Ligand based structural studies of the CB1 cannabinoid receptor.

The structural characterization of G-protein coupled receptors (GPCRs) is quite important as these proteins represent a vast number of therapeutic targets involved in drug discovery. However, solving the three-dimensional structure of GPCR has been a significant obstacle in structural biology. A variety of reasons, including their large molecular weight, intricate interhelical packing, as well as their membrane-associated topology, has hindered efforts aimed at their purification. In the absence of pure protein, available in the native conformation, classical methods of structural analysis such as X-ray crystallography and nuclear magnetic resonance spectroscopy cannot be utilized successfully. Alternative methods must therefore be explored to facilitate the structural features involved in drug-receptor interactions. The methods described herein detail the use of covalent probes, or affinity labels, capable of binding covalently to a target GPCR at its binding site(s). Our approach involves the incorporation of a number of reactive moieties in different regions of the ligand molecule each of which is expected to react with different amino acid residues. Information obtained from such work coupled with computer modeling and validated by the use of site-directed mutagenesis of GPCRs allows for three-dimensional mapping of the receptor binding site. It also sheds light on the different possible binding motifs for the various classes of agonists and antagonists and identifies amino acid residues involved with GPCR activation or inactivation.

Concentration-dependent tubular secretion of acetazolamide and its inhibition by salicylic acid in the isolated perfused rat kidney.

An isolated perfused rat kidney (IPK) technique was used to study the effect of salicylic acid (SA) on the excretion of acetazolamide (AZ). Initial experiments were conducted in the absence of interactants at three nominal AZ concentrations (50, 100, and 250 micrograms/ml). Over the concentration range studied, AZ demonstrated net tubular secretion in the IPK. Significant decreases in excretion ratio (4.97 +/- 0.79-2.66 +/- 1.1) and secretory clearance (0.809 +/- 0.23-0.541 +/- 0.28) were observed with increasing AZ concentration, consistent with saturation of tubular secretion. Using a facilitated model for renal secretion, values of tubular transport parameters were obtained from a plot of excretion ratio vs. unbound AZ concentration: tmax = 118 +/- 29.4 micrograms/min, KM = 53.4 +/- 22.4 micrograms/ml, and tmax(A) = 6.31 +/- 2.82 micrograms/min. In the presence of SA (200 micrograms/ml), renal secretion of AZ was inhibited, as demonstrated by significant decreases in renal clearance (0.731 +/- 0.21-0.147 +/- 0.03) and excretion ratio (3.77 +/- 0.82-0.378 +/- 0.07). Although these findings were indicative of a reabsorption component to AZ excretion in the IPK that had not been previously proposed, the results were consistent with a previous investigation of concomitant administration of AZ and SA in humans (Br. J. Clin. Pharmacol. 27, 866, 1989), thereby endorsing utilization of the IPK as a screening tool for renal clearance mechanisms in humans.

A novel electrophilic high affinity irreversible probe for the cannabinoid receptor.

In order to explore the structural requirements for cannabinoid activity we have been involved in the design and synthesis of stereochemically defined high affinity probes for the cannabinoid receptor. This effort has involved the development of irreversible ligands which will allow us to obtain detailed information on the cannabinoid receptor active site(s). The irreversible ligands, which incorporate highly reactive functional groups in a strategic position of the ligand, may form covalent bonds with amino acid residues at the receptor active site or in the neighborhood of this site. We shall discuss the biochemical properties of one of these probes, which incorporates the electrophilic isothiocyanate group into the structure of the highly potent cannabinoid agonist (-)-1',1'-dimethylheptyl-delta 8-THC. This ligand, (-)-7'-isothiocyanato-1',1'-dimethylheptyl-delta 8-THC (7'-NCS-DMH-delta 8-THC), was evaluated for its affinity for cannabinoid binding sites using rat forebrain membrane preparations and found to have an apparent IC50 value of 660 pM. Incubation of the membrane preparation with a ligand concentration of five times the apparent IC50 resulted in the irreversible occupation of nearly all of the receptor specific binding sites.

Syntheses of racemic and nearly optically pure ether lipids and evaluation of in vitro antineoplastic activities.

In addition to rac-2-O-methyl-1-O-octadecylglycero-3-phosphocholine (rac-ET-18-OCH3, rac-Edelfosine, 1), three racemic ether lipid analogs, 4, 5, and 6, were synthesized where N,N-dimethylamino, N-methylpyrrolidino, and N-methylmorpholino groups, respectively, have been substituted for the trimethylammonio group. The two enantiomers, (R)-ET-18-OCH3 (2) and (S)-ET-18-OCH3 (3), and all four possible chiral methylcholine analogs, 7, 8, 9, and 10, of (R)-ET-18-OCH3 (2) were also synthesized. Three human leukemic cell lines (CEM, HUT 78, and Namalwa) were used to assess the in vitro antineoplastic properties of these 10 ether lipid analogs. At ether lipid concentrations of 5-50 micrograms/mL, dose- and time-dependent cytotoxicities were demonstrated up to 24 h. CEM and HUT 78, both T cell derived, were more sensitive to the ether lipids than Namalwa, which is B cell derived. rac-ET-18-OCH3 (1) with its R and S enantiomeric forms, 2 and 3, respectively, exhibited modest stereoselectivity in HUT 78 and Namalwa with 1 and 2 slightly more cytotoxic than 3. Ether lipid (EL) analogs 4, 5, and 6 demonstrated significantly greater cytotoxicity in normal peripheral lymphocytes, 4 and 6 exhibited a modest increase in cytotoxicity in HUT 78 and Namalwa (P < 0.05), and 5 demonstrated greater cytotoxicity (P < 0.05) in Namalwa than the parent EL 1. The calculated 24 h ID50 values suggest that the beta-methyl analogs, 9 and 10, were more cytotoxic than the alpha-methyl analogs, 7 and 8, in all the tested cancer cell lines.

Characterization of methotrexate elimination and interaction with indomethacin and flurbiprofen in the isolated perfused rat kidney.

The mechanism of methotrexate (MTX) renal elimination and the interaction of MTX with the nonsteroidal anti-inflammatory drugs, indomethacin and flurbiprofen, were characterized in the isolated perfused rat kidney. Perfusion studies elucidating MTX renal disposition were performed in perfusate (4.0% bovine serum albumin) at initial MTX concentrations of 1, 5, 12 and 25 micrograms/ml and in controls without MTX. Interaction studies were performed at clinically relevant indomethacin and flurbiprofen concentrations of 2.5 and 10 micrograms/ml, respectively, and a MTX concentration of 25 micrograms/ml (representative of an oncolytic MTX dose). MTX unbound fractions were concentration and interactant independent. Kidney viability was within normal limits for this technique among all perfusions. MTX renal clearance was nonlinear, increasing from 0.310 to 0.434 ml/min over the concentration range studied. Corresponding excretion ratios increased from 0.933 to 1.52, indicative of MTX renal elimination involving the processes of filtration, secretion and reabsorption. Excretion ratio results were supported by tubular transit rate calculations. Interaction studies indicated that there was secretory inhibition of MTX as evidenced by a decrease in both excretion ratio and tubular clearance at 25 micrograms/ml of MTX. Therefore, the secretory component was significantly inhibited by indomethacin and flurbiprofen after concomitant administration of oncolytic doses of MTX.

Characterization of human platelet GMP-140 as a heparin-binding protein.

Human platelet GMP-140 has been identified as a heparin-binding protein. Purified platelet GMP-140 bound to Heparin-Sepharose CL-6B and was eluted by approximately 0.5 M sodium chloride. Radioiodinated GMP-140 bound specifically and saturably to heparin immobilized on Matrex-Pel 102 beads. Binding of radioiodinated GMP-140 to heparin-Matrex-Pel 102 beads was divalent cation-independent and was strongly inhibited by excess fluid phase GMP-140 and heparin and by other sulfated glycans such as fucoidin and dextran-sulfate. Binding was not inhibited by chondroitins 4- and 6-sulfate or mannose 6-phosphate.

A variant of Glanzmann's thrombasthenia characterized by abnormal glycoprotein IIb/IIIa complex formation.

