Pharmacological activities of trimetoquinol and 1-benzyl halogen-substituted analogues on rat beta-adrenoceptor subtypes.

The beta-adrenoceptor activity profile of trimetoquinol and its 1-benzyl halogen-substituted analogues was studied in rat tissues containing primarily beta 1 (atria)-, beta 2 (trachea)- and atypical beta/beta 3 (distal colon and brown adipose tissue)-adrenoceptors. Functional biological activity resided in the (-)-isomer of trimetoquinol which was 112-, 275-, 372- and 513-fold more potent than (+)-trimetoquinol in trachea, right atria, distal colon and brown adipose tissue, respectively. (+/-)-Trimetoquinol was equally or slightly less active than (-)-trimetoquinol. The 1-benzyl halogen-substituted analogues of trimetoquinol exhibited differential activation of beta-adrenoceptor subtypes. In functional assays, 3'-iodotrimetoquinol was a potent activator of all beta-adrenoceptor subtypes. 3',5'-Diiodotrimetoquinol was 10-fold more potent as an agonist in tissues containing atypical beta/beta 3-adrenoceptors than those tissues containing beta 1- and beta 2-adrenoceptor sites. Furthermore, this drug was a partial agonist as compared to (+/-)-trimetoquinol and 3'-iodotrimetoquinol on beta 1-adrenoceptors. Pharmacological properties of the compounds on rat beta 3-adrenoceptors expressed in Chinese hamster ovary (CHO) cells were consistent with results observed in functional assays. 3',5'-Diiodotrimetoquinol possessed the greatest potency for activation of adenylyl cyclase. Rank order of affinity for rat beta 3-adrenoceptor was 3'-iodotrimetoquinol = 3',5'-diiodotrimetoquinol > (+/-)-trimetoquinol > (-)-isoprenaline. These results suggest that 3',5'-diiodotrimetoquinol is a promising drug for further chemical modification in the development of selective beta 3-adrenoceptor ligands.

Gangliosides have a bimodal effect on DNA synthesis in U-1242 MG human glioma cells.

GM1, GD1a, and GT1b inhibit both PDGF-stimulated and serum-stimulated DNA synthesis in Swiss 3T3 cells and the human glioma cell line U-1242 MG in a dose-dependent manner. The ganglioside inhibitory effect is counteracted in a dose-responsive fashion by serum such that ganglioside-induced inhibition is essentially abolished in 10% serum. Because of the potentially important role that gangliosides play in growth regulation of human gliomas, this phenomenon was studied in detail using U-1242 MG cells. Stimulation of DNA synthesis by low doses of serum in U-1242 MG cells is inhibited in a dose-responsive fashion by ganglioside GM1. However, serum itself counteracts the inhibitory effect of ganglioside in a dose responsive way. Kinetic analyses demonstrate that GM1 competes with some components of serum for sites on U-1242 MG cells (Kb of GM1 = 12.5 microM). On the other hand, GM1, GD1a, and GT1b stimulate DNA synthesis in quiescent U-1242 MG cells in both sparse and confluent conditions, indicating that ganglioside-stimulated DNA synthesis is dependent on the phase of cellular growth rather than cellular density. This growth stimulatory effect of gangliosides is more potent on quiescent, confluent cells than quiescent, sparse cells. These results demonstrate that exogenously added gangliosides can have opposite (bimodal) effects on progression of human glioma cells through the cell cycle depending upon the growth phase of the cells.

Comparison of the effects of antimitotic drugs on alpha-tubulin mRNA, microtubules and nicotinic receptor-mediated catecholamine secretion in adrenal chromaffin cells in culture.

1. Four hour treatments of adrenal chromaffin cells with colchicine (10, 100 microM and 1 mM), tubulozole (10 microM) or podophyllotoxin (100 microM) decreases alpha-tubulin mRNA content. Vinblastine (10 microM) and taxol (10 microM), however, do not decrease alpha-tubulin mRNA content. 2. Immunocytochemical techniques demonstrate that 4 hr treatments with all of the antimitotic drugs (colchicine, podophyllotoxin, taxol, tubulozole and vinblastine) produce abnormal microtubule arrays. 3. The effects of 4 hr treatments with the antimitotic drugs on adrenal catecholamine secretion are all qualitatively the same; each drug selectively inhibits adrenal nicotinic acetylcholine receptor-stimulated catecholamine release, while having no inhibitory actions on release stimulated through noncholinergic mechanisms. 4. These studies demonstrate that autoregulation of tubulin synthesis occurs in cultured adrenal chromaffin cells. 5. All of the antimitotic drugs selectively inhibit nicotinic receptor-mediated adrenal catecholamine release under treatment conditions that affect adrenal microtubules. These results support the possibility that the actions of the antimitotic drugs on adrenal nicotinic receptors may involve microtubules.

