release of LH.

This study was undertaken to determine whether cyclic AMP mediates the extracellular Ca2+-independent component of luteinizing hormone (LH) release. Gonadotropin releasing hormone (GnRH) increased cAMP production in female pituitaries incubated in Ca2+-free medium containing 3-isobutyl-1-methylxanthine (IBMX), but was ineffective in male pituitaries. The increases in female pituitaries were inhibited by flufenamate (flu). GnRH-stimulated LH secretion from male pituitaries was completely inhibited in Ca2+-free medium, whereas only a partial inhibition was obtained from female pituitaries, a response prevented by cycloheximide. Infusions of flu completely inhibited the extracellular Ca2+-independent release of LH, while dibutyryl cAMP (dbcAMP) partially restored this component of LH secretion. The dbcAMP-restored response was dependent upon protein synthesis. These results suggest that (i) the extracellular Ca2+-independent component of LH release is indirectly mediated by cAMP through the stimulation of de novo protein synthesis, and (ii) extracellular Ca2+ is required for the activation of adenylate cyclase in male but not in female gonadotrophs.

Disinhibitory effect of phencyclidine in the hippocampus in vitro: PCP receptors implicated.

The effects of phencyclidine (PCP) and two dioxolane stereoisomers, dexoxadrol and levoxadrol, on hippocampal inhibition were compared. Field potentials were recorded in the CA1 pyramidal cell layer in the rat hippocampal slices in vitro. Recurrent inhibition of the population spikes evoked orthodromically by stimulation of the Schaffer collaterals was induced by antidromic conditioning stimulation at appropriate time intervals before the orthodromic stimulation. The drugs were applied by micropressure ejection in concentrations which did not affect the unconditioned population spike. After PCP or dexoxadrol administration, the orthodromically evoked population spike was much less reduced by the antidromic conditioning stimulation than before, suggesting that the recurrent inhibition was diminished. Levoxadrol had only negligible effect. Since dexoxadrol has many PCP-like pharmacological properties but levoxadrol does not, we concluded that PCP attenuates hippocampal recurrent inhibition by activating the PCP receptors. It is suggested that this action results in depression of excitatory synaptic transmission from axon collaterals to the inhibitory interneuron with possible involvement of the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptor.