Role of beta-adrenergic receptor subtypes in lipolysis.

In vitro lipolysis stimulated by low (-)-isoprenaline concentrations (< or =30 nM) in epididymal white adipocytes from Sprague-Dawley rats was inhibited at least 60-80% by the specific beta1-antagonists LK 204-545 and CGP 20712A (1 microM), suggesting that at these low (10 nM) concentrations of (-)-isoprenaline lipolysis was primarily (80%) but not solely mediated via beta1-adrenergic receptors. Low concentrations (100 nM) of (-)-noradrenaline and formoterol also confirmed a role for beta1-adrenergic receptors in mediating lipolysis at low concentrations of these agonists. At higher agonist concentrations, beta3-adrenergic receptors were fully activated and were the dominant beta-adrenergic receptor subtype mediating the maximum lipolytic response, and the maximum response was not affected by the beta1-antagonists, demonstrating that the beta3-receptor is capable of inducing maximum lipolysis on its own. Studies of lipolysis induced by the relatively beta2-selective agonist formoterol in the presence of beta1-blockade (1 microM CGP 20712A) demonstrated the inability of the beta2-selective antagonist ICI 118-551 to inhibit the residual lipolysis at concentrations of ICI 118-551 < or = 1 microM. Higher concentrations of ICI 118-551 inhibited the residual formoterol-induced lipolysis competetively, but with low affinity (approximately 500-fold lower than its beta2-adrenergic receptor pA2, 7.80 +/- 0.21), suggesting that formoterol was not acting via beta2-adrenergic receptors. These data are consistent with beta1-adrenergic receptors playing an important role in lipolysis at physiological but not pharmacological concentrations of catecholamines and that beta2-adrenergic receptors play no obvious direct role in mediating beta-adrenergic receptor agonist-induced lipolysis in vitro. Finally, racemic-SR 59230A, unlike the pure (S, S)-isomer (a beta3-selective antagonist), was found to be a nonselective antagonist at the three beta-adrenergic receptor subtypes, showing that the other enantiomers have different selectivity.

LK 204-545, a highly selective beta1-adrenoceptor antagonist at human beta-adrenoceptors.

LK 204-545 ((+/-)-1-(2-(3-(2-cyano-4-(2-cyclopropyl-methoxy-ethoxy)phenoxy)-2-hydro xy-propyl-amino)-ethyl)-3-(4-hydrxy-phenyl) urea), an antagonist that possesses high beta1-/beta2-selectivity in the rat, and a range of cardio-selective and non-selective beta-adrenoceptor antagonists were examined to compare their radioligand binding affinities for human beta1-, beta2- and beta3-adrenoceptors transfected into CHO cells. LK 204-545 and CGP 20712A displayed the highest beta1-/beta2- (approximately 1800 and approximately 650, respectively) and beta1-/beta3-selectivity (approximately 17000 and approximately 2200, respectively) at human beta-adrenoceptors with LK 204-545 being approximately 2.75-fold more beta1-/beta2-selective and approximately 8-fold beta1-/beta3-selective than CGP 20712A. The high potency of LK 204-545 at transfected human beta1-adrenoceptors and in functional models of rat beta1-adrenoceptors together with its high selectivity, identify it as a useful ligand for studying beta1-adrenoceptors and suggest that it may be the preferred ligand for human beta-adrenoceptor studies.

Pharmacology and subcellular distribution of [3H]rilmenidine binding sites in rat brain.