Nicotinic and nonnicotinic receptor-mediated actions of vinblastine.

Vinblastine has been demonstrated to inhibit nicotinic acetylcholine receptor (nAChR) activity in adrenal chromaffin cells and superior cervical ganglia and to alter agonist binding affinity to nAChR of the electric organ of Torpedo californica. In cultured chromaffin cells, vinblastine (IC50, 8.9 microM) is significantly more potent than hexamethonium (IC50, 16 microM) and decamethonium (IC50, 18 microM) and significantly less potent than d-tubocurarine (IC50, 2 microM), pentolinium (IC50, 0.6 microM), and mecamylamine (IC50, 0.1 microM) in inhibiting nAChR-stimulated catecholamine release. These results demonstrate that vinblastine has moderately potent anti-nAChR activity on adrenal nAChR. On the other hand, vinblastine does not interfere with phrenic nerve stimulation of rat diaphragm musculature in concentrations up to 200 microM. However, in relatively high doses, vinblastine (10-200 microM) produces an increase in baseline tension of diaphragm muscle. This effect is concentration related (EC50, approximately 88 microM), reversible, and independent of phrenic nerve stimulation. The elevation in baseline tension is unaffected by nAChR blockade via d-tubocurarine, but is dependent upon the presence of extracellular calcium. The results suggest that vinblastine's antinicotinic actions are selective for neuronal-type nAChR and do not extend to nAChR of mammalian skeletal muscle. High concentrations of vinblastine appear to elicit contractures of skeletal muscle that are unrelated to nAChR.

Ischaemically induced alterations in electrical activity and mechanical performance of isolated blood perfused canine myocardial preparations.

STUDY OBJECTIVE: The aim was to investigate the direct effect of ischaemic insult on the electrical activity or mechanical performance of the sinoatrial node, atrioventricular node or Purkinje fibres (ventricular papillary muscle) of the dog heart. DESIGN: The three isolated tissues independently mounted in a water jacketed glass container were cross circulated through their respective arteries at a constant pressure (120 mm Hg) with arterial blood from a heparinised supporting dog under sodium pentobarbitone anaesthesia. Rate of automatic rhythmical contractions (automaticity) or tension development was measured before, during, and after ischaemia (30 min). EXPERIMENTAL PREPARATIONS: Seven sinoatrial nodes, five atrioventricular nodes, and 11 papillary muscles were prepared from the heart of mongrel dogs, weighing 11.5-15 kg. MEASUREMENTS AND MAIN RESULTS: The automaticity of the sinoatrial node and atrioventricular node exhibited a high degree of resistance to ischaemia, whereas both the automaticity (Purkinje fibres) and the tension development of papillary muscle were more susceptible to ischaemia and ultimately disappeared during ischaemia. Upon resumption of the blood supply, contractility rapidly returned to preischaemic levels. However, force-frequency analyses revealed that even after reperfusion, papillary muscle had lost the ability to respond normally to stimulus frequencies greater than 2 Hz. CONCLUSIONS: The findings suggest (1) that in the special excitatory and conductive system, the more distal the segment is to the sinoatrial node, the more vulnerable it is to ischaemia; (2) that the myocardium impaired by ischaemic insult lacks functional reserve, as evidenced by enlargement of the differences between the alternate tensions of pulsus alternans; and (3) that cessation of the blood supply does not predominantly damage those tissues whose functions are mainly regulated by a slow inward calcium current.

Inhibition of prolactin release from anterior pituitary lactotrophs in culture by sulfur-containing analogs of dopamine.

The effects of permanently charged and uncharged analogs of dopamine were examined for their ability to inhibit basal prolactin release from primary cultures of rat pituitary lactotrophs. The charged quaternary trimethyldopamine and the charged dimethylsulfonium analogs were active (IC50's were 4.3 and 31 microM, respectively) while the permanently uncharged monoethylsulfide was devoid of significant activity. Dopamine and dimethyldopamine, which are able to exist in both charged and uncharged forms, are more potent (IC50's were 36 and 44 nM, respectively) but all compounds were capable of approaching the same maximum degree of prolactin release inhibition. Haloperidol, a dopamine receptor antagonist, was able to block the actions of each of the agonists. The data suggest that (a) dopamine agonists do not have to be in the uncharged form in order to activate the dopamine receptor that regulates prolactin release, (b) the uncharged monomethylsulfide analog of dopamine is incapable of activating the dopamine receptor, and (c) the nitrogen on the side chain of dopamine can be replaced by another atom and still retain prolactin release inhibiting activity.

Prostaglandin B1 can modify the pressor responses to sympathetic nerve stimulation.

PGB1, a metabolite of PGA1, has the ability to enhance peripheral vascular resistance and elevate blood pressure in animals whose vascular tone is low. The effect is not central in origin and apparently is not the result of changes in cholinergic or alpha-adrenoceptor sensitivity or changes in vascular smooth muscle susceptibility per se.

