Inhibition of endothelial-bound angiotensin converting enzyme, in vivo.

1. We determined apparent Ki constants of two inhibitors, captopril and CL242,817, for pulmonary endothelial-bound angiotensin converting enzyme (ACE) in anaesthetized rabbits. [3H]-benzoyl-Phe-Ala-Pro was used as the substrate. The apparent kinetic parameters Km and Amax (product of Vmax and microvascular plasma volume) were measured, as was the ratio (Amax/Km) (measured under first order reaction conditions) before and 30s after the i.v. administration of captopril 10 nmol kg-1 or CL242,817, 35 nmol kg-1. 2. Under mixed order reaction conditions, ([S] greater than or equal to Km), apparent Km values increased from 12.2 +/- 1.9 microM to 32.9 +/- 3.3 microM (P less than 0.05) in the captopril-treated rabbits and from 9.3 +/- 2.3 microM to 45.8 +/- 9.8 microM (P less than 0.05) in the CL242,817-treated rabbits, indicative of competitive inhibition. However, apparent Amax values decreased from 10.3 +/- 2.1 to 4.5 +/- 0.8 mumol min-1 (P less than 0.05) and 8.9 +/- 1.7 to 4.8 +/- 0.5 mumol min-1 (P less than 0.05), respectively. 3. Under first order reaction conditions ([S] much less than Km), the Amax/Km ratio decreased from 763 +/- 100 to 125 +/- 38 ml min-1 (P less than 0.05) and 1009 +/- 149 to 126 +/- 44 ml min-1 (P less than 0.05) in the captopril- and CL242,817-treated groups respectively. 4. When the single pass transpulmonary binding of 80pmol [3H]-RAC-X-65 (an ACE inhibitor) was measured in additional rabbits, a significant (P < 0.05) decrease in RAC-X-65 binding was observed 30s after captopril (80% decrease) or CL242,817 (85% decrease), a result expected for a loss of catalytically active enzyme mass due to tightly bound captopril or CL242,817. 5. These results indicate that, in vivo, both captopril and CL242,817 are competitive, tight binding inhibitors of lung ACE. Furthermore, they suggest means for evaluating the interaction of other potential ACE inhibitors with the pulmonary endothelial membrane-bound enzyme, in vivo, possibly in phase I clinical trials.

Disruption of muscarinic receptor-G protein coupling is a general property of liquid volatile anesthetics.

The influences of 3 volatile anesthetics, chloroform, enflurane and isoflurane, on muscarinic acetylcholine receptors in rat brainstem were determined. Each of the volatile anesthetics increased [3H]methylscopolamine [( 3H]MS) binding affinity, but did not affect the number of [3H]MS binding sites. Carbamylcholine affinity for brainstem muscarinic receptors was not altered after equilibration of brainstem membranes with any of these anesthetics. The ability of guanine nucleotides to depress the high affinity binding of two agonists, carbamylcholine and [3H]oxotremorine-M, was decreased or eliminated after equilibration of brainstem membranes with any of the anesthetics. In each of these actions, these anesthetics resemble halothane and diethyl ether. These results indicate that interference with muscarinic receptor-G protein interactions is a common property of liquid volatile anesthetics and may represent a general mechanism for the disruption of signal transmission between cells during anesthesia.

Diethyl ether effects on muscarinic acetylcholine receptor complexes in rat brainstem.

The influence of diethyl ether on muscarinic acetylcholine receptor-G protein interactions was studied using membranes isolated from rat brainstem. Membranes were equilibrated with diethyl ether (0.5 to 10%) for 20 min before, and then during, the binding assay. The affinity, but not the number, of [3H]N-methylscopolamine [( 3H]MS) binding sites was increased in the presence of diethyl ether (KD in air = 0.41 nM, KD in 2% diethyl ether = 0.21 nM). This increase in affinity reflected a decrease in the rapid dissociation rate constant (air k-1 = 13 X 10(-3) min-1, 2% diethyl ether k-1 = 7 X 10(-4) min-1) rather than a change in the association rate constant. Diethyl ether had no effect on the binding affinity of the muscarinic agonist carbamylcholine. However, the binding of a radiolabeled muscarinic agonist, [3H]oxotremorine-M [( 3H]Oxo-M), to high affinity binding sites decreased about 25% in the presence of 2% diethyl ether. The ability of a guanine nucleotide to depress the high affinity binding of both carbamylcholine and [3H]Oxo-M was decreased or eliminated by diethyl ether. Diethyl ether appears to interfere with muscarinic receptor-G protein interactions, perhaps by stabilizing receptor-G protein complexes or inhibiting the binding of guanine nucleotides.

Halothane attenuation of muscarinic inhibition of adenylate cyclase in rat heart.

Halothane stimulated basal adenylate cyclase activity in rat cardiac membranes. Maximal stimulation (54%) was obtained after equilibrating the membranes with 2% halothane. Halothane did not affect the fractional stimulation of adenylate cyclase activity produced by either forskolin or isoproterenol. However, halothane decreased carbamylcholine inhibition of adenylate cyclase activity stimulated by both forskolin and isoproterenol. Maximal depression of carbamylcholine inhibition of stimulated cyclase activity was obtained after equilibration with 1% halothane. These results are consistent with evidence from ligand binding studies and indicate that halothane disrupts muscarinic receptor-G-protein interactions.

Effects of halothane on high affinity agonist binding and guanine nucleotide sensitivity of muscarinic acetylcholine receptors from brainstem of rat.

