Selectivity of action of 8-alkylamino analogues of N6-cyclopentyladenosine in vivo: haemodynamic versus anti-lipolytic responses in rats

1. A1 adenosine receptor agonists with reduced intrinsic activity may be therapeutically useful as result of an increased selectivity of action. In this study the tissue selectivity of three 8-alkylamino substituted analogues of N6-cyclopentyladenosine (CPA) was investigated for haemodynamic and anti-lipolytic effects using an integrated pharmacokinetic-pharmacodynamic approach. 2. Chronically instrumented male Wistar rats received intravenous infusions of 4.0 mg kg(-1) 8-methylaminoCPA (8MCPA), 12.0 mg kg(-1) 8-ethylaminoCPA (8ECPA), 20.0 mg kg(-1) 8-butylaminoCPA (8BCPA) or vehicle during 15 min. During experimentation, serial arterial blood samples were drawn for the determination of agonist concentrations and plasma non-esterified fatty acid (NEFA) levels. Blood pressure and heart rate were monitored continuously. In addition to the CPA analogues, each rat received a rapid bolus infusion of CPA to determine the maximal effects of the full agonist. 3. The concentration-time profiles of the CPA analogues could be described by a bi-exponential function. Values for clearance, volume of distribution at steady state and elimination half-life were 44+/-5, 48+/-6 and 39+/-2 ml min(-1) kg(-1), 0.97+/-0.09, 0.84+/-0.10 and 1.05+/-0.07 1 kg(-1) and 25+/-2, 28+/-2 and 40+/-2 min for 8MCPA, 8ECPA and 8BCPA, respectively (mean+/-s.e.mean, n=6-8). 4. Different models were used to derive the concentration-effect relationships for heart rate and NEFA, yielding estimates of potency (EC50) and intrinsic activity (Emax) for both effects of the compounds in vivo. On heart rate the compounds acted as partial agonists, with Emax values of -173+/-14, -131+/-11 and -71+/-6 beats min(-1) for 8MCPA, 8ECPA and 8BCPA, respectively. These Emax values were significantly lower than the maximal effect of CPA (-208+/-8 beats min(-1)). With regard to the anti-lipolytic effect all three compounds were full agonists and lowered NEFA levels to the same extent as CPA (69%). The estimated Emax values were 63+/-5, 63+/-4 and 68+/-2%, respectively. 5. Furthermore, the compounds were more potent in causing anti-lipolytic than cardiovascular effects. The EC50 values for the NEFA and heart rate lowering effects were 37+/-15, 68+/-22 and 659+/-108 ng ml(-1) and 164+/-22, 341+/-76 and 975+/-190 ng ml(-1) for 8MCPA, 8ECPA and 8BCPA, respectively (mean+/-s.e.mean, n=6-8). 6. This study demonstrates that partial agonists for the A1 adenosine receptor have increased selectivity of action in vivo. The 8-alkylamino analogues of CPA may be useful anti-lipolytics with less pronounced haemodynamic side effects.     

Differential role of vasoactive prostanoids in porcine and human isolated pulmonary arteries in response to endothelium-dependent relaxants

The pig is increasingly being used in medical research, both as a model of the human cardiovascular system, and as a possible source of organs for xenotransplantation. However, little is known about the comparative functions of the vascular endothelium between porcine and human arteries. We have therefore compared the effects of two endothelium-dependent vasorelaxants, acetylcholine (ACh) and the Ca2+-ATPase inhibitor, cyclopiazonic acid (CPA) on the porcine and human isolated pulmonary artery using isometric tension recording. ACh and CPA produced endothelium-dependent relaxations of both the human and porcine pulmonary arteries. In the porcine pulmonary artery, the cyclo-oxygenase inhibitor, flurbiprofen had no effect on relaxations to ACh (Emax: control 67.8+/-8.8% versus 72.4+/-9.5% (n=11)) or CPA (Emax: control 79.6+/-5.0% versus 94.0+/-10.6% (n=7)). The nitric oxide synthase inhibitor, L-NAME converted relaxations to both ACh and CPA into contractile responses (maximum response: ACh 30.0+/-11.1% (n = 10); CPA 80.4+/-26.2% (n = 8) of U46619-induced tone). These contractile responses in the presence of L-NAME were abolished by flurbiprofen. In the human pulmonary artery, L-NAME and flurbiprofen partly attenuated relaxations to ACh (Emax: control: 45.1+/-12.1%; flurbiprofen: 33.4+/-13.5%; L-NAME: 10.1+/-7.2%) and CPA (Emax: control: 78.1+/-5.5%; flurbiprofen: 69.6+/-7.2%; L-NAME 37.9+/-10.7% of U46619-induced tone). These responses were abolished by the combination of both inhibitors. We have demonstrated that while the release of nitric oxide is important in responses to endothelium-dependent vasorelaxants in both human and porcine pulmonary arteries, in the human arteries, there is an important role for vasorelaxant prostanoids whilst in the porcine arteries, vasoconstrictor prostanoids are released.     