Glanzmann's thrombasthenia is a congenital bleeding abnormality characterized by absent platelet aggregation due to the failure of fibrinogen to bind to activated thrombasthenic platelets. In the majority of cases, this defect is caused by the absence or marked reduction of a specific fibrinogen-binding aggregation receptor, the GP IIb/IIIa complex. E.T., an 18-year-old female with a life-long history of bleeding and easy bruising, had the normal clinical features of Glanzmann's thrombasthenia. Surprisingly, sodium dodecyl sulphate-polyacrylamide gel electrophoresis of her platelets showed no apparent abnormality of the GP IIb/IIIa complex. Control platelets washed in the presence of 2 mM EDTA and control and patient platelets washed in the presence of 2 mM calcium ions showed normal reactivity with anti-GP IIb, anti-GP IIIa, and anti-GP IIb/IIIa complex specific monoclonal antibodies as evaluated by flow cytometry. In contrast, patient's platelets washed in the presence of 2 mM EDTA reacted with anti-GP IIb, anti-GP IIIa, but not with the complex-specific monoclonal antibodies. The increased susceptibility of the patient's GP IIb/IIIa complex to EDTA dissociation was confirmed by crossed immunoelectrophoresis (CIE). CIE analysis further indicated that the patient's GP IIb/IIIa complex did not bind fibrinogen. The combined results suggest that this patient has Glanzmann's thrombasthenia due to an abnormal association of the GP IIb/IIIa complex which results in the failure of the complex to bind fibrinogen.

Bernard-Soulier syndrome.

Bernard-Soulier syndrome (BSS) is a rare autosomal bleeding disorder characterized clinically by prolonged skin bleeding time, normal clot retraction and thrombocytopenia with large and morphologically abnormal platelets, and biochemically by the absence of platelet membrane glycoproteins (GP) Ib, V and IX. GP Ib and GP IX exist in the platelet membrane as a heterodimer complex which acts as the major receptor mediating platelet adhesion to blood vessel subendothelium. Studies with BSS platelets have proved particularly rewarding in the investigation of the GP Ib-IX complex as a multifunctional receptor protein. The transmembrane complex contains binding domains for von Willebrand factor, thrombin, fibrin and quinine/quinidine drug-dependent antibodies as well as an attachment site on the cytoplasmic side of the membrane for a platelet cytoskeleton. In addition, the internal segment of the beta-chain of GP Ib contains a cyclic AMP-dependent protein kinase-associated phosphorylation site which appears to regulate platelet reactivity. Limited proteolytic cleavage of the complex, in particular the GP Ib alpha-chain, has allowed immunological and functional characterization of three distinct domains; a 45 kDa segment at the N-terminal end of the alpha-chain of GP Ib, which contains binding sites for von Willebrand factor and thrombin, a 90 kDa highly glycosylated region of GP Ib alpha and a membrane-associated region consisting of the remnant of GP Ib alpha disulphide-linked to GP Ib beta and non-covalently-complexed with GP IX. This membrane-associated region contains the antigenic epitope(s) for quinine/quinidine drug-dependent antibodies. It is highly probable that the future study of platelets from patients with the Bernard-Soulier syndrome will further clarify the role of the GP Ib-IX complex in platelet physiology.

A Gompertz age-specific mortality rate model of aging: modification by dietary restriction in rats.

A Gompertz age-specific mortality rate model characterizing actuarial effects of food restriction in rats was developed based on mortality data from the study of Yu et al. (J. Gerontol. 40: 657-670, 1985). Results indicated that in the presence of adequate nutrition, food restriction reduces the aging rate parameter in a consistent manner, regardless of the age at which food restriction is initiated.

Cardiovascular interactions of lidocaine with verapamil or diltiazem in the dog.

Lidocaine in low and high doses was given by sequential infusions to isoflurane-anesthetized dogs (1.75 +/- 0.03% end-tidal concentration) with or without concurrent infusions of diltiazem or verapamil, to assess changes in cardiovascular function. When lidocaine was administered alone, the low plasma levels (approximately 2 micrograms/ml) caused only a modest reduction in left ventricular dP/dt. The higher plasma lidocaine levels (approximately 6 micrograms/ml) reduced both left ventricular dP/dt and cardiac index, and increased pulmonary capillary wedge pressure and systemic vascular resistance. Diltiazem or verapamil, when administered alone at plasma concentrations of approximately 150-200 ng/ml, prolonged atrioventricular conduction, decreased heart rate and cardiac index, and, in the case of verapamil, also decreased left ventricular dP/dt and mean arterial pressure. When lidocaine was added to diltiazem or verapamil, the low plasma levels of lidocaine depressed cardiac function in the presence of either calcium channel blocking drug. In the presence of these levels of verapamil or diltiazem, only one of six verapamil-treated animals and three of six diltiazem-treated animals were able to maintain a mean arterial pressure greater than 50 mmHg with the higher dose of lidocaine. Caution may be advised if the addition of lidocaine, by whatever route, is indicated in subjects who have recently received intravenous verapamil or diltiazem.

