Widely distributable and retainable in-situ gelling material for treating myocardial infarction.

Intramyocardial hydrogel injection is a promising therapy to prevent negative remodeling following myocardial infarction (MI). In this study, we report a mechanism for in-situ gel formation without external stimulation, resulting in an injectable and tissue-retainable hydrogel for MI treatment, and investigate its therapeutic outcomes. A liquid-like polymeric solution comprising poly(3-acrylamidophenylboronic acid-co-acrylamide) (BAAm), polyvinyl alcohol (PVA), and sorbitol (S) increases the viscous modulus by reducing the pre-added sorbitol concentration is developed. This solution achieves a sol-gel transition in-vitro in heart tissue by spontaneously diffusing the sorbitol. After intramyocardial injection, the BAAm/PVA/S with lower initial viscous modulus widely spreads in the myocardium and gelate compared to a viscoelastic alginate (ALG) hydrogel and is retained longer than the BAAm/S solution. Serial echocardiogram analyses prove that injecting the BAAm/PVA/S into the hearts of subacute MI rats significantly increases the fraction shortening and ejection shortening and attenuates the expansion of systolic LV diameter for up to 21 d after injection compared to the saline injection as a control, but the ALG injection does not. In addition, histological evaluation shows that only the BAAm/PVA/S decreases the infarct size and increases the wall thickness 21 d after injection. The BAAm/PVA/S intramyocardial injection is better at restraining systolic ventricular dilatation and cardiac failure in the rat MI model than in the control groups. Our findings highlight an effective injectable hydrogel therapy for MI by optimizing injectability-dependent distribution and retention of injected material. STATEMENT OF SIGNIFICANCE: In-situ gelling material is a promising strategy for intramyocardial hydrogel injection therapy for myocardial infarction (MI). Since the sol-gel transition of reported materials is driven by external stimulation such as temperature, pH, or ultraviolet, their application in vivo remains challenging. In this study, we first reported a synthetic in-situ gelling material (BAAm/PVA/S) whose gelation is stimulated by spontaneously reducing pre-added sorbitol after contacting the heart tissue. The BAAm/PVA/S solution spreads evenly, and is retained for at least 21 d in the heart tissue. Our study demonstrated that intramyocardial injection of the BAAm/PVA/S with more extensive distribution and longer retention had better effects on preventing LV dilation and improving cardiac function after MI than that of viscoelastic ALG and saline solution. We expect that these findings provide fundamental information for the optimum design of injectable biomaterials for treating MI.

Modulation of ß-adrenergic receptor in rat lung after gamma irradiation.

OBJECTIVES: To study the effect of γ irradiation on ß-adrenergic receptors of the lung. MATERIALS AND METHODS: Healthy Sprague-Dawley rats were used as an animal model. Cell membrane proteins of lung tissue were harvested after the whole lung received 20 Gy of 60Co γ irradiation. 125I-labeled iodopindolol (125I-IPIN) was used as a ligand of ß-adrenergic receptors. The numbers of the ß-adrenergic receptors were determined by radioligand-receptor binding assay (RBA). Data were compared with irreversible blockage using antagonist bromoacetylalprenololmenthan (BAAM). RESULTS: The post-radiation RBA assay showed that the number of ß-adrenergic receptors in lung tissue decreased at a steady rate. It decreased to 48% of the normal level at the 15th day after irradiation. At 40 days after radiation the level of ß-adrenergic receptors started to increase at a steady rate and reached to the normal level around 70 days after radiation. There were significant differences in receptor synthesis, degradation and regeneration rates between irradiation group and BAMM group. CONCLUSIONS: The whole lung irradiation could severely affect the levels of ß-adrenergic receptors. The potential clinical implications of radiation-induced changes of ß-adrenergic receptors warrant further investigation.

Synthesis and characterization of high-affinity 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-labeled fluorescent ligands for human ß-adrenoceptors.