N6,C8-distributed adenosine derivatives as partial agonists for adenosine A1 receptors

The synthesis and biological evaluation of N6, C8-disubstituted derivatives of adenosine as potential partial agonists for adenosine receptors is described. Via three routes, two series of compounds were prepared, viz., N6-cyclopentyladenosine derivatives 3a-e and C8-(cyclopentylamino)adenosine analogs 3e and 9a-d, respectively. The X-ray structure determination of one of these compounds, N6-ethyl-8(cyclopentylamino)adenosine (9b), was carried out (orthorhombic, space group P2(1)2(1)2(1) (No. 19) with a = 11.039(3), b = 8.708(2), and c = 24.815(12) angstrom, Z=4,R1=0.0974,R2(W) = 0.2455). Due to intramolecular hydrogen bonding, the ribose moiety of this compound is in an anti conformation. The compounds were tested in vitro in radioligand binding studies, yielding their affinities for A1 and A2a adenosine receptors. All compounds appeared A1 selective, with affinities in the high nanomolar, low micromolar range. On A1 receptors the so-called GTP shift was also determined, i.e., the ratio between the affinities measured in the presence and absence of 1 mM GTP. All GTP shifts (values between 1.1 and 3.8) were lower than the GTP shift for CPA (6.0). This GTP shift appeared indicative for partial agonism in vivo, since the N6-cyclopentyladenosine derivatives showed lower intrinsic activities than the prototypic full agonist N6-cyclopentyladenosine on the decrease in heart rate in conscious, normotensive rats.     

Modelling of the pharmacodynamic interaction of an A1 adenosine receptor agonist and antagonist in vivo: N6-cyclopentyladenosine and 8-cyclopentyltheophylline

1. The purpose of this investigation was to develop a pharmacokinetic-pharmacodynamic model for the interaction between an adenosine A1 receptor agonist and antagonist in vivo. The adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA) and the antagonist, 8-cyclopentyltheophylline (CPT) were used as model drugs. The CPA-induced reduction in mean arterial pressure and heart rate were used as measurements of effect. 2. Four groups of eight rats each received 200 micrograms kg-1 of CPA i.v. in 5 min during a steady-state infusion of CPT at a rate of 0, 57, 114 or 228 micrograms kg-1 h-1. The haemodynamic parameters were continuously measured and frequent blood samples were taken to determine the pharmacokinetics of the drugs. 3. CPT had no influence on the pharmacokinetics of CPA and the baseline values of the haemodynamic variables. Furthermore, no clear antagonism by CPT was observed of the CPA-induced reduction in mean arterial pressure. However, CPT antagonized the effect on heart rate, and with increasing CPT concentrations, a parallel shift of the CPA concentration-effect relationship to the right was observed. 4. An agonist-antagonist interaction model was used to characterize the interaction quantitatively. On the basis of this model, the pharmacodynamic parameters of both CPA and CPT could be estimated. For CPA the values were (mean +/- s.e.): Emax = 198 +/- 11 b.p.m., EC50 = 2.1 +/- 0.7 ng ml-1, Hill factor = 2.3 +/- 0.6 and for CPT: EC50 = 3.7 +/- 0.3 ng ml-1 and Hill factor = 3.1 +/- 0.1. 5. It is concluded that the competitive agonist-antagonist interaction model may be of value to characterize quantitatively the pharmacodynamic interactions between adenosine A1 receptor ligands in vivo.     

Synthesis and structure-activity relationships of 3,7-dimethyl-1-propargylxanthine derivatives, A2A-selective adenosine receptor antagonists

A series of 8-substituted derivatives of 3,7-dimethyl-1-propargylxanthine (DMPX) was synthesized and investigated as A2A adenosine receptor antagonists. Different synthetic strategies for the preparation of DMPX derivatives and analogues were explored. A recently developed synthetic procedure starting from 3-propargyl-5,6-diaminouracil proved to be the method of choice for the preparation of this type of xanthine derivatives. The novel compounds were investigated in radioligand binding studies at the high-affinity adenosine receptor subtypes A1 and A2A and compared with standard A2A adenosine receptor antagonists. Structure-activity relationships were analyzed in detail. 8-Styryl-substituted DMPX derivatives were identified that exhibit high affinity and selectivity for A2A adenosine receptors, including 8-(m-chlorostyryl)-DMPX (CS-DMPX, Ki A2A = 13 nM, 100-fold selective), 8-(m-bromostyryl)-DMPX (BS-DMPX, Ki A2A = 8 nM, 146-fold selective), and 8-(3,4-dimethoxystyryl)-DMPX (Ki A2A = 15 nM, 167-fold selective). These and other novel compounds are superior to the standard A2A adenosine receptor antagonists KF17837 (4) and CSC (5) with respect to A2A affinity and/or selectivity.     

