Alan S. Fairhurst

Interactions of D600 (methoxyverapamil) and local anesthetics with rat brain α-adrenergic and muscarinic receptors

Abstract D600 (methoxyverapamil) was found to inhibit the specific binding assayed in rat brain homogenates of the antagonist agents [ 3 H]WB 4101 and [ 3 H]QNB to the α-adrenergic and muscarinic receptors respectively. The IC 50 concentrations of D600 in standard binding experiments were 1.7 × 10 −6 M and 1.4 × 10 −5 M, with calculated k 1 values of 0.98 × 10 −6 M and 8.83 × 10 −6 M.Scatchard analyses showed these inhibitions to be competitive. Lidocaine and tetracaine also inhibited radioligand binding to these receptors, with K 1 values of 5.25 × 10 −4 M and 4.85 × 10 −5 M for the α-receptor and 8.2 × 10 t-5 M and 6.94 × 10 −6 M for the muscarinic receptor; these inhibitions also appeared to be competitive. Increasing the Ca 2+ concentration in the assays to 10 mM did not influence the effects of D600 or the anesthetics. Analyses of inhibitions of muscarinic receptor binding produced by D600 and lidocaine over a range of pH indicated that the inhibitory species of D600 is the uncharged form, whereas the charged form of lidocaine is inhibitory. Interactions of D600 and lidocaine with the agonist site on the muscarinic receptor were studied by measuring the effects of these agents on the displacement of [ 3 H]QNB by the muscarinic agonist carbachol. Comparison of these results with a theoretical model indicates that carbachol, [ 3 H]QNB, and D600 or lidocaine competitively displace one another at the same agonist site. The binding of labeled naloxone to the opiate receptor was also inhibited by D600, the IC 50 g being 4 × 10 t−6 M. These inhibitory effects of D600 and the local anesthetics on different receptors suggest that these agents may act by a common mechanism, namely by perturbing membrane structures. These results suggest caution in interpreting experiments in which D600 and verapamil are used analytically as Ca antagonists to assess the involvement of Ca in a biological system.

Effect of Carbamazepine on the In Vitro Uptake and Release of Norepinephrine in Adrenergic Nerves of Rabbit Aorta and in Whole Brain Synaptosomes

The effects of carbamazepine, in vitro, on adrenergic neuronal and whole brain synaptosomal uptake and release of tritiated norepinephrine (3H‐NE) were assessed. At 104 M, carbamazepine inhibited :,H‐NE uptake by 22% in rabbit thoracic aorta and in brain synaptosomes. At the same concentration, carbamazepine inhibited stimulation‐induced release of 3H‐NE by 42.69c and inhibited isometric contraction in rabbit ear artery helical strips by 31.6%. At 10‐5M, carbamazepine exhibited a 17.6% inhibition of 3H‐NE uptake in brain synaptosomes in the absence of effects on transmitter release. Cocaine, 10‐4 M, and imipramine, 10‐4 M, inhibited uptake by 88% and 85%, respectively, in aorta, and cocaine, 10‐4 M, inhibited synaptosomal uptake by 67.7%. Since antiepileptic blood levels of carbamazepine range between 1.3 and 3.0 × 10‐5 M, it was concluded that the observed effects of carbamazepine are insufficient to account for the anticonvulsant action of the drug. However, the blockade of 3H‐NE uptake by brain synaptosomes at 10‐5 M serves to explain the recently described analeptic activity of this agent.

Naloxone enhancement of acetic acid-induced writhing in rats.

Abstract Long-lasting visceral pain was produced in rats by the intraperitoneal injection of dilute acetic acid, and the response to this stimulus measured by observing the number of writhes over a subsequent 50 min. period. Prior treatment of the animals with naloxone or saline showed that naloxone increased the frequency of writhing. The enhancement by naloxone of the response to this nociceptive stimulus suggests that the binding of an endogenous opioid to the opiate receptors increases during prolonged nociceptive stimulation.

