Effect of cocaine on rat pineal melatonin synthesis in vivo and in vitro.

Moderate concentrations (10 microM) of cocaine increased melatonin content and N-acetylserotonin and serotonin-N-acetyltransferase activity in rat pineal glands freshly placed in organ culture. Pineals cultured for 48 hours or taken from ganglionectomized animals did not respond to cocaine. Both procedures markedly reduced pineal noradrenalin (NA). Cocaine (5, 10, 20 mg/kg) given to adult intact rats stimulated pineal melatonin synthesis but only in animals exposed to constant light for 24 hours. Pineal denervation and/or adrenal demedullation neither completely eliminated NA from blood nor prevented cocaine-induced stimulation of melatonin synthesis in these light-primed animals.

Beta adrenoreceptors in the rabbit bladder detrusor muscle.

This study examines the beta adrenergic receptors of the rabbit detrusor smooth muscle, employing [125I]iodocyanopindolol (ICYP) as a ligand for the binding of beta adrenergic receptors. Saturation binding experiments on the isolated membrane fraction yielded a KD for ICYP of 14.7 pM and a maximum binding of 147.6 fmol/mg of protein. Displacement of labeled ICYP by a series of beta adrenergic agents yielded the following KD values for the combined high and low affinity binding sites: I-propranolol, 0.76 nM; ICI 118,551, 1.7 nM; zinterol, 38.0 nM; metoprolol, 3.5 microM; and practolol, 61.4 microM. When these displacement experimental results were compared to KD values from other reported binding studies with ICYP for beta adrenoreceptors, both the order of potency and the KD values indicated primarily beta-2 adrenergic receptor subtypes. Computer program Scatfit analysis of the displacement curves indicated a single slope and affinity constant for all five beta adrenergic agents. Hofstee plots for zinterol, ICI 118,551 and metoprolol, however, were not linear and indicated that minor populations of beta-1 adrenoreceptors were also present as both high and low affinity binding sites could be defined. It is concluded that the primary receptor population is beta-2 and that this tissue is heterogenous with a small population of beta-1 adrenoreceptors representing approximately 13 to 23% of the total beta adrenoreceptor population.

Identification of muscarinic receptors in rat cerebral cortical microvessels.

Microvessels isolated from rat cerebral cortex consist mainly of capillaries (greater than 85%). Fresh, intact microvessel preparations have been analyzed by radioligand binding techniques for muscarinic receptors. Scatchard analysis of specific quinuclidinyl benzilate (QNB) binding indicates that microvessels possess a large number of muscarinic sites (914 fmol/mg protein) of high affinity (KD = 0.034 nM). The association and dissociation rate constants (0.37 min-1 nM-1 and 0.0067 min-1, respectively) yield an equilibrium KD of 0.018 nM. Displacement of [3H]QNB by muscarinic ligands and control substances is typical of muscarinic receptors. The results indicate that cerebral microvessels possess a large population of muscarinic receptors.

Spare cholinergic receptors in the urinary bladder.

The concentration-effect relationships of carbachol were studied for three different responses of detrusor muscle of rabbit urinary bladder: the mechanical response, the increase of 45Ca uptake and the displacement of [3H]quinuclidinyl benzilate (QNB) from muscarinic receptors. The EC50 for carbachol in production of contractile responses of the detrusor muscle was observed to be 0.27 microM. The 45Ca uptake by detrusor muscle strips was studied by the lanthanum-residual wash out method. Carbachol increased 45Ca uptake with an EC50 of 12.7 microM. [3H]QNB binding was performed on homogenates of rabbit bladder detrusor muscle. By Scatchard analysis, the receptor density was found to be 47.2 fmol/mg of protein and the KD 0.074 nM. The contractile responses, the 45Ca uptake and the specific [3H]QNB binding were all blocked by atropine and so are taken to represent muscarinic receptor-mediated phenomena. Carbachol displacement of [3H]QNB yielded an EC50 of 135 microM and a Kl of 42 microM. It is concluded that the mechanical response requires a much lower concentration of carbachol than that needed for stimulating 45Ca uptake or for occupying [3H]QNB binding sites and that there appears to be approximately a 150-fold muscarinic receptor excess.

Tissue distribution and elimination kinetics of polybrominated biphenyls (PBB) from rat tissue.