We have previously reported that in rat brain membranes, [3H]rilmenidine, in addition to labelling alpha2-adrenoceptors and the I2B-subtype of imidazoline receptor binding site (I2B-RBS), may label an additional I-RBS population, distinct from previously classified I1-RBS and I2-RBS. In this study, using crude or fractionated rat brain membranes we examined the possible association of [3H]rilmenidine-labelled I-RBS with the A- and B-isoforms of monoamine oxidase (MAO) by studying the inhibition of [3H]rilmenidine binding by a number of MAO inhibitors; and comparing the maximal binding density (Bmax) and subcellular distribution of [3H]rilmenidine binding sites with that of MAO-A and MAO-B catalytic sites labelled by [3H]RO41-1049 and [3H]RO19-6327 and 12-RBS labelled by [3H]2-BFI. Inhibition of [3H]rilmenidine binding by all MAO inhibitors tested produced very shallow curves (slope 0.29-0.56). Clorgyline and moclobemide (selective MAO-A inhibitors) displayed moderate affinities (60-140 nM), while pargyline (non-selective MAO-inhibitor), RO41-1049 (selective MAO-A inhibitor) and RO19-6327 (selective MAO-B inhibitor) exhibited very low affinities (> 2 microM) for 50-75% of [3H]rilmenidine-labelled I-RBS in crude brain membranes and even lower affinity for the remaining binding. Under identical buffer conditions, the Bmax of [3H]rilmenidine-labelled I-RBS (1.45+/-0.14 pmol/mg protein) was considerably lower than those of MAO-A (13.10+/-0.15 pmol/mg) and MAO-B (10.35+/-0.50 pmol/mg) sites. These results suggest that [3H]rilmenidine does not interact directly with the active catalytic site of either MAO enzyme and could at best only associate with a subpopulation of MAO molecules. Binding studies on five fractions of rat cortex homogenates-nuclear (N), heavy (M) and light (L) mitochondrial, microsomal non-mitochondrial (P), and soluble cytosolic (S) fractions-revealed that 45% of total [3H]rilmenidine binding was present in the P fraction cf. 20 and 23% in the M and L fractions, in contrast to [3H]RO19-6327 and [3H]2-BFI which bound 11-13% in the P fraction and 36-38% and 35-44% in the M and L fractions, respectively. Binding of all ligands in the N fraction was 6-15% of total. These studies reveal that [3H]rilmenidine-labelled I-RBS, unlike the I2-RBS, are not predominantly associated with mitochondrial fractions containing the MAO enzymes (and cytochrome oxidase activity), but appear to be distributed in both the mitochondrial and plasma membrane fractions in rat cerebral cortex.

[3H]Rilmenidine-labelled imidazoline-receptor binding sites co-localize with [3H]2-(benzofuranyl)-2-imidazoline-labelled imidazoline-receptor binding sites and monoamine oxidase-B in rabbit, but not rat, kidney.

The distribution and relative densities of imidazoline-receptor binding sites (I-RBS) and monoamine oxidase (MAO)-A and -B enzyme(s) in rat and rabbit kidney were compared autoradiographically using fixed nanomolar concentrations of [3H]rilmenidine and [3H]2-(benzofuranyl)-2-imidazoline ([3H]2-BFI) to label I-RBS, and [3H]RO41-1049 and [3H]RO19-6327 to label MAO-A and -B isoenzymes, respectively. In rat kidney, high densities of I-RBS labelled by [3H]rilmenidine were observed in the cortex and outer stripe (120-280 fmol/mg tissue), in contrast to low I-RBS densities labelled by [3H]2-BFI (<4 fmol/mg). A relatively high density of [3H]RO41-1049 binding to MAO-A enzyme was present in all regions of the rat kidney (160-210 fmol/mg) compared with a low density of [3H]RO19-6327 binding to MAO-B (< 25 fmol/mg). Comparison of MAO-A and -B distributions with that of [3H]rilmenidine-labelled I-RBS strongly suggests a lack of association in rat kidney. Similarly, the extremely low densities of [3H]2-BFI-labelled I2-RBS in rat kidney contrasts with the density of MAO-A, but is consistent with the low density of MAO-B. Rabbit kidney cortex and outer stripe contained high relative densities of [3H]rilmenidine-labelled I-RBS (200-215 fmol/mg) and [3H]2-BFI-labelled I2-RBS (45-60 fmol/mg) with lower densities in the inner stripe and inner medulla (< or = 100 and 30 fmol/mg respectively). A high density of MAO-A binding was observed in the inner stripe (515 fmol/mg) with lower levels in the cortex and outer stripe (100-240 fmol/mg), while high densities of MAO-B binding were observed in the cortex and outer stripe (290-450 fmol/mg) with lower levels in the inner stripe (65 fmol/mg). The correlation between the localization of [3H]rilmenidine-labelled I-RBS and [3H]RO19-6327-labelled MAO-B in rabbit kidney (r = 0.87, P = 0.057) suggest that [3H]rilmenidine may label a binding site co-existent with MAO-B, but not MAO-A (n.s.), in this tissue, but rilmenidine did not inhibit [3H]RO41-1049 or [3H]RO19-6327 binding. The distribution of [3H]2-BFI-labelled I2-RBS overlapped the combined distributions of both MAO-A and -B isoenzymes, suggesting that [3H]2-BFI may label sites on both enzymes in the rabbit, but [3H]2-BFI binding only correlated with [3H]RO19-6327 (r = 0.84, P = 0.07), not [3H]RO41-1049 binding (n.s.). Moreover, 2-BFI only inhibited [3H]RO19-6327, not [3H]RO41-1049 binding. These data are consistent with reports that I2-RBS are located on MAO-B and allosterically influence the catalytic site. The relationship of [3H]rilmenidine- and [3H]2-BFI-labelled I-RBS and the identity of non-MAO-associated [3H]rilmenidine-labelled I-RBS requires further investigation.

The effect of levcromakalim (BRL 38227) on bladder function in patients with high spinal cord lesions.