Muscarinic receptor subsensitivity in the longitudinal muscle of the rat ileum following chronic anticholinesterase treatment with diisopropylfluorophosphate

Abstract Chronic administration of diisopropylfluorophosphate to rats at a dose regimen producing 70 per cent inhibition of ileal cholinesterase was accompanied by a decrease in the sensitivity of the isolated ileum to oxotremorine and carbachol. The same treatment produced an increase in the sensitivity of the ileum to acetylcholine during the first ten days of anticholinesterase treatment, but after ten days of treatment the sensitivity of the ileum to acetylcholine was reduced. When [ 3 H]quinuclidinyl benzilate binding was measured in ileal longitudinal muscle homogenates from anticholinesterase-treated rats, a decrease in binding was observed which was due primarily to an increase in the dissociation constant of [ 3 H]quinuclidinyl benzilate. Chronic diisopropylfluorophosphate treatment also increased the K i of various cholinergic ligands as determined by competitive displacement of [ 3 H]quinuclidinyl benzilate binding. Scatchard analysis of agonist displacement ol [ 3 H]quinuclidinyl benzilate binding revealed two agonist binding sites which were present in approximately equal concentrations. Chronic diisopropylfluorophosphate treatment caused an increase in the dissociation constant of the high affinity site but produced only small effects on the dissociation constant of the low affinity site and on the relative concentration of the two sites. These results indicate that the tolerance to chronic anticholinesterase treatment is, in part, a receptor-mediated phenomenon.

A calcium antagonist drug binding site in skeletal muscle sarcoplasmic reticulum: Evidence for a calcium channel

The sarcoplasmic reticulum (S.R.) of rabbit skeletal muscle has been found to contain a single, high affinity binding site for the Ca antagonist drug [3H]-nitrendipine. Two subfractions of the reticulum were studied, the heavy (HSR) and light (LSR) preparations, which exhibited similar nitrendipine equilibrium dissociation constants (KD) of 1nM. Crude cardiac and brain membranes assayed under the same conditions exhibited KD values of 0.2-0.3nM. The concentration of binding sites per mg. protein (Bmax) in HSR was found to be very high, namely 6.7 picomoles/mg, some four times greater than that of LSR. [3H]-nitrendipine binding to HSR was reversible and inhibited by the Ca antagonists flunarizine and verapamil, and by the intracellular Ca release antagonist TMB-8 (8-diethylamino-octyl 3,4,5-trimethylbenzoate hydrochloride). However, unlabelled nitrendipine at 2 X 10(-5)M had no effect on contraction of isolated electrically stimulated rabbit lumbrical or rat diaphragm muscles, nor did it affect the neuromuscular junction as studied in rat phrenic nerve-diaphragm preparations. Also, little effect of 2 X 10(-5)M nitrendipine was seen on net 45Ca uptake by HSR. These results suggest that [3H]-nitrendipine binding to skeletal muscle S.R. resembles that of brain membranes, which also contain a high affinity binding site for [3H]-nitrendipine and which similarly are pharmacologically insensitive to this dihydropyridine type of Ca channel blocking agent. Since HSR is also enriched in calsequestrin and terminal cysternae from which Ca is released in vivo, it seems likely that the [3H]-nitrendipine binding sites in S.R. are associated with Ca channels in the S.R.

Effects of dihydropyridine calcium channel blocking drugs on rat brain muscarinic and α-adrenergic receptors

The dihydropyridine (DHP) Ca2+ channel blocking drugs nicardipine, nitrendipine, nimodipine, felodipine, nifedipine and nisoldipine were examined for activity in inhibiting specific (-)-[3H] QNB and [3H]WB4101 binding to the muscarinic and alpha-adrenergic receptors, respectively, of rat brain. Muscarinic receptor binding was affected most by nicardipine, with felodipine having less activity; the other DHP drugs were essentially inactive at 3 X 10(-5) M. The (+)-stereoisomer nicardipine (KI = 4.07 X 10(-7) M) was 27 times more potent than the (-)-isomer in inhibiting [3H]QNB binding, and this inhibition was found to be competitive. This inhibitory effect of nicardipine was not mediated via interaction with the high-affinity DHP binding site assumed to be associated with a Ca2+ channel. (+)-Nicardipine inhibited the binding of [3H]nitrendipine to this DHP binding site of brain, with a K1 of 9.01 X 10(-11) M, and was 10 times more potent than the (-)-isomer. Thus, the muscarinic receptor was 4200 times less sensitive to (+)-nicardipine than was this DHP binding site. Nicardipine was also the most potent DHP drug inhibiting [3H]WB4104 binding to the alpha-adrenergic receptor, although the other drugs were also somewhat active, in the rank order sequence listed above. This effect of nicardipine on the adrenergic receptor was also stereoselective, with (+)-nicardipine (KI = 3.46 X 10(-7) M) being about 3 times more potent than the (-)-isomer, in producing competitive inhibition of radioligand binding. These data suggest that the effects on brain receptors occur as a result of direct, stereospecific effects of DHP drugs on these receptors and are not due to Ca2+ channel blocking activity of these drugs.