The growing practice of exploiting noninvasive fluorescence-based techniques to study G protein-coupled receptor pharmacology at the single cell and single molecule level demands the availability of high-quality fluorescent ligands. To this end, this study evaluated a new series of red-emitting ligands for the human ß-adrenoceptor family. Upon the basis of the orthosteric ligands propranolol, alprenolol, and pindolol, the synthesized linker-modified congeners were coupled to the commercially available fluorophore BODIPY 630/650-X. This yielded high-affinity ß-adrenoceptor fluorescent ligands for both the propranolol and alprenolol derivatives; however, the pindolol-based products displayed lower affinity. A fluorescent diethylene glycol linked propranolol derivative (18a) had the highest affinity (log K(D) of -9.53 and -8.46 as an antagonist of functional ß2- and ß1-mediated responses, respectively). Imaging studies with this compound further confirmed that it can be employed to selectively label the human ß2-adrenoceptor in single living cells, with receptor-associated binding prevented by preincubation with the nonfluorescent ß2-selective antagonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118551) ( J. Cardiovasc. Pharmacol.1983, 5, 430-437. ).

Responsiveness, affinity constants and beta-adrenoceptor reserves for isoprenaline on aortae from normo-, pre- and hypertensive rats.

The objective of this study was to determine the responsiveness, affinity constants and beta-adrenoceptor reserves for isoprenaline on the isolated aorta in the maturation of normotensive and hypertensive rats. The effects of a very slowly reversible antagonist, bromoacetylal-prenololmenthane (BAAM), on the relaxant responses of the aortae of 5- and 14-week-old Wistar Kyoto normotensive rats (WKY) and spontaneously hypertensive rats (SHRs) to isoprenaline were determined. Five-week-old SHRs are pre-hypertensive and the aortic rings are less responsive to isoprenalinethan age-matched WKY (pD2 values: WKY, 8.40; SHRs, 8.03). Similar relaxant responses to forskol in were obtained on the aortae of 5- and 14-week-old WKY and SHRs. The K(A) value for isoprenaline at the aortic beta2-adrenoceptors of the 5-week-old WKY was 2.1 x 10(-7) M, and similar values were obtained on the aortae of 5-week-old SHR and 14-week-old WKY and SHRs. In the maturation of the WKY aortae from 5 to 14 weeks, there was a reduction in the maximum response, a major loss of sensitivity and a loss of beta2-adrenoceptor reserve for isoprenaline. On 5-week-old SHR aorta, the sensitivity to isoprenaline was 2.5-fold lower, and the beta2-adrenoceptor reserve was less than on age-matched WKY. In the development of hypertension on the SHR aorta from 5 to 14 weeks, there was a reduction in the maximum response to isoprenaline. At 14 weeks, the sensitivity and the beta-adrenoceptor reserve to isoprenaline were similar, but the maximum responses were lower on the SHR than WKY. As there are differences in pre-hypertensive SHR and age-matched WKY aortic responses to isoprenaline, it is no longer valid to consider that the loss of responsiveness to isoprenaline in hypertension is solely owing to the hypertension. There are no changes in affinity, but major changes in the sensitivity, maximum responses and aortic beta2-adrenoceptor reserves to isoprenaline in the maturation of normotensive and pre-hypertensive aortae.

Influence of receptor reserve on beta-adrenoceptor-mediated responses in human lung mast cells.