The concentration of polybrominated biphenyl (PBB) in serum in a large number of organs was determined in a population of rats for 36 weeks a single dose of PBB. Groups were killed at 6, 12, 24 and 36 weeks after exposure to PBB (1 mg/100g body wt, i.p.). Growth, weight gain and appearance of the rats and their internal organs were normal. Complex and varied relationships were found in tissue concentrations with time after PBB administration. Serum and fat had apparent first-order elimination kinetics with calculated half-times of 23.1 and 69.3 weeks, respectively. For five other tissues, apparent t 1/2s ranged from 9.0-63 weeks, while for four others, kinetics could not be determined from these 4 time points. It is likely that a substantial residue of PBB will still remain in the body of the rat at the end of its life span because of the persistence of PBB in lipid-rich tissues (adipose, adrenal, and brain).

Dihydroergocryptine: a "pseudo-irreversible" alpha-adrenergic antagonist in the guinea pig vas deferens.

The ergot alkaloid, dihydroergocryptine, exhibits some of the characteristics of a competitive alpha-adrenergic antagonist. Dihydroergocryptine physiological antagonism is surmountable by high concentrations of alpha-adrenergic agonists and [3H]-dihydroergocryptine readily binds and dissociates from crude membranes with the characteristics expected of an alpha-adrenoreceptor ligand. However, during physiological studies, dihydroergocryptine antagonism is not readily reversible by washing. To explain this apparently paradoxical behavior of dihydroergocryptine, the characteristic of [3H]-dihydroergocryptine accumulation and efflux in the guinea pig vas deferens were studied. Vas deferens segments accumulated 0.99 pmol [3H]-dihydroergocryptine/mg protein. Most of the radioligand was extractable by acid-ethanol. About 5-6% of the radioligand remained bound to extracted tissue residues and appeared to be associated with crude membrane fractions prepared from vas deferens segments. Kinetic analysis of [3H]-dihydroergocryptine efflux from vas deferens segments indicated the presence of three compartments of radioligand in this tissue. A large compartment of [3H]-dihydroergocryptine emptied slowly and may represent radioligand accumulated into the intracellular space. [3H]-Dihydroergocryptine also was released from a compartment which exhibited the size and kinetics characteristic of alpha-adrenoreceptor sites on guinea pig vas deferens crude membranes. A small compartment of [3H]-dihydroergocryptine was nonexchangeable and nonextractable by acid-ethanol; this nonextractable radioligand may be bound covalently to membrane sites and/or other tissue components.

Dopamine-beta-hydroxylase: a modulator of beta adrenergic receptor activity.

The demonstrated proportionality between norepinephrine and dopamine-beta-hydroxylase released exocytotically from sympathetic nerve endings led us to explore the possible physiologic significance attached to this proportionality. The beta adrenoreceptor mediated desensitization of the hyperpolarization response to norepinephrine observed in pinealocyte membranes was rapidly reversed following the infusion of dopamine-beta-hydroxylase (DBH). This infusion also resulted in an augmented cyclic 3',5'-adenosine monophosphate (cAMP) response to isoproterenol in vitro. We suggest that the dissociation of the concentrations of norepinephrine and dopamine-beta-hydroxylase released into the synaptic cleft as a result of continuous sympathetic nerve stimulation produces a relative dopamine-beta-hydroxylase deficiency which is associated with the beta receptor desensitization. The observation that DBH restored the membrane sensitivity to NE in vitro in rat pineals, suggests that it either augments the rate at which agonist recognizes the beta receptor and couples to adenylate cyclase, or directly increases the activity of adenylate cyclase itself. The increase in cAMP generated in the presence of DBH supports such a hypothesis.

Clinically significant adverse effects in a Phase 1 testing program.

Twelve years' experience with a Phase I drug testing program in normal prison volunteers is reported. Involved in 805 protocol studies were 29,162 participants over 614,534 subject days. During this period there were 64 significant medical events of which 58 were adverse drug reactions and 6 were complications. One subject has residual hip changes due to an infectious complication, another on placebo died from cerebrovascular hemorrhage while asleep. There was complete recovery from all adverse drug reactions and the other 4 complications encountered. Thus a clinically significant medical event occurred once every 9,602 days subject exposure or about once every 26.3 years of individual subject participation.

Purine nucleoside and nucleotide interactions on normal and subsensitive alpha adrenoreceptor responsiveness in guinea-pig vas deferens.