Modification of Ryanodine Toxicity by Dantrolene and Halothane in a Model of Malignant Hyperthermia

Ryanodine toxicity in animals has been suggested to constitute a model of malignant hyperthermia. Dantrolene is known to block the development of malignant hyperthermia triggered by halothane in susceptible swine. The authors studied the influences of dantrolene and halothane on the effects of ryanodine in vitro in isolated rat diaphragm muscle segments, and in vivo in mice, to explore the validity of this model. In the diaphragm experiments, dantrolene was found to block or delay the development of contractures produced by ryanodine and to delay the potentiation of ryanodine-induced contractures caused by halothane. In mice, ryanodine at various dosages was injected and animals surviving after one hour were examined. Such survivors appeared grossly to be normal, and may constitute a model for the malignant hyperthermia patient. They were found to be susceptible to halothane and to succinylcholine, being killed by treatment with these two agents at dosages that were not lethal to control mice. Pretreatment of mice for 48 hours with orally administered dantrolene, followed by injection of ryanodine and then halothane anesthesia, decreased the lethality of ryanodine but did not reduce the number of deaths caused by the subsequent exposure to halothane. That the effects of ryanodine in vitro and in vivo are diminished and potentiated by dantrolene and halothane, respectively, would suggest that the ryanodine toxicity model of malignant hyperthermia may have validity and is worthy of further study. A prediction from this model is that the terminal cisternae of skeletal muscle sarcoplasmic reticulum may be altered in MH.

Effect of ryanodine on skeletal muscle reticulum calcium adenosine triphosphatase (CaATPase).

Abstract Ryanodine activates the CaATPase of a heavy (2000–8000 g ) sub-unit of skeletal muscle sarcoplasmic reticulum, with K A (for half-maximal activation) of 1·9 × 10 −5 M in the presence of 5 × 10 −3 M MgATPand 10 −6 M freeCa. K A is only 3·7 × 10 −6 M when the muscle fraction is exposed to ryanodine in the absence of Mg, and K A in the presence of 5 mM free Mg is 3.3 × 10 −5 M, with Mg competitively inhibiting the effect of the alkaloid. Ryanodine does not affect ADP-ATP exchange rate or the steady state level of phosphoprotein, but activates CaATPase by altering Ca permeability and decreasing intravesicular free Ca. The effect of ryanodine increases with temperature, is not affected by extensive dialysis and appears irreversible, with 1 mole of drug bound per 10 7 g of membrane protein; the drug has little effect on standard fragmented sarcoplasmie reticulum (FSR) preparations.

Effects of alkanols and halothane on rat brain muscarinic and α-adrenergic receptors

Effects of the primary alcohols ethanol, butanol, pentanol and of halothane were measured on the binding functions of muscarinic and α-adrenergic receptor preparatoins in rat brain homogenates, with the use of the antagonists 3H-quinuclidinyl benzilate and 3H-WB-4101. IC50 concentrations of the alkanols for the muscarinic and α-receptors respectively were: ethanol, 2.0 M and 1.4 M; butanol, 0.24 M and 0.16 M; heptanol, 3.7 × 10−3 M and 2.6 × 10−3. The plot of IC50 values versus number of carbon atoms in the alkanol was linear and of the same slope as the plot of membrane fluidity changes, thus indicating the importance of the membrane/water partition coefficient of the alkanol. Halothane at clinical concenrations had no effect on the receptors, although significant inhibitino of radioligand binding was produced by 2.5 M halothane, and inhibition was complete in presence of 17.5 mM anesthetic. From the correlation of receptor binding inhibitions with membrane fluidity changes reported by other workers, it is suggested that the activity of membrane receptors may be modulated by the fluidity of their membranes.

Altered tracheal smooth muscle activities in an animal model of human myotonic dystrophy

Abstract Although the 20,25 Diazacholesterol-treated rat exhibits several pathological changes involving skeletal muscle and other tissues resembling those seen in human myotonic dystrophy patients, this animal has not hitherto been examined as a model for studying smooth muscle involvement in this human disease. Rats were therefore treated chronically with 20, 25-D and comparisons made with control animals of dose-response curves of tracheal strip preparations to carbamylcholine, before and after partial irreversible blockade of cholinergic receptors by Dibenamine. Tracheas from myotonic animals exhibited diminished contractility and an increased ED 50 . The dissociation constant for the carbamylcholine-cholinergic receptor interaction was decreased and the percentage receptor occupancy for a given contractile response was increased. These results indicate that tracheal smooth muscle function is altered after 20,25-D administration and suggest that such animals may constitute a model for studying smooth muscle function in human myotonic dystrophy.