Effect of adenosine receptor agonists on spontaneous and K(+)-evoked acetylcholine release from the in vivo rat cerebral cortex

Repeated applications of elevated K+ (100 mM) in artificial cerebrospinal fluid (CSF) were used to evoke an efflux of acetylcholine (ACh) from the in vivo rat cerebral cortex using a cortical cup technique. Elevated K+ reproducibly increased the levels of ACh in cup superfusates by a factor of 3-5-fold above basal levels (27.2 +/- 9.7 nM). The adenosine A1 receptor agonist N6-cyclopentyl adenosine (CPA), at a concentration of 10(-8) M, depressed basal, but not K(+)-evoked ACh efflux. 10(-6) M CPA increased basal, but did not alter K(+)-evoked, ACh efflux. The A2 selective agonist CGS 21680 did not alter either basal, or K(+)-evoked, ACh efflux. The inhibitory effects of 10(-8) M CPA on ACh efflux would be consistent with the presence of adenosine A1 receptors on cholinergic nerve terminals in the cerebral cortex. At a higher concentration (10(-6) M) CPA elevated basal release, possibly by activating low affinity A2 receptors. The failure of CGS 21680 (10(-8) M) to alter basal ACh release suggests an absence of high affinity A2 receptors in these terminals. Whereas elevated K+ in cup superfusates consistently enhanced ACh efflux from the cerebral cortex, this increase was not affected by either CPA or CGS 21680. High K(+)-evoked release of cerebral cortical ACh may be an inappropriate model for the study of adenosine's actions on neurotransmitter release.     

Structure-based design of new constrained cyclic agonists of the cholecystokinin CCK-B receptor

New constrained cyclic pseudopeptide cholecystokinin-B (CCK-B) agonists have been designed on the basis of conformational characteristics of the potent and selective CCK-B agonist Boc-Trp-(NMe)Nle-Asp-Phe-NH2 (Ki = 0.8 nM, selectivity ratio CCK-A/CCK-B > 6000) (Goudreau et al. Biopolymers, 1994, 34, 155-169). These compounds are among the first successful examples of macrocyclic constrained CCK4 analogs endowed with agonist properties and as such may be of value for the development of nonpeptide CCK-B agonists. The affinities and selectivities of these compounds for CCK-B and CCK-A receptors have been determined in vitro by measuring the displacement of [3H]pCCK8 binding to guinea pig cortex and pancreas membranes, respectively. The most potent compound, 8b, N-(cycloamido)-alpha-Me(R)Trp-[(2S)-2-amino-9- ((cycloamido)carbonyl)nonanoyl]-Asp-Phe-NH2, has a Ki value of 15 +/- 1 nM for guinea pig cortex membranes with a good CCK-B selectivity ratio (CCK-A/CCK-B = 147). Furthermore, 8b behaved as a potent and full agonist in a functional assay which measures the stimulation of inositol phosphate accumulation in CHO cells transfected with the rat CCK-B receptor (EC50 = 7 nM). The in vivo affinity of 8b for mouse brain CCK-B receptors was determined following intracerebroventricular injection (ID50 approximately 29 nmol/kg). 8b was also shown to cross the blood-brain barrier (0.16%), after intravenous administration in mice. 8b also increased gastric acid secretion measured in anesthetized rats after intravenous injection. Therefore, 8b appears to be an interesting pharmacological tool and is currently under investigation as a lead for further development of nonpeptide CCK-B agonists.     