Interaction of nitroglycerin with human blood components.

Nitroglycerin is rapidly lost from solution when incubated with red blood cells or whole blood. The assumption that the loss is enzymatic in nature may not be true, since no major metabolite is detected during this incubation. Explanation on the basis of a chemical reaction is also difficult, since the products of the chemical hydrolysis of nitroglycerin are the same as the metabolic products. After an initial rapid loss, nitroglycerin disappearance at 37 degrees follows an apparent first-order process in the concentration range of 10--480 ng/ml when incubated with washed red blood cells suspended in normal saline solution. The half-life for the reaction of the apparent first-order phase varies with the initial concentration and increases as the concentration increases (4 min at 10 ng/ml, 52 min at 480 ng/ml), suggesting a mixed kinetic mechanism. Metabolites of nitroglycerin (1,2- and 1,3-dinitroglycerin) react similarly to nitroglycerin in terms of an apparent initial, fast step, a secondary first-order dependence, and concentration-dependent rate effects; however, the rate of the reaction is much slower (t 1/2 = 33 min at 10 ng/ml) for the metabolite. These data suggest the possibility of a physical mechanism for the loss of nitroglycerin. Since the loss to red blood cells can be rapid, it seems that the mechanism should be delineated, and that the rate of disappearance be considered in an analysis of the pharmacodynamics of the drug.

Nitroglycerin tolerance and cyclic GMP generation in the longitudinal smooth muscle of the guinea-pig ileum.

The effects of in vitro nitroglycerin tolerance and methylene blue pretreatment on the ability of nitroglycerin and nitroprusside to promote relaxation and tissue accumulation of cyclic GMP were examined in the carbachol-contracted longitudinal smooth muscle of the guinea-pig ileum. Nitroglycerin and nitroprusside produced concentration-dependent increases in cyclic GMP levels. However, only nitroglycerin increased cyclic GMP levels before the onset of relaxation. Nitroglycerin tolerance produced approximately 200- and 5-fold shifts to the right of nitroglycerin and nitroprusside relaxation curves, respectively. Methylene blue pretreatment produced approximately 5-fold shifts to the right of both nitroglycerin and nitroprusside relaxation curves. Whenever there was an inhibition of nitroglycerin- or nitroprusside-induced relaxation, there was a corresponding reduction of cyclic GMP generation. Methylene blue inhibited the ability of 8-bromoguanosine 3',5'-monophosphoric acid to promote smooth muscle relaxation, suggesting that it also may impair the subsequent actions of cyclic GMP. This study provides the first demonstration of nitroglycerin tolerance in a nonvascular smooth muscle and provides evidence that cyclic GMP mediates the relaxant effects of nitroglycerin in the longitudinal smooth muscle of the guinea-pig ileum. However, because nitroprusside promoted tissue accumulation of cyclic GMP subsequent to relaxation, an exclusive role for cyclic GMP-mediated relaxation for this drug in the longitudinal smooth muscle appears unlikely.

Assessment of emetic and antiemetic activity.

Compounds having acute emetic or antiemetic activities have been quantitatively evaluated and compared with the reference standards, apomorphine and chlorpromazine, respectively. The bioassay for an emetic required the establishment of mean threshold emetic doses in groups of dogs for both the reference standard apomorphine and the test compound. The bioassay for antiemetics was based upon the degree to which the test drug elevated the threshold emetic dose for apomorphine. That effect was contrasted with the change in emetic threshold produced by equimolar concentrations of chlorpromaine.

Acute toxicity of prostaglandin Bx in male, albino, ICR mice.

Median lethal doses were estimated for PGBx administered to mice by the intraperitoneal and intravenous routes. The incidence of lethality (and therefore the LD50) was time dependent over a 96 hour period. The animals were active, were responsive and took nourishment during the 4-day post-injection period so that they apparently died from the direct effects of PGBx (or its metabolites) and not as a consequence of depression-induced dehydration or starvation.

Vascular tolerance to nitroglycerin and cyclic GMP generation in rat aortic smooth muscle.