Hormesis, Gompertz functions, and risk assessment.

A historical survey of the literature indicates that benefits derived from low doses of toxic substances have been reported over many centuries. Hippocrates, Paracelsus, Arndt, Schulz, and Hahnemann (founder of homeopathy) have all reported that low doses of toxic substances may be "stimulatory" or otherwise beneficial. Assessment of mortality data from modern-day bioassay studies indicates that low-dose animal exposure to a variety of toxic agents can, through an unknown mechanism, also induce beneficial changes which promote health and prolong life (longevity hormesis). This nonspecific and apparently reversible phenomenon has been modeled kinetically through use of age-specific mortality rate and a generalized Gompertz function; the basic assumption is that mortality in an interval is a function of the weighted sum of intensities of physiologic injury during that interval. It was assumed that longevity-enhancing hormetic reduction in population injury may be decremented from life-shortening injury produced through the aging process and concomitant toxicity. At low exposure levels, a net reduction in age-specific mortality rate can sometimes be observed. The implications for risk assessment are significant. It is tacitly assumed in generating virtually all estimates of risk that toxic manifestations observed at higher doses are the sole effects elicited at lower doses. This appears to be qualitatively incorrect.

A Gompertz age-specific mortality rate model of aging, hormesis, and toxicity: fixed-dose studies.

Based on the proposition that the logarithm of age-specific mortality rate (Gompertzian) is a linear measure of the mean intensity of injury for a homogeneous mammalian population in a uniform environment, a model was developed which characterizes mortality experience resulting from both toxic and hormetic actions. The mortality-reducing component (longevity hormesis) was assumed to be reversible; toxic effects, on the other hand, were assumed to accumulate irreversibly. Following chronic low-dose administration of selected toxic substances, it was demonstrated (in certain cases) that longevity hormesis could enhance lifespan, even in the presence of concomitant toxicity. Even when toxicity was evident, hormesis could ameliorate some of the mortality. The assumption that high-dose chronic toxicity studies can generate realistic estimates of risk at low doses is challenged.

Relationship of oestrogen and progesterone and their oviductal receptors in laying and non-laying 5-year-old hens.

This study focuses on the relationship of ageing to reproductive function in the domestic hen. Reproductive function was assessed by egg-laying records of hens from the same flock of 4, 5 and 6 years of age. Egg laying decreased with advancing age and the percentage of non-laying hens increased. During an ovulatory cycle, plasma levels of oestrogen and progesterone (including the preovulatory surge) were similar in both the 5-year-old and 1-year-old laying hens. In both young and old non-laying hens, the preovulatory surge of progesterone was absent. Levels of oestrogen were similar for all hens, but the oestrogen-dependent oviduct was atrophied in non-laying 5-year-old hens. This reduction in oviductal size was correlated with reduced levels of magnal nuclear oestrogen receptor and cytosol progesterone receptor. These results suggest a refractoriness of the oviduct to oestrogen stimulation in the ageing hen.

A simple technique for silicone rubber perfusion of the microcirculation of the mouse kidney.

A technique was described in which mouse kidneys were perfused with a silicone rubber compound. The technique involved cannulation of the left ventricle of the heart and subsequent perfusion of the entire systemic circulation of the mouse. Constant pressure was maintained to prevent distortion artifacts in the resultant renal microvascular cast. The method is adaptable to perfusion of other organs or animals.

The effect of tonicity on lens volume.

Rat lenses incubated in hypotonic medium adapted to the medium by first undergoing osmotic swelling and then returning after 1 to 2 days to their originial volume. Two processes--osmotic swelling and volume regulation--appear to be working simultaneously. The former is indicated by a decrease in the concentration of cell potassium to the expected level and the latter by a limitation of lens volume associated with a decrease in the potassium content of the lens. These findings are consistent with those observed in other types of cells and may be of significance in understanding the etiology of cataracts.