A1 adenosine receptor modulation of electrically-evoked contractions in the bisected vas deferens and cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon both electrically-evoked and phenylephrine-induced contractile responses were investigated in the bisected vas deferens and the cauda epididymis of the guinea-pig. Electrical field-stimulation (10 s trains of pulses at 9 Hz, 0.1 ms duration, supramaximal voltage) elicited biphasic and monophasic contractile responses from preparations of bisected vas deferens and cauda epididymis, respectively; these responses were abolished by tetrodotoxin (300 nM). 2. In the prostatic half of the vas deferens the A1 selective adenosine receptor agonists, N6-cyclopentyladenosine (CPA) and (2S)-N6-[2-endo-norbornyl]adenosine ((S)-ENBA) and the non-selective A1/A2 adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) inhibited electrically-evoked contractions (pIC50+/-s.e.mean values 6.15+/-0.24, 5.99+/-0.26 and 5.51+/-0.24, respectively). The responses to CPA were blocked by the A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine, DPCPX (100 nM). 3. In the epididymal half of the vas deferens NECA potentiated (at < or = 100 nM) and inhibited (at > or = 1 microM) electrically-evoked contractions. In the presence of the non-selective alpha-adrenoceptor antagonist phentolamine (3 microM), the alpha1-adrenoceptor antagonist, prazosin (100 nM), or at a reduced train length (3 s) NECA inhibited electrically-evoked contractions (pIC50 values 6.05+/-0.25, 5.97+/-0.29 and 5.71 +/-0.27, respectively). CPA (at 10 microM) also inhibited electrically-evoked contractions in this half of the vas deferens. In the presence of prazosin (100 nM), CPA also inhibited electrically-evoked contractions (pIC50 6.14+/-0.67); this effect was antagonized by DPCPX (30 nM, apparent pK(B) 8.26+/-0.88). In the presence of the P2 purinoceptor antagonist, suramin (300 microM), CPA (up to 1 microM) potentiated electrically-evoked contractions. 4. NECA, CPA and APNEA potentiated electrically-evoked contractions in preparations of cauda epididymis (pEC50 values 7.49+/-0.62, 7.65+/-0.74 and 5.84+/-0.86, respectively), the response to CPA was competitively antagonized by DPCPX (100 nM) with an apparent pK(B) value of 7.64+/-0.64. 5. The alpha1-adrenoceptor agonist phenylephrine elicited concentration-dependent contractile responses from preparations of bisected vas deferens and cauda epididymis. NECA (1 microM) potentiated responses to phenylephrine (< or = 1 microM) in the epididymal, but not in the prostatic half of the vas deferens. In preparations of epididymis NECA (1 microM) shifted phenylephrine concentration response curves to the left (4.6 fold). In the presence of a fixed concentration of phenylephrine (1 microM), NECA elicited concentration-dependent contractions of preparations of the epididymal half of the vas deferens and of the epididymis (pEC50 values 7.57+/-0.54 and 8.08+/-0.18, respectively). NECA did not potentiate responses to ATP in either the epididymal half of the vas deferens or the epididymis. 6. These studies are consistent with the action of stable adenosine analogues at prejunctional A1 and postjunctional A1-like adenosine receptors. The prejunctional A1 adenosine receptors only inhibit the electrically-evoked contractions of purinergic origin (an effect predominant in the prostatic half of the vas deferens). At the epididymis, where electrically-evoked contractions are entirely adrenergic, the predominant adenosine receptor agonist effect is a potentiation of alpha1-adrenoceptor-, but not of ATP-induced contractility.     

American Pediatric Society John Howland Award 1998: presentation and acceptance

In the dog saphenous vein (DSV), phenylephrine (PE) responses through alpha-1 adrenoceptors receptors are antagonized by both alpha-1 and alpha-2 receptor antagonists. Furthermore, pretreatment with chloroethylclonidine (CEC) eliminates prazosin binding but reduces rauwolscine binding by half (). In new functional experiments, the effects of preincubation with phenoxybenzamine (PBZ), an irreversible alpha adrenoceptor antagonist, on responses to PE and two selective alpha-2 adrenoceptor agonists were evaluated. Also, the ability of prazosin or rauwolscine to prevent irreversible losses of responses to these agonists when coincubated with PBZ was determined. Preincubation in PBZ (10-300 nM) concentration dependently reduced PE Emax and the calculated fraction of residual receptors (q). Preincubation in PBZ (10-300 nM) increased KB values for prazosin (30 and 100 nM) but did not alter the KB value for rauwolscine (50 nM) acting at the residual receptors from control values. Coincubation of PBZ with prazosin partially prevented these PBZ actions (Emax partly restored) on responses to PE, but coincubation of rauwolscine (/=300 nM caused >50% reduction in Emax values of responses but did not alter the EC50 values for either agonist. Coincubation of rauwolscine with PBZ protected responses to alpha-2 agonists against PBZ (1 microM) effects. This study shows that PE initiates contractions at atypical alpha-1 adrenoceptors represented by all sites of PE action. Rauwolscine antagonizes PE actions but does not protect against PBZ inactivation. Typical alpha-2 adrenoceptors are distinguished from the unusual alpha-1 adrenoceptors by their lesser sensitivity to PBZ and their protection by rauwolscine from PBZ.     

Characterization of novel ligands for wild-type and natural mutant diazepam-insensitive benzodiazepine receptors

A series of benzodiazepine receptor ligands with different chemical structures were evaluated for their affinities at diazepam-sensitive and diazepam-insensitive binding sites for [3H]Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-[1,5a][1,4] benzodiazepine-3-carboxylate) in cerebellar GABAA receptors. Rats of Wistar strain and of alcohol-sensitive (ANT) and alcohol-insensitive (AT) lines were used. The ANT rats possess a single point mutation in their GABAA receptor alpha 6 subunit, which makes their diazepam-insensitive sites sensitive to benzodiazepine agonists, unlike those of AT and Wistar rats. All compounds evaluated displayed high-affinity binding to diazepam-sensitive sites (Ki < 50 nM). In contrast, a wider range of affinities were observed at diazepam-insensitive sites which depended upon the basic structure and substitutions. The 7- and 8-halogen substituted imidazobenzodiazepines and 12-halogen substituted diimidazoquinazolines displayed the highest affinities (Ki < 15 nM), while intermediate to low affinities (100 < Ki < 4000 nM) were displayed by imidazoquinazolines, thienopyrimidines, one oxoimidazoquinoxaline, and some cyclopyrrolones. The imidazoquinoxalines evaluated displayed the lowest affinity (Ki > 10000 nM). The oxoimidazoquinoxaline, 6-chloro-3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-4,5-dihydro-5-isop ropyl-4-oxo-imidazo[1,5-a]quinoxaline (NNC 14-0578) and suriclone represent the first benzodiazepine receptor full agonists to bind with relatively high affinity (Ki approximately 100 nM) to diazepam-insensitive sites. The 5 position substituted methoxybenzyl, dimethylallyl, and 4-fluorobenzyl oxoimidazoquinoxaline analogs demonstrated a 58-336-fold higher affinity for ANT than AT diazepam-insensitive sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of probucol on low density lipoprotein oxidation and femoral atherosclerosis