The effects of the potassium channel opener levcromakalim (BRL 38227) 7.5 micrograms kg-1 were examined on urodynamic variables and blood pressure during inflow and voiding cystometry in six high spinal cord lesion patients. Levcromakalim administration significantly increased the duration of bladder contraction (197 +/- 128 s to 267 +/- 167 s, P < 0.05) and also reduced blood pressure (126 +/- 13/67 +/- 9 mm Hg to 104 +/- 25/52 +/- 12 mm Hg) but was without effect on other urodynamic parameters. Because of concerns about hypotensive responses, further studies involving higher doses of levcromakalim should be considered only if the drug was administered intravesically.

A simple and reliable method for construction of parallel multibarrel microelectrodes.

A modification to the method of construction of parallel or "piggy-back" electrodes for extracellular single-unit recording combined with iontophoresis is described that facilitates the alignment of the two components of the array. The method involves the use of an orthogonal viewing device (a pair of mirrors mounted symmetrically at 45 degrees to the horizontal), which produces a pair of virtual images of the electrode components that can be viewed with a microscope. A slight displacement between the electrodes is easily detected as an uneven separation between the electrode images. The positions of the electrode components are adjusted until there is no visible separation of the electrodes or their virtual images.

Validation of a solid-phase extraction high-performance liquid chromatographic assay for doxazosin.

The determination of doxazosin by high-performance liquid chromatography with fluorescence detection is described. Propanolol was used as the internal standard. Plasma samples were treated with methanol to precipitate the proteins. Doxazosin was isolated with C18 reversed-phase extraction columns. The determination limit is 1 ng/ml of plasma, while the extraction columns can be reused frequently. The method is applied to clinical trial samples.

Diurnal blood pressure variation in quadriplegic chronic spinal cord injury patients.

1. Measurement of blood pressure and heart rate over a 24 h period was performed in 10 quadriplegic spinal cord injury patients and 10 immobilized, neurologically intact orthopaedic subjects by using the Spacelabs 90207 automated ambulatory monitoring system. 2. Systolic and diastolic blood pressure fell significantly at night in orthopaedic subjects but not in quadriplegic patients, and night-time blood pressures were similar in both groups. 3. Cumulative summation of differences from a reference value (cusum analysis) confirmed a markedly diminished diurnal blood pressure variation in the quadriplegic patients. 4. These findings could not be accounted for on the basis of blood pressure variations during chronic postural change. 5. Heart rate fell significantly at night in both groups. 6. The findings suggest that the increase in blood pressure during waking hours in neurologically intact subjects is a consequence of a diurnal variation in sympathetic activity (absent in quadriplegic patients with sympathetic decentralization) which is independent of changes in physical activity.

Differential forskolin activation of rat heart and lung adenylate cyclase. Dependence on membrane-protein interactions.

We have investigated whether the greater ability of forskolin to activate adenylate cyclase (EC 4.6.1.1) from rat heart compared with rat lung is due to interactions between G-proteins and catalytic units, isoforms of catalytic units or membrane-protein interactions. Interactions between Gs and catalytic units were found to be similar in both tissues with 10 microM Gpp(NH)p increasing activity up to 5-fold. While MnCl2 increased the response of the lung enzyme to forskolin, it reduced the response of the cardiac enzyme and uncoupled Gs from the cardiac catalytic units indicating that Gs interactions potentiate the response to forskolin. After enzyme solubilisation with n-octyl-beta-D-glucopyranoside, the response to forskolin was identical in heart and lung whether assayed with magnesium or manganese chloride, and not significantly different from the heart membrane enzyme. Overall, the results show that the relatively poor response of lung adenylate cyclase to forskolin is due to specific inhibitory interactions between the enzyme and lung membrane constituents.

Forskolin-mediated activation of cardiac, liver and lung adenylate cyclase in the rat. Relation to [3H]forskolin binding sites.

We investigated whether differences in binding sites for [3H]forskolin could account for the low potency of forskolin on adenylate cyclase (EC 4.6.1.1) from rat lung compared with heart or liver adenylate cyclase. Forskolin (0.1 mM) increased basal adenylate cyclase activity 41-fold in heart, 27-fold in liver, but only 3-fold in lung. The low potency in lung could not be accounted for by any lack of enzyme or stimulatory nucleotide-binding protein, since sodium fluoride (10 mM) increased basal activity 9-12-fold in all three tissues. The effectiveness of forskolin on adenylate cyclase appears to be related to the presence of specific [3H]forskolin binding sites. [3H]Forskolin binding in both heart and liver membranes was consistent with single binding sites with dissociation constants of 0.74 +/- 0.25 microM and 1.43 +/- 0.21 microM respectively. No such binding sites were detected in lung membranes. The binding was of low affinity (greater than 100 microM) and showed no tendency to saturate. These results are not consistent with the hypothesis that the nucleotide-binding protein influences stimulation of adenylate cyclase by forskolin, rather [3H]forskolin binding sites appear to be an important determinant of the effect of forskolin in different tissues.