1. The effects of the beta-adrenoceptor agonists isoprenaline and salbutamol on IgE-mediated histamine release from human lung mast cells (HLMC) were evaluated. Both agonists (10(-10)-10(-5) M) inhibited histamine release in a dose-dependent manner and isoprenaline (pD2, 8.3+/-0.1, mean+/-s.e.mean) was more potent than salbutamol (7.3+/-0.1). Moreover, the mean data indicated that salbutamol was a partial agonist when compared with isoprenaline. However, there was a large degree of interexperimental variability because, in 11 of 32 experiments, salbutamol was a full agonist and, in 21 of 32 experiments, a partial agonist relative to isoprenaline. These data suggest that different HLMC preparations possess variable receptor reserves. 2. The effect of the irreversible beta-adrenoceptor antagonist, bromoacetylalprenolol menthane (BAAM), on the inhibition of IgE-mediated histamine release by both isoprenaline and prostaglandin E2 (PGE2) was assessed. Whereas BAAM (100 nM) antagonized the isoprenaline inhibition of histamine release from activated HLMC, BAAM had no effect on the PGE2 inhibition. Pretreatment of HLMC with the beta2-selective competitive antagonist, ICI 118551 (100 nM), protected against the loss in responsiveness to isoprenaline following treatment with BAAM. 3. Concentrations of 1, 10 and 100 nM of BAAM caused dose-dependent rightward shifts in the dose-response curve for the isoprenaline inhibition of histamine release. Furthermore, there was a dose-dependent reduction in the maximal inhibitory response obtained with isoprenaline following treatments with increasing concentrations of BAAM. Although the rightward shifts in the isoprenaline dose-response curves, with a given concentration of BAAM, were similar in all experiments, there was some variability in the depression of the maximal response in individual experiments. Thus, in 6 of 16 experiments, BAAM (1 nM) did not depress the maximal response to isoprenaline, whereas in 10 of 16 experiments there was a depression (7 to 49% reduction) in the maximal response. These data suggest that different HLMC preparations possess variable receptor reserves. 4. Isoprenaline was more potent as an inhibitor in those HLMC preparations in which there was a larger receptor reserve (i.e. preparations in which the maximal inhibitory response to isoprenaline was unaffected by pretreatment with 1 nM BAAM). 5. The influence of receptor reserve on the inhibition by salbutamol of histamine release from HLMC was evaluated. There was a good correlation (r=0.77) between receptor reserve and the maximal response (relative to isoprenaline) obtained with salbutamol. Thus, HLMC preparations with larger receptor reserves were more responsive to salbutamol. 6. Receptor reserve influenced the desensitization of beta-adrenoceptor-mediated responses in HLMC. Cells were incubated (24 h) with isoprenaline (1 microM), washed and then the ability of a second isoprenaline (10(-10)-10(-5) M) exposure to inhibit histamine release was assessed. The pretreatment caused a reduction in the isoprenaline inhibition of histamine release although the extent of desensitization was highly variable, ranging from essentially negligible levels in some preparations to substantial reductions (93% desensitization) in the ability of isoprenaline to inhibit histamine release. There was a reasonable correlation (r=0.59) between receptor reserve and desensitization. Preparations that possessed a larger receptor reserve were more resistant to desensitization. 7. Collectively, these data suggest that a receptor reserve exists for the beta-adrenoceptor-mediated inhibition of histamine release from HLMC but that the size of this reserve varies between HLMC preparations. Moreover, the size of this receptor reserve may influence the sensitivity of HLMC to beta-adrenoceptor agonists and the susceptibility of individual HLMC preparations to desensitization.

Affinity constants and beta-adrenoceptor reserves for isoprenaline on cardiac tissue from normotensive and hypertensive rats.

To determine whether there are differences in cardiac beta-adrenoceptor responsiveness, isoprenaline affinity constants and fractional beta-adrenoceptor occupancy-response relationships for isoprenaline in the early stages of established hypertension, we studied the effects of bromoacetylalprenololmenthane (BAAM) and ([3,5-diamino-6-chloro-N-(1[N-beta-(2-hydroxyl-3-alpha-naphthoxypropy lamino)ethylcarbamoyl]-1-methylethyl)-pyrazine-2-carboxamide (ICI 147 798), slowly reversible beta-adrenoceptor antagonists, on the isoprenaline responses of the left ventricular papillary muscle and the left and right atria of 6-month-old Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). The papillary muscles, but not the right and left atria, of the SHR were less responsive to isoprenaline than those of the WKY. The isoprenaline pD2 values (the negative logarithms of the molar concentrations of agonist producing 50% of the maximum response) were 7.72 and 8.00 on the SHR and WKY papillary muscles, respectively. On the WKY papillary muscle the isoprenaline KA values were 2-3 x 10(-6) M, which is as expected for isoprenaline at beta1 or beta2-adrenoceptors. Isoprenaline had 100-fold greater affinity on the WKY and SHR left atria than on the papillary muscles; the isoprenaline KA values were 2-4 x 10(-8) M. On the WKY papillary muscle and left atrium, isoprenaline had to occupy 3-4% of the beta-adrenoceptors to produce a 50% maximum response; on the WKY papillary muscle and left atrium isoprenaline had to occupy 25-35% and 55%, respectively, of the beta-adrenoceptors to produce a 90% maximum response. The SHR papillary muscles and left atrium had smaller beta-adrenoceptor reserves for isoprenaline than did the WKY tissues. We were unable to obtain isoprenaline KA values on the WKY right atrium. The isoprenaline KA value on the SHR right atrium was 1-4 x 10(-8) M. Because the isoprenaline KA values for the left and right atria are markedly different from those previously reported for isoprenaline at beta1 or beta2-adrenoceptors, we suggest that atypical beta-adrenoceptors might be present on the atria of WKY and SHR. We have also demonstrated a lower beta-adrenoceptor reserve on SHR papillary muscle and atria in the early stages of established hypertension.