Our knowledge of the traits possessed by extraintestinal isolates of Escherichia coli, necessary for growth and survival in urine, is limited. To identify such determinants, transposon (TnphoA'1,4) mutant libraries of a clinical isolate (CP9) were generated and screened for derivatives exhibiting decreased growth in urine in vitro, and for mutants with active lacZ fusions that were induced in urine relative to laboratory medium. Using this approach we identified two genes, guaA (CPA24) and argC (CPI-1), which were previously unrecognized as being important for growth in human urine. Unexpectedly, not only does CPA24 (guaA) not grow in human urine in vitro, but it is sensitive to its effects, undergoing a 2-3 log loss of viability over 6 h. By contrast, CPA24 neither grows nor is killed in M9 minimal medium and artificial urine. Therefore, we postulate that lack of guanine or its derivatives in urine, and the inability of CPA24 to synthesize these compounds de novo, prevents CPA24 from synthesizing other guanine (or derivatives)-dependent products that are critical for growth and survival in urine. Although it seems logical that decreased growth in urine in vitro should correlate with diminished urovirulence, this concept was tested by challenging mice with CPA24 in vivo in a mouse model of urinary tract infection (UTI). Indeed, CPA24 was found to be significantly less virulent compared with its wild-type parent CP9. CPI-1(argC) was identified because of the significant induction of its argC::lacZ fusion in urine. Subsequent testing in urine demonstrated that its growth was significantly diminished in all urine samples tested (four females, three males). Polyamine synthesis is dependent upon, in part, the arginine biosynthetic pathway. Therefore, we tested whether the induction of argC in urine and/or the decreased growth of CPI-1 was a result of low levels of polyamines or arginine in urine. The results suggest that low levels of arginine, but not polyamines, in human urine are responsible. When tested in vivo in the mouse model of UTI, CPI-1 was also found to be significantly less virulent than CP9. In summary, we have established that guaA and argC are the first genes, which we are aware of, that have been shown to contribute to the growth of E. coli in urine in vitro and both have diminished urovirulence in vivo. These results support the concept that urine can be used in vitro as a screening tool to identify urovirulence traits.     

The effect of artemisinine, its derivatives and mefloquine against chloroquine-resistant strains of Plasmodium falciparum in vitro

A 48 h in vitro test of the efficacy of artemisinine, dihydroartemisinine, artemether, mefloquine and chloroquine was carried out against 3 chloroquine-resistant strains of Plasmodium falciparum, strains K1 and T996 from Thailand and LS21 from India. A sigmoid Emax model was fitted to all in vitro inhibition data for each combination of drug and strain. Strains K1 and LS21 were strongly resistant to chloroquine, whereas T996 was partially resistant. Artemisinine, dihydroartemisinine and artemether were active against all strains, with complete growth inhibition at 10(-7) M. Artemether and dihydroartemisinine were both more potent than artemisinine, with 50% effective (EC50) values of 0.57-1.6 nM and 0.36-3.1 nM respectively; the EC50 of artemisinine was 1.5-6.1 nM for the 3 strains. The EC50 values for mefloquine were 46-185 nM. At higher concentrations, strains K1 and LS21 were fully inhibited, while with strain T996 mefloquine did not fully inhibit even at the highest concentration, 1.28 x 10(-6) M. It is concluded that artemisinine and its derivatives were highly effective against the 3 chloroquine-resistant strains, one of which showed borderline resistance to mefloquine.     

Pharmacological characterization of the receptors involved in the beta-adrenoceptor-mediated stimulation of the L-type Ca2+ current in frog ventricular myocytes