The influence of halothane on muscarinic receptors with a high affinity for agonists was studied using [3H]oxotremorine-M. [3H]Oxotremorine-M bound with high affinity (KD = 2.8 nM) to a subpopulation of muscarinic receptors in the brainstem of rat, representing 32% of the total receptor pool. Agonist affinity for binding sites for [3H]oxotremorine-M was not affected by a guanine nucleotide (5'-guanylylimidodidiphosphate; Gpp(NH)p), although the level of binding was decreased, presumably due to the conversion of receptors to lower affinity conformations. However, only 58% of 3 nM binding of [3H]oxotremorine-M was sensitive to Gpp(NH)p. Halothane had two effects on the binding of [3H]oxotremorine-M: halothane (1) decreased the level of binding of [3H]oxotremorine-M without affecting agonist affinity for the surviving sites, and (2) lowered the sensitivity of the binding of [3H]oxotremorine-M to Gpp(NH)p by a factor of 120. The decrease in binding of [3H]oxotremorine-M binding was nonselective with regard to the sensitivity of the receptors to the guanine nucleotide, insofar as Gpp(NH)p inhibited the binding of [3H]oxotremorine-M to the same extent in the presence and absence of halothane. These results suggest that halothane (1) converts both G protein-coupled and -uncoupled muscarinic receptors to states of lower agonist affinity and (2) lowers the affinity of receptor-G protein complexes for guanine nucleotides.

High-affinity agonist binding to rat brainstem muscarinic receptors is eliminated by low pH.

The effects of hydrogen ion concentration on high-affinity agonist binding to muscarinic receptors were determined in rat brainstem membranes using [3H]oxotremorine-M as a probe. [3H]Oxotremorine-M bound with high affinity to a small subpopulation of brainstem muscarinic receptors (10% of the receptors labelled by [3H]methylscopolamine). [3H]Oxotremorine-M binding was constant between pH 7.0 and 9.0; the number of high-affinity sites decreased below pH 7.0 and at pH 5.0 no binding was detected. This decrease was irreversible; when brainstem membranes were incubated for 1 h at low pH and then returned to pH 8.0, agonist binding was not restored. In contrast, the total number of receptors, i.e. the number of [3H]antagonist binding sites, was not affected by prolonged incubation at low pH. Agonist affinity for the surviving [3H]oxotremorine binding sites and the sensitivity of agonist binding to guanine nucleotides were not altered in media of low pH (pH 5.5). These findings suggest that [3H]oxotremorine-M binds only to receptors which are functionally coupled to guanine nucleotide-dependent regulatory proteins and that this coupling is irreversibly inactivated in media of low pH.

Effect of pH on muscarinic acetylcholine receptors from rat brainstem.

The effect of hydrogen ion concentration on ligand binding to muscarinic acetylcholine receptors was studied in membranes isolated from rat brainstem. The binding of [3H]methylscopolamine was constant between pH 7 and 10. The affinity, but not the number, of [3H]methylscopolamine binding sites decreased below pH 7; at pH 4 little binding was detected. When brainstem membranes were incubated at various pH levels from 3 to 11 for 1 h and then returned to pH 8, [3H]methylscopolamine binding affinity was restored to control levels. Carbamylcholine binding affinity was also depressed in media of low pH. However, this decrease was permanent after a 1-h incubation at pH 4 (i.e. carbamylcholine affinity was not restored on raising the pH to 8). The capacity of a guanine nucleotide to affect carbamylcholine was also abolished by a 1-h incubation at pH 4, and was not restored by raising the pH. The guanine nucleotide-dependent regulatory protein may be irreversibly inactivated or dissociated from the receptor at low pH. The receptor's binding subunit, on the other hand, appears to be much less sensitive to hydrogen ion concentration.

Halothane effects on muscarinic acetylcholine receptor complexes in rat brain.

Muscarinic acetylcholine receptors in membranes from rat cerebral cortex or brainstem were equilibrated with halothane (0.5 to 5%). Halothane did not affect the number of [3H]methylscopolamine [( 3H]MS) binding sites. [3H]MS binding affinity, however, was increased in the presence of halothane (KD, air = 0.41 nM; KD, 2% halothane = 0.26 nM). This increase reflected a decrease in the dissociation rate constant (from 13 X 10(-3) min-1 to 6.5 X 10(-3) min-1) rather than a change in the bimolecular rate constant of association (1.8 and 1.9 X 10(7) M-1 min-1 in the absence and presence of 2% halothane respectively). Carbamylcholine affinity for brainstem or cortical muscarinic receptors was not affected by halothane. The ability of a guanine nucleotide to lower carbamylcholine affinity for brainstem receptors, however, was eliminated after equilibration with 2% halothane.

In vivo determinations of Ki values for angiotensin converting enzyme inhibitors.

We present two methods for calculating Ki values of angiotensin converting enzyme (ACE) inhibitors, such as captopril, in anesthetized or conscious rabbits. Both methods are based on indicator-dilution type determinations of single pass transpulmonary metabolism of the ACE substrate benzoyl-phe-ala-pro (BPAP). The first method involves two determinations of Michaelis-Menten constants Km and Amax (product of Vmax and lung capillary plasma volume) of endothelial-bound ACE for BPAP. Thirty seconds before the second determination of kinetic constants, the inhibitor is administered iv (e.g. captopril, 12 nmol/kg). Comparisons of the apparent Km and Amax values, obtained after the inhibitor to the control values obtained from the first determination, provide Ki values. With the second method, the ratio Amax/Km is obtained, under first-order reaction conditions, before and 30 sec after administration of inhibitor. These apparent and control ratios are used to calculate Ki values. In both methods, plasma levels of the inhibitor at the time of the determination of apparent kinetic constants are estimated by injecting radio-labelled inhibitor (e.g. 3H-captopril), analyzing radioactivity in arterial samples and correcting for plasma protein binding. These methods are potentially applicable to the clinical evaluation of new ACE inhibitors, in vivo, under normal or pathologic conditions.