The effects of bromoacetylalprenololmenthane on the responses to isoprenaline of the left ventricle from normo-, prehyper- and hypertensive rats.

1. We have studied the effects of bromoacetylalprenololmenthane (BAAM), a very slowly reversible beta-adrenoceptor antagonist, on the responses of the left ventricle of 5 and 22 week old Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) to isoprenaline. At 5 weeks the SHRs were prehypertensive and at 22 weeks they had hypertension-induced hypertrophy of the left ventricle. 2. The mean potency of isoprenaline on the left ventricle from 5 week old WKY was similar to that obtained on left ventricle with BAAM at 10(-6) M for 30 min, there was a parallel rightward shift of the isoprenaline concentration-response curves. Treatment with a higher concentration of BAAM (10(-5)M) caused non-parallel rightward shifts of the concentration-response curves, with a depression of the isoprenaline maximum responses. These data were used to derive affinity (KA) values for isoprenaline. The mean isoprenaline KA value on the left ventricle from 5 week old WKY was 2.44 x 10(-6)M, and similar KA values were obtained on the left ventricles from 22 week old WKY and 5 and 22 week old SHRs. On all the left ventricles tested, isoprenaline produced a half maximal response by occupying less than 1%, and a near maximal response by occupying about 5% of the available beta(1)-adrenoceptors. 4. This study has shown that there are no differences in the cardiac responses to isoprenaline at beta(1)-adrenoceptors, isoprenaline KA values or the beta(1)-adrenoceptor reserve for isoprenaline on the SHR left ventricle in prehypertension or in the early stages of hypertension-induced hypertrophy.

Relationship between desensitization and downregulation of beta-adrenoceptors in cardiac tissues after prolonged in vivo infusion of T-0509, a beta 1-adrenoceptor agonist.

To examine the contribution of beta-adrenoceptor (beta AR) downregulation to desensitization of beta ARs by chronic administration of a beta AR agonist, we compared the adenylyl cyclase (AC) activities in two kinds of cardiac ventricular membranes with decreased available beta ARs: one was derived from rats infused with a selective beta 1 AR agonist, T-0509 [(-)-(R)-1-(3,4-dihydroxyphenyl)- 2-[(3,4-dimethoxyphenethyl)-amino]ethanol hydrochloride], in vivo (40 micrograms/kg/hr, s.c. for 6 days); and the other was obtained from treatment of control membranes with an irreversible beta AR antagonist, bromoacetyl alprenolol methane (BAAM). T-0509 infusion decreased the densities of beta 1 ARs and beta 2 ARs by 26% and 32%, respectively, and reduced the maximal isoproterenol-stimulated AC activity by 53%. The amount of Gs alpha and Gi alpha proteins in the membranes was not significantly changed by T-0509 infusion. To make preparations that mimic the T-0509-induced downregulation, we treated the control membranes with 100 nM BAAM in vitro. The BAAM treatment decreased the Bmax value of [125I]iodocyanopindolol for beta 1 ARs and beta 2 ARs by 29% and 36%, respectively, whereas it reduced the maximal effect of isoproterenol on AC activity only by 37%. These results suggest that downregulation of beta ARs cannot fully account for the desensitization by chronic treatment of T-0509 and that other mechanism(s) can play a significant role in the loss of responsiveness.