1. The whole-cell patch-clamp was used for studying the effects of various beta1- and beta2-adrenoceptor agonists and antagonists on the L-type Ca current (Ica) in frog ventricular myocytes. 2. Dose-response curves for the effects of isoprenaline (non selective beta-agonist), salbutamol (beta2-agonist), dobutamine (beta1-agonist) on ICa were obtained in the absence and presence of various concentrations of ICI 118551 (beta2-antagonist), metoprolol (beta1-antagonist) and xamoterol (partial beta1-agonist) to derive EC50 (i.e. the concentration of beta-agonist at which the response was 50% of the maximum) and Emax (the maximal response) values by use of a Michaelis equation. Schild regression analysis was performed to examine whether the antagonists were competitive and to determine the equilibrium dissociation constant (K(B)) for the antagonist-receptor complex. 3. Isoprenaline increased ICa with an EC50 of 20.0 nM and an Emax of 597%. ICI 118551 and metoprolol competitively antagonized the effect of isoprenaline with a K(B) of 3.80 nM and 207 nM, respectively. 4. Salbutamol increased ICa with an EC50 of 290 nM and an Emax of 512%. ICI 118551 and metoprolol competitively antagonized the effect of salbutamol with a K(B) of 1.77 nM and 456 nM, respectively. 5. Dobutamine increased ICa with an EC50 of 2.40 microM and an Emax of 265%. ICI 118551 and metoprolol competitively antagonized the effect of dobutamine with a K(B) of 2.84 nM and 609 nM, respectively. 6. Xamoterol had no stimulating effect on ICa. However, xamoterol competitively antagonized the stimulating effects of isoprenaline, salbutamol and dobutamine on ICa with a K(B) of 58-64 nM. 7. We conclude that a single population of receptors is involved in the beta-adrenoceptor-mediated regulation of ICa in frog ventricular myocytes. The pharmacological pattern of the response of ICa to the different beta-adrenoceptor agonists and antagonists tested suggests that these receptors are of the beta2-subtype.     

Agonist activity of antimigraine drugs at recombinant human 5-HT1A receptors: potential implications for prophylactic and acute therapy

The actions of several serotonergic ligands in use or under development for the treatment of migraine headaches were examined at recombinant human 5-HT1A receptors stably expressed in Chinese Hamster Ovary cells. Affinities (K(i)s) at this site were determined in competition binding experiments with [3H]-8-OH-DPAT ([3H](+/-)8-hydroxy-N,N-dipropylaminotetralin), whilst agonist efficacy was measured by stimulation of [35S]-GTP gamma S (guanylyl-5'-[gamma[35S]thio]-triphosphate) binding. Of the prophylactic antimigraine drugs tested, methysergide and lisuride behaved as efficacious agonists (Emax > or = 90% relative to 5-HT) whereas pitozifen and (-)propranolol acted as a partial agonist (60%) and an antagonist, respectively. This suggests that there is no correlation between agonism at 5-HT1A receptors and prophylactic antimigraine action. In contrast, serotonin, dihydroergotamine, sumatriptan, naratriptan and alniditan, which are effective in acute interruption of migraine attacks, each displayed high efficacy (Emax = 100, 100, 92.6, 79.3, 79.1% respectively) and marked affinity (Ki = 18.7, 0.6, 127, 26.4 and 3.0 nM respectively) at 5-HT1A receptors. EC50 values for agonist stimulation of [35S]-GTP gamma S binding correlated with respective Ki values at 5-HT1A receptors (r = 0.93) and the stimulation of [35S]-GTP gamma S binding by these compounds was antagonised by the selective 5-HT1A antagonist WAY 100,635 (N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl) cyclo-hexanecarboxamide; 100 nM). These data suggest that agonism at 5-HT1A receptors may be involved in some actions of drugs used in acute antimigraine therapy. In comparison with the above compounds, novel ligands targeted at 5-HT1B/1D receptors, such as GR125,743 (N-[4-methoxy-3-(4-methyl-piperazin-1-yl)phenyl] -3-methyl-4-(4-pyridyl)benzamide) and GR 127,935 (N-[4-methoxy-3-(4-methylpiperazin-1-yl)-phenyl]-2'-methyl-4'-(5-m ethyl-1, 2,4-oxadiazol-3-yl)-biphenyl-4-carboxamide), only weakly activated [35S]-GTP gamma S binding (32.4 and 32.1% efficacy) and displayed moderate affinity at 5-HT1A receptors (Kis 53.1 and 49.8 nM) suggesting that they constitute useful tools to differentiate 5-HT1A and 5-HT1B/1D receptor-mediated actions. In conclusion, the present data indicates that several antimigraine agents exhibit marked 5-HT1A receptor activity and that although this is unlikely to be important for prophylactic action it may be relevant to the ancilliary properties of drugs used for acute migraine treatment.     

Current progress in the development of a totally implantable Gyro centrifugal artificial heart

We studied the contractile responses to endothelin-1 (ET-1) of aortic strips from female transgenic rats, TGR(mRen2)27, heterozygous for the Ren-2 mouse gene, during the phases of developing (blood pressure in rats aged 5 weeks; 156 +/- 8 mmHg), steady (blood pressure in rats aged 11 weeks: 206 +/- 27 mmHg), and reversed (blood pressure in rats aged 35 weeks: 151 +/- 17 mmHg) hypertension. These responses were compared with those of aortae from sex- and age-matched, genetically homogeneous, normotensive Sprague-Dawley (SD) rats. Aortic strips from both transgenic and SD rats were deprived of endothelium before isometrically recording developed tension to cumulatively added ET-1. Aortic strips from 5- and 11-week-old female transgenic TGR(mRen2)27 (hfTG) rats responded to ET-1 with higher Emax values and lower EC50 values than those of age-matched SD rats. Conversely, aortic strips from 35-week-old hfTG rats exhibited lower Emax and higher EC50 values than aortic strips from SD rats. Within the hfTG rats, aortic strips from 11-week-old rats showed increased Emax and decreased EC50 of ET-1 as compared with either 5- or 35-week-old hfTG rats. These data are in keeping with the hypothesis that ET-1 contributes to the hypertension of hfTG rats and suggest that an altered vascular responsiveness to the peptide may be implicated in the changes of their systolic blood pressure occurring with ageing in this animal model.     