Recent reports have suggested that cyclic guanosine 3', 5'-monophosphate (cGMP) may mediate drug-induced vascular smooth muscle relaxation. This hypothesis was examined by correlating increases in cGMP formation with vascular smooth muscle relaxation after sensitivities to nitroglycerin and nitroprusside were altered. Both nitroglycerin and nitroprusside produced a concentration-dependent increase in rat aortic cGMP levels, which preceded relaxation. A specific inhibition of nitroglycerin relaxation was accomplished by pretreating helical rat aortic strips with 5.5 X 10(-4) M nitroglycerin for 1 hr (in vitro nitroglycerin tolerance). Both nitroglycerin and nitroprusside were inhibited by pretreating tissues with 10(-5) M methylene blue for 1 hr. Whenever nitroglycerin or nitroprusside relaxation was inhibited, there was a corresponding inability to generate cGMP. Vascular tissues derived from nitroglycerin-tolerant animals (in vivo tolerance) exhibited a profile of cGMP reduction that closely resembled that of the in vitro model. Vascular smooth muscle relaxation induced by 8-bromoguanosine-3', 5'-monophosphoric acid was not inhibited by either in vitro nitroglycerin tolerance or methylene blue, suggesting that the subsequent action of cGMP, once formed, is not impaired. The results of this study support the contention that cGMP generation may be an important step in the promotion of vascular smooth muscle relaxation by nitroglycerin and nitroprusside.

Haloperidol causes irreversible damage to rat anterior pituitary lactotropes in culture.

Haloperidol, in concentrations in excess of 10(-5)M promotes the release of prolactin from rat anterior pituitary cells in monolayer culture. Histologic evidence revealed that 10(-4)M concentrations cause an irreversible cytotoxic effect that resulted in a loss of cell membrane integrity and a massive release of intracellular prolactin into the surrounding medium. Paradoxically, at 10(-5)M, haloperidol significantly inhibited prolactin release and promoted an accumulation of intracellular secretory granules. Lower concentrations had no significant effect. Although it is nominally classified as a dopamine receptor blocker and can (in lower concentrations) inhibit dopamine's suppression of prolactin release, haloperidol in higher concentration enhances prolactin release by a cytotoxic mechanism unrelated to dopamine receptor interaction.

Loss of nitroglycerin from aqueous solution into plastic intravenous delivery systems.

The mechanism of potential loss of nitroglycerin stored in plastic and glass containers was studied from an equilibrium and kinetic approach. Plastic strips equilibrated with dilute aqueous solutions of neat nitroglycerin showed that the drug was lost by absorption. Drug loss was followed by an electron-capture GLC assay. The same assay of control solutions in glass showed no drug loss in 48 hr at pH 5.7. The kinetics of nitroglycerin absorption and desorption were determined using synthesized 14C-labeled drug. Absorption can be quantified using a diffusion model, where the concentration in the aqueous phase falls with time. Curve fitting yielded an average diffusion coefficient in plastic of 2.05 x 10(-9) cm2/sec and a partition coefficient of 104 (plastic-water) at 30 degrees. Temperature-dependence studies of absorption showed that the diffusion coefficient followed an Arrhenius relationship with an energy requirement of 19.6 kcal/mole, whereas effects on the partition coefficient were negligible. Nitroglycerin desorption from plastic disks under sink conditions into water can be quantified by assuming a diffusion model where the concentration at the surface of a plane sheet remains constant. Nonlinear least-squares curve fitting generated a diffusion coefficient of 1.14 x 10(-9) cm2/sec for the desorption process at 30 degrees.

Effect of phenylbutazone on the metabolic disposition of warfarin in the dog.

Phenylbutazone has been shown to modify the physiologic disposition of warfarin in the dog. Dogs pretreated with phenylbutazone for a week, and permitted to remain drug-free for an additional week, eliminated warfarin from plasma 2-3 times faster than non-pretreated controls with an average half-life of 7.8 hours. In-vitro hepatic microsomal metabolism of warfarin and cytochrome P-450 content were greater for hepatic microsomes derived from phenylbutazone pretreated animals.

Phenylbutazone-warfarin interaction in the dog.

The administration of phenylbutazone together with warfarin to dogs resulted in an elevation of the free fraction of warfarin in the plasma from 2-6 to 8-0% thus providing direct support for the notion that phenylbutazone induced inhibition of warfarin binding to plasma proteins. This inhibition as evaluated by a kinetic method was accompanied by a two-fold decrease in the plasma half-life of warfarin from 18-4 h in control animals to 9-6 h in phenylbutazone-treated animals. Marked increases in warfarin-induced hypoprothrombinaemia were observed when at doses up to 8 mg kg-1 (orally) it was given with phenylbutazone (50 mg kg-1, orally). The unbound fraction of warfarin in canine plasma ranged from 1-7 to 4-3% indicating individual differences in the extent of the plasma binding of warfarin in the dog.

Topical application of a chlordane-containing ectoparasiticide: effect on the plasma half-life of warfarin in dogs.

Brief exposure of dogs to topical chlordane solutions resulted in a significant and long-lasting decrease in the biological half-life of orally administered warfarin. The effect is presumed to be an expression of chlordane's well-documented inductive effect on hepatic microsomal drug metabolizing enzymes and its long-term storage in fat depots. The facility with which chlordane is absorbed through the intact skin of dogs may render casually-treated animals unsuitable for subsequent pharmacologic study for long periods of time.