Loss of maximum attenuation and receptor reserve for isoprenaline at the beta 2-adrenoceptors of the portal veins of hypertensive rats.

OBJECTIVE: To test the hypothesis that vascular beta 2-adrenoceptor hyporesponsiveness in spontaneously hypertensive rats (SHR) is not induced by increased blood pressure or venous hypertrophy. DESIGN: We compared the attenuating effects or isoprenaline, sodium nitroprusside and verapamilon the portal veins from Wistar-Kyoto (WKY) rats and SHR. studied the effects of slowly reversible beta-adrenoceptor antagonists, bromoacetylalprenololmenthane (BAAM) and ICI 147798, on the isoprenaline responses in order to determine the affinity and fractional beta 2-adrenoceptor occupancy-response relationships for isoprenaline. RESULTS: The SHR portal veins did not develop hypertrophy. There was a small reduction in the sensitivity to isoprenaline and a marked reduction in the maximum attenuation of hypertension caused by isoprenaline. The sensitivity and efficacy of sodium nitroprusside and verapamil were not altered by hypertension. BAAM and ICI 147798 inhibited the isoprenaline responses and reduced the maximum attenuation to isoprenaline. In the WKY rat portal vein the dissociation constant (KA) values for isoprenaline were independent of BAAM concentration, and was 1.78 +/- 0.32 x 10(-7) mol/l. Similar isoprenaline KA values were obtained from the ICI 147798 data and in the SHR portal vein. In the WKY rat portal vein, from the BAAM data, it was calculated that isoprenaline produced 50, 95 and 100% maximum responses by occupying 6 +/- 1, 20 +/- 3 and 43 +/- 5% (n = 21) of the available beta 2-adrenoceptors, respectively. Similar occupancy-response relationships were obtained in the WKY rat portal vein from the ICI 147798 data. At each level of isoprenaline response the receptor reserve was significantly smaller in the SHR than it was in the WKY rat portal vein. Thus, from the BAAM data, isoprenaline produced 50, 95 and 100% maximum responses by occupying 14 +/- 3, 33 +/- 6 and 58 +/- 7% (n = 15) of the available SHR portal vein beta 2-adrenoceptors, respectively. CONCLUSIONS: The SHR portal vein displays a selective beta 2-adrenoceptor hyporesponsiveness in the absence of a raised blood pressure or hypertrophy. This beta 2-adrenoceptor-associated hyporesponsiveness consisted of a marked loss of maximum attenuation in response to isoprenaline and of beta 2-adrenoceptor reserve for isoprenaline responses.

Analysis of the role of receptor number in defining the intrinsic activity and potency of partial agonists in neuroblastoma x glioma hybrid NG108-15 cells transfected to express differing levels of the human beta 2-adrenoceptor.

Many agonist ligands are known experimentally to display a range of efficacies and potencies in different tissues and preparations. To analyze the role of the levels of receptor expression and availability in the intrinsic activities and potencies of agonists, the function of a number of beta-adrenoceptor ligands was examined in clones of neuroblastoma x glioma hybrid NG108-15 cells transfected to express differing levels of the human beta 2-adrenoceptor, as well as after treatment of these cell lines with the irreversible beta-adrenoceptor antagonist bromoacetyl alprenolol menthane (BAAM). Clone beta N22 expressed approximately 10-fold higher levels of the receptor than did clone beta N17. In measurements of agonist stimulation of adenylyl cyclase activity in membranes of these cells or agonist stimulation of the formation of the complex of Gs alpha and adenylyl cyclase, which acts as the high affinity binding site for [3H]forskolin in whole cells, a series of beta-adrenoceptor agonists, including dichloroisoprenaline, ephedrine, dobutamine, and salbutamol, displayed higher intrinsic activity and showed concentration-response curves that were substantially to the left (lower EC50 values) in clone beta N22, compared with clone beta N17. Treatment of clone beta N22 cells with varying concentrations of BAAM reduced the intrinsic activity of these ligands and shifted the concentration-response curves for these agents to the right. In clone beta N22 cells and membranes, reduction in the observed intrinsic activity for ephedrine required elimination of a smaller fraction of the beta 2-adrenoceptor reserve than for salbutamol and reduction in the effect of the full agonists isoprenaline and epinephrine was noted only with high fractional elimination of the receptor pool. The effect of isoprenaline was substantially reduced, however, by BAAM treatment of clone beta N17 cells, where the beta 2-adrenoceptor number approached extremely low levels. Analysis of the data using the formalisms of Whaley et al. [Mol. Pharmacol. 45:481-489 (1994)] showed that prediction of alterations in agonist potency with receptor number for full agonists can be adequately extended to partial agonists.