The arterial blood supply of the human patella. Its clinical importance for the operating technique in vascularized knee joint transplantations

1 This study aimed to assess the effect of cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic reticulum calcium (Ca) pump, against contractile responses produced by selective tachykinin NK1 and NK2 receptor agonists, [Sar9]substance P (SP) sulfone and [beta Ala8]neurokinin A (NKA) (4-10), respectively, on the circular muscle of guinea-pig colon. All experiments were performed in the presence of atropine (1 microM) and indomethacin (10 microM). 2 In organ bath experiments, a submaximally equieffective concentration of the two agonists (10 nM) was selected: [Sar9]SP sulfone (10 nM) produced a biphasic contraction, the two amplitudes averaging 75 +/- 2 and 43 +/- 3% of the maximal response to KCl (80 mM) at 1 and 15 min from application of the agonist, respectively. CPA (3 microM for 60 min) slightly reduced the phasic response to [Sar9]SP sulfone (16 +/- 4% inhibition) and markedly suppressed the tonic component (89 +/- 3% inhibition). 3 The contraction produced by [beta Ala8]NKA (4-10) (10 nM) was more sustained than that induced by the NK1 receptor agonist: it averaged 69 +/- 5 and 73 +/- 4% of the response to KCl at 1 and 15 min from application of the agonist, respectively. CPA slightly and evenly depressed the response to [beta Ala8]NKA (4-10) (18 +/- 7 and 21 +/- 5% inhibition at 1 and 15 min). 4 In the presence of tachykinin NK1 and NK2 receptor antagonists (SR 140333 and MEN 10627, respectively, 1 microM each) and of L-nitroarginine (100 microM), KCl (40 mM) produced a distinct phasic and tonic contraction which was suppressed by 1 mM nifedipine. CPA (3 microM) did not affect the phasic contraction to KCl but abolished the tonic component of the response. 5 In the presence of 1 microM nifedipine, the response to [beta Ala8]NKA (4-10) was slightly depressed (32 +/- 6% inhibition) in its early component only, while the response to [Sar9]SP sulfone was abolished. CPA produced a slight inhibition (15 +/- 9 and 33 +/- 10% at 1 and 15 min, respectively) of the nifedipine-resistant response to [beta Ala8]NKA (4-10), an effect similar to that observed in the absence of nifedipine. Therefore, a large part of the response to [beta Ala8]NKA (4-10) persisted in the presence of both CPA and nifedipine. 6 In the sucrose gap, a prolonged superfusion with [Sar9]SP sulfone (0.1 microM for 5 min) produced sustained depolarization with superimposed spikes and contraction. CPA (3 microM) produced transient depolarization and contraction. In the presence of CPA, the initial responses (depolarization, spikes and contraction) to [Sar9]SP sulfone were unaffected but the sustained component of contraction was absent; the latter effect was accompanied by a suppression of spikes while the sustained depolarization was present. 7 We conclude that, during sustained depolarization produced by the NK1 receptor agonist, blockade of the sarcoplasmic reticulum Ca pump by CPA produces a faster Ca-dependent inactivation of Ca channels, thereby eliminating spikes and abolishing the tonic component of contraction. Ca mobilization/reuptake from a CPA-sensitive store seems to be of minor importance for regulating the NK2 receptor-mediated contractile responses.     

Ciprofloxacin pharmacokinetics in burn patients

Cyproterone acetate (CPA), a synthetic progestin recently found to induce genotoxic effects in hepatocytes from female rats and from humans of both genders, and two structural analogues, chlormadinone acetate (CMA) and megestrol acetate (MGA), have been compared for their capacity to induce DNA repair synthesis as measured by quantitative autoradiography. Exposure of primary human hepatocytes for 20 h to concentrations of CPA, CMA and MGA ranging from 2 to 50 microM induced positive responses in cultures from donors of both genders and the amounts of DNA repair elicited by the three progestins were similar. Under the same experimental conditions substantial differences were observed in the amounts of DNA repair elicited by the three progestins in primary hepatocytes from female rats, their potency decreasing in the following order CPA > CMA > MGA, and the three compounds failed to induce DNA repair in hepatocytes from male rats. These results, which agree with previous findings, suggest that for these sex steroids extrapolation to humans of results obtained in rats might be questionable.     