Adenosine and adenosine 5'-monophosphate (AMP) augment contractile responses to norepinephrine (NE) in isolated guinea-pig vas deferens. Dipyridamole slightly enhances, while theophylline antagonizes, adenosine effects on responses to NE. Adenosine triphosphate (ATP) and the nonhydrolyzable analog, adenosine 5'-(beta,gamma-imido)triphosphate (AppNp) depress responses to NE. Adenine and adenosine diphosphate (ADP) are ineffective in influencing alpha adrenoreceptor responsiveness. Repetitive stimulation of isolated vas deferens with maximal concentrations of NE markedly reduce the contractile response to test concentrations of 6 micron NE. Spontaneous resensitization of responses to NE to control levels occurs within 25 to 35 minutes after the end of desensitization treatment. Adenine nucleosides and nucleotides promote a more rapid rate of alpha adrenoreceptor resensitization, with a potency order: AMP greater than adenosine greater than ADP. Adenine and ATP did not influence the rate of alpha adrenoreceptor resensitization. The adenine nucleotides ADP, ATP and the analog AppNp elicit concentration-dependent contractions of guinea-pig vas deferens. Theophylline antagonizes this contractile activity to adenine nucleotides. AMP, adenosine and adenine are devoid of agonistic activity. In the presence of NE, however, AMP and adenosine produce contractile responses of isolated vas deferens strips, and the agonistic activity of ADP, ATP and AppNp is profoundly enhanced. Agonistic actions of purines in the presence of NE are antagonized by phentolamine much more effectively than by theophylline. The results suggest the existence of a purinergic receptor mediating excitatory responses of guinea-pig vas deferens. Furthermore, there appears to be mutual interaction between purinergic and alpha adrenoreceptor mechanisms. That adenyl derivatives are capable of augmenting subsensitive alpha adrenoreceptor responsiveness suggests that adenine nucleosides or nucleotides, released during sympathetic transmission, may be required for maintenance of normal alpha adrenoreceptor sensitivity.

Ultrastructural changes in guinea-pig myocardium after acute ouabain treatment.

Ultrastructural changes in the region adjacent to the nuclear pole were identified in the ventricular myocardium of ouabain-treated guinea pigs after 1 to 4 hours of treatment. Significant changes include expansion of the Golgi complex, an increase in the number of coated vesicles and an increase in the number of cytoplasmic particulates including glycogen particles and ribosomes. The coated vesicles appear to coalesce with the internal terminal regions of T-tubules and thereby may contribute to increase tubular mass or surface. In contrast to acute ouabain treatment, the morphologic change identified with the administration of another positive inotropic drug, isoproterenol, consists of some increase in cytoplasmic particulates, but no significant change in the Golgi apparatus or in the number of coated vesicles compared with control hearts. It is concluded that the administration of ouabain leads to activation of the Golgi complex, possibly associated with the synthesis of new T-tubular membrane components.

Accumulation of radioactive cardiac glycosides by various brain regions in relation to the dysrhythmogenic effect.

Ouabain was administered at a loading dose of 3 mug/kg followed by an infusion at a rate of 1 mug/kg-1 min-1 in order to produce severe dysrhythmia in dogs within 60 minutes. Similarly, digitoxin at a loading dose of 9 mug/kg followed by an infusion at a rate of 3 mug kg-1 min-1 was administered to compare its effect with that of ouabain. 2 During the 60 min experimental period, the plasma concentrations gradually rose with the continuous infusion of these drugs. However, in comparison to the 60 min plasma value of 119+/-20 pmol/ml for ouabain and 177+/-68 pmol/ml for digitoxin, the cerebrospinal fluid (CSF) concentrations for these drugs at this time were less than 5 pmol/ml. 3 Upon termination of the experiment at 60 min it was found that kidney, liver, heart, adrenal, and the non-neural tissue in the brain such as pituitary and choroid plexus concentrated ouabain and digitoxin to give high tissue to plasma ratios. However, various neural areas of the brain (cerebellum, mesencephalon, hypothalamus, pons, and medulla) showed no preferential localization or uptake of these two glycosides. 4 Concentration of ouabain and digitoxin by the choroid plexus does not seem to affect the ionic composition of the CSF. 5 It was concluded that sampling the large areas of neural tissue above could provide no evidence for local accumulation of digitalis glycosides that might account for a central nervous system origin of digitalis-induced cardiac arrhythmias.

The effects of digitoxin and its metabolites on the transmembrane potential and contractile characteristics of guinea-pig ventricle strips.

The effects of 10(-7) and 10(-6) M digitoxin, and some of its metabolites, digitoxigenin-bis-digitoxoside, digitoxigenin-mono-digitoxoside and digitoxigenin on the transmembrane potential and contractile characteristics of guinea-pig right ventricle strips were studied to define the role of the sugar side-chain in these cleavage products of digitoxin. Digitoxin and digitoxigenin produced their maximum inotropic responses, without induction of arrhythmias, at about 30 min. However, the bis and mono compounds produced arrhythmias within 12 min, so the inotropy recorded may not be the maximum response. Digitoxin with 3 sugar residues and the bis compound with 2 sugar residues produced a prolongation in the action potenital duration. In contrast, the mono compound with one sugar residue and the digitoxigenin with no sugar residue produced a shortening of the action potential duration. There may be a relationship between the number of sugar moieties and the action potential duration; digitoxin and its bis derivative increased the action potential duration and mono- and digitoxigenin decreased the action potential duration. There also appeared to be an unusual relationship between the number of sugar moieties and induction of arrhythmias; arrhythmogenicity occurred at the doses employed with only the bis- and mono-digitoxoside. Finally, there appeared to be no simple relationship between chemical structure and inotropic potency.