Role of auto-inhibitory feed-back in cardiac sympathetic transmission assessed by simultaneous measurements of changes in 3H-efflux and atrial rate in guinea-pig atrium.

Guinea-pig right atria were labelled with [3H]-noradrenaline or [3H]-dopamine before superfusion in a flow-cell. Choice of label did not significantly alter either the relationship between 3H-efflux and number of electrical field pulses or the inhomogeneity of labelling. The relationship between 3H-efflux and frequency of 4 field pulses (0.125-2 Hz) was hyperbolic and similar to the tachycardia-frequency relationship measured simultaneously. No evidence was found for a U shaped 3H-efflux-frequency relationship (Story, McCulloch, Rand & Standford-Starr, 1981). Phentolamine (1 microM) did not alter the 3H-efflux or atrial rate responses to 4 field pulses at stimulus levels that gave 50-60% of the maximum rate response. In the presence of neuronal uptake inhibition (desipramine, DMI 0.1 microM), rate and 3H-efflux responses to 4 field pulses were enhanced at all frequencies and were further increased by phentolamine. In the absence of DMI, prolonged trains of field pulses (8 and 12 pulses) at low frequency (0.25 Hz) were not sufficient to activate auto-inhibitory feed-back. At 2 Hz phentolamine enhanced both 3H-efflux and rate responses at 12 field pulses. We conclude that in guinea-pig right atrium auto-inhibitory feed-back plays little role in the modulation of transmitter release at levels of stimulation that cause 50-60% of maximum tissue response. This is because neuronal uptake normally prevents synaptic concentrations of noradrenaline from activating prejunctional alpha 2-adrenoceptors. Stimulation sufficient to induce a near-maximal response or the presence of neuronal uptake inhibition are necessary to evoke autoinhibitory feed-back.

Role of autoinhibitory feedback in cardiac sympathetic transmission.

The relationship between two indices of transmitter release measured simultaneously and the frequency of 4 field pulses (0.125-2 Hz) were obtained from superfused guinea pig right atria after labelling with 3H-noradrenaline. The relationships between 3H-efflux or rate responses and frequency were hyperbolic. Autoinhibitory feedback did not play a role since phentolamine (1 microM) did not alter the 3H-efflux or rate responses to 4 field pulses that gave 50-60% of the maximum rate response. In the presence of neuronal uptake block (desipramine (0.1 microM) phentolamine enhanced 3H-efflux and rate responses to 4 field pulses at all frequencies. In the absence of desipramine prolonged trains of field pulses (8-12 pulses) at low frequency (0.25 Hz) were not sufficient to activate autoinhibitory feedback. At 2 Hz phentolamine enhanced both responses at 12 field pulses. We conclude that in the right atrium autoinhibitory feedback plays little role in the modulation of transmitter release at levels of stimulation that cause 50-60% of maximum tissue response. The presence of neuronal uptake inhibition or high stimulus strengths are necessary to evoke autoinhibitory feedback.

A simple method for the assay of urinary metanephrines using high performance liquid chromatography with fluorescence detection.

A method is described which extends a high performance liquid chromatographic assay for urinary catecholamines to the assay of urinary metanephrines. The amines are separated from urine after acid hydrolysis of conjugates by ion-exchange chromatography, and then further purified by solvent extraction. The final extracts are suitable for direct HPLC assay using the endogenous fluorescence of the amines for detection. Twenty-four hour excretion of the amines in 35 hospital in-patients was found to be (mean and range: 169 micrograms/24 h normetanephrine (39-423), 102 micrograms/24 h metanephrine (19-290) and 138 micrograms/24 h 3-methoxytyramine (32-234)).

No stereoselective first-pass hepatic extraction of propranolol.

The plasma level: time profile for l-propranolol and total propranolol concentrations were examined in normotensive subjects after intravenous and oral dl-propranolol. l-Propranolol concentrations in plasma accounted for about 60% of total propranolol. This was attributed to lower volume of distribution for the isomer. Mean plasma clearance of 1-propranolol was similarly affected while apparent plasma half-life for the l-isomer and total propranolol were of the same order. Oral bioavailability of 1- and total propranolol averaged 40.7 +/- 8.5% and 42.4 +/- 12.9%. Food and hydralazine increased oral bioavailability of total and l-propranolol by similar magnitudes. We conclude that difference in the kinetics of l- and total propranolol concentrations in plasma are small and probably of no clinical significance. Presystemic clearance of propranolol in man does not appear to be stereospecific.