Synthesis of 2-amido-2,3-dihydro-1H-phenalene derivatives as new conformationally restricted ligands for melatonin receptors

Tetrahydroanthracene, tetrahydrophenanthrene, and tetrahydrophenalene moieties were used to design novel constrained melatoninergic agents. Compounds 1 and 2 were synthesized from the cyclization of the aryl succinic acids 6a,b followed by catalytic reduction, Curtius degradation to the amino derivatives, and acetylation. The phenalene derivatives 3 were prepared by cyclization of the aza lactones of the corresponding alpha-N-acetyl amino acids. The ketone derivatives were reduced directly by catalytic hydrogenation to produce the compounds 3. The different compounds were evaluated in vitro in binding assays using 2-[125I] iodomelatonin and chicken brain membranes. Melatonin and 2-acetamido-8-methoxytetralin were used as the reference compounds. The results showed the superiority of the dihydrophenalene framework 3 over those of tetrahydroanthracene and tetrahydrophenanthrene. 3a had relatively good affinity for melatonin receptors (Ki = 28.7 nM). Introduction of an additional methoxy group gave a derivative (3c) with nanomolar affinity (Ki = 0.7 nM), confirming the existence of a secondary binding site in the receptor which has been described previously. An increase in the affinity was also observed with the propionamido derivative 3e (Ki = 6.0 nM). The potential agonist properties of the compound 3e were evaluated on the dermal melanocytes of Xenopus laevis tadpoles. At the concentration of 2.3 nM (5 x Ki), melatonin gave a melanophore index value of 1. Similarly to melatonin, 3e was shown to be a potent agonist of the melanosome aggregation.     

The heterocyclic substituted pyridine derivative (+/-)-2-(-3-pyridinyl)-1-azabicyclo[2.2.2]octane (RJR-2429): a selective ligand at nicotinic acetylcholine receptors

The present report describes in vitro studies demonstrating that the heterocyclic substituted pyridine compound (+/-)-2-(3-pyridinyl)-1-azabicyclo[2.2.2]octane (RJR-2429) is extremely potent in activating human muscle nicotine ACh receptor (nAChR) (EC50 = 59 +/- 17 nM; Emax = 110 +/- 09% vs. nicotine). RJR-2429 is markedly less potent in activating nAChRs in the clonal cell line PC12, with EC50 = 1100 +/- 230 nM and Emax = 85 +/- 20% when compared with nicotine. The activation of a putative alpha 3 beta 4-containing nAChR in PC12 by RJR-2429 reveals a potency intermediate between nicotine and epibatidine (EC50 of 20,000 nM for nicotine and 30 nM for epibatidine). Dose-response curves for agonist-induced ileum contraction indicate that RJR-2429 is equipotent with nicotine, having an EC30 of approximately 2 microM. RJR-2429 binds with high affinity to alpha 4 beta 2 receptor subtype (Ki = 1.0 +/- 0.3 nM), and chronic exposure results in significant up-regulation of the high-affinity [3H]nicotine binding sites. In addition, RJR-2429 does not activate nAChRs present in rat thalamic preparations but is a potent inhibitor of this receptor subtype. It antagonizes nicotine-stimulated ion flux in thalamic synaptosomes with an IC50 of 154 +/- 37 nM. It also is a potent partial agonist at nAChRs mediating dopamine release from rat synaptosomal preparations (EC50 = 2 +/- 1 nM; Emax = 40%; epibatidine and nicotine have EC50 values of 0.4 and 100 nM, respectively). A model for the structure-activity profile of RJR-2429, nicotine and epibatidine was derived by molecular forcefield and quantum mechanics calculations and may provide important clues for the development of ligands selective for nAChR subtypes as probes in the life sciences or as potential therapeutic tools.     

Processing of adenosine receptor agonists in rat and human whole blood

A stability study of adenosine receptor agonists in rat and human whole blood was performed. The compounds were incubated at 37 degrees in fresh blood, and aliquots of the incubation mixture were hemolyzed at regular time intervals and analyzed with HPLC. N6-cyclopentyladenosine (CPA) and N6-cyclobutyladenosine (CBA) were degraded, whereas N6-cyclohexyladenosine, N6-cycloheptyladenosine and N6-sulfophenyladenosine were not. 2-Chloroadenosine had a half-life very similar to that of CPA. However, the 2'-, 3'-, and 5'-deoxyribose derivatives of CPA remained intact. The nucleoside transport inhibitor nitrobenzylthioinosine attenuated CBA and CPA metabolism in rat blood as did the inhibitor of adenosine deaminase erythro-9-(2-hydroxy-3-nonyl)adenine, albeit at relatively high concentrations. Complete blockade of CBA and CPA degradation was achieved by a preincubation of rat and human blood with the adenosine kinase (AK) inhibitor 5'-amino-5'-deoxyadenosine. We conclude that the two adenosine analogues are metabolized by AK both in rat and in human whole blood.