Cardiac NaK ATPase activity during positive inotropic and toxic actions of ouabain.

In order to define pharmacological actions of ouabain in the dog heart, ouabain uptake and subcellular distribution and its effect on NaK ATPase (MG2+ dependent, Na+-K+-activated adenosinetriphosphate phosphohydrolase, E.C. 3.6.1.3), have been investigated in 21 open-chest dogs. A continuous infusion of ouabain (0.036 mug/kg/min) after a loading dose (20 mug/kg) produced a relatively constant plasma concentration of approximately 10(-8) M (6 ng/ml) ouabain, which induced a sustained positive inotropic response for the 300 min experimental period. In these hearts much greater binding of ouabain was noted in the NaK ATPase and microsomal fractions than in other myocardial fractions. No statistically significant inhibition of NaK ATPase activity was noted. Doubling the loading and infusion doses of ouabain raised the plasma level of ouabain to approximately 3 X 10(-8) M and produced various types of arrhythmia within an hour, which persisted for the rest of the 5 h experimental period. Under this experimental protocol there was a significant inhibition of NaK ATPase activity and increased binding of ouabain to this enzyme. This study does not support the hypothesis that there is a causal relationship between inotropic response to ouabain and NaK ATPase inhibition. It was concluded that NaK ATPase inhibition might be causally related to the development of ouabain toxicity.

The uptake and subcellular distribution of radio-labelled metabolites of digitoxin in the guinea-pig isolated perfused heart.

1 Comparisons were made of the uptake and inotropic effects of concentrations of 0.1 muM of digitoxin and its cleavage products digitoxigenin-bis-digitoxoside, digitoxigenin-mono-digitoxoside and digitoxigenin in the isolated perfused hearts of guinea-pigs. 2 Digitoxin produced the greatest inotropic responses in this series, while the sequence of cleavage products produced progressively smaller responses. 3 The uptake of digitoxin was significantly higher than that of the three metabolites, and the uptake of metabolites became progressively less with cleavage. The highest binding in each case was found in the microsomal fraction. 4 The uptake of all four digitaloids was reduced when the potassium in the perfusion medium was increased.

The uptake and subcellular distribution of radio-labeled metabolites of digoxin in the isolated perfused guinea-pig heart.

Cardiac glycosides like digitoxin and digoxin with three digitoxoside sugar residues have been reported to undergo step-wise degradation to yield the corresponding genins, and the importance of the digitoxoside side-chain for the pharmaco-dynamics property of cardiac glycosides has also been suggested. A sytematic study was therefore undertaken on the cleavage products of digoxin. Digoxigenin-bis-digiitoxoside (with two sugar residues) digoxigenin-monodigitoxoside (with one sugar residue) and digoxigenin (with no sugar) were compared with the parent compound, digoxin. The radio-labeled compounds were perfused through isolated guinea pig hearts using 10-7 M concentration in the perfusion medium for a fixed period of 64 min followed by an 8 min period of wash-out with normal medium. The uptake and sub-cellular distribution of the drugs were thereafter measured scintillation counting. All the compounds produced postive inotropic responses, the mondigitoxoside producing the greatest effect, digoxigenin next in order of inotropic response magnitude, the bis-digitoxoside produced the least effect, and digoxin was intermediate between the genin and bis-digitoxoside. The uptake of the monodigitoxoside was the highest, and in general, the quantitative uptake was related to the inotropic response. The greatest binding of each digitoxoside was found in the microsomal fraction. Both mechanical activity and uptake of all four drugs were uniformly reduced by an increase in potassium concentration in the perfusion medium.

Ventral medial hypothalamus: involvement in hypoglycemic convulsions.

After the ventral medial hypothalamus of mice was lesioned with gold thioglucose, the dose of insulin required to produce convulsions in 50 percent of the animals was doubled compared to that in nonlesionad controls. No dose of insulin, up to 50 milliunits per gram, produced convulsions in more than 60 percent of the lesioned mice, even though blood glucose levels fell to approximately 24 milligram percent.