Ocular delivery of the beta-adrenergic antagonist alprenolol by sequential bioactivation of its methoxime analogue.

Ocular delivery of alprenolol, a beta-adrenergic antagonist, by site-specific bioactivation of its methoxime analogue results in significant and prolonged decrease of the intraocular pressure in rabbits after topical administration. Alprenolone methoxime is stable in isotonic phosphate vehicle but undergoes enzymatic hydrolysis to the corresponding ketone in the eye. The ketone is then converted to alprenolol by a carbonyl reductase present in the iris-ciliary body. The benefit of this chemical delivery system approach includes the facile release of a potential antiglaucoma agent only at the site of the action; thus, unwanted systemic effects of the drug can be avoided.

Minimal cardiac electrophysiological activity of alprenoxime, a site-activated ocular beta-blocker, in dogs.

A closed chest catheter technique was used in dogs to examine the potential cardiac effects of alprenoxime, a potent new ocular antihypertensive agent. Alprenoxime was designed to undergo metabolic activation to the beta-blocker, alprenolol, specifically within the eye using hydrolase and reductase enzymes that reside in the iris-ciliary body. Previous studies in rabbits confirmed that intraocular pressure (IOP) significantly decreased after topically instilling ophthalmic drops of alprenoxime, while heart rates remained essentially unchanged after intravenous dosing. To further explore the safety and ocular specificity of this potential antiglaucoma drug, several cardiac electrophysiologic parameters were monitored during alprenoxime infusion in anesthetized dogs. In contrast to the pharmacologically significant increases (33-144%) measured after alprenolol or other previously tested beta antagonist infusion, the identical dose of alprenoxime had no effect on sinus cycle length (SCL), conduction times through the bundle of His and atrium (H and AH), or any other monitored cardiac electrophysiologic parameter. No changes greater than 6% from baseline were detected with alprenoxime infusion. Similarly, no beta-antagonist cardiac activity could be detected in isoproterenol stimulated dogs after alprenoxime. The results demonstrate that alprenoxime has no significant cardiac activity at doses much greater than potential therapeutic levels. The study provides further support that the new agent could be safely used in treating glaucoma.

Receptor availability defines the extent of agonist-mediated G-protein down-regulation in neuroblastoma x glioma hybrid cells transfected to express the beta 2-adrenoceptor.

Sustained exposure of neuroblastoma x glioma hybrid, NG108-15, cells transfected to express the human beta 2-adrenoceptor (clone beta N22) to isoprenaline or iloprost (an agonist at the endogenously expressed IP prostanoid receptor) resulted in a substantial and selective down-regulation of the alpha subunit of the G-protein Gs. Treatment of these cells with the irreversible beta-adrenoceptor antagonist bromoacetyl alprenolol menthane diminished both the potency and the maximal ability of isoprenaline but not of iloprost to cause Gs alpha down-regulation. These results demonstrate that the extent of agonist-mediated Gs alpha down-regulation is dependent upon the availability of receptor to agonist.

Alprenolol and bromoacetylalprenololmenthane are competitive slowly reversible antagonists at the beta 1-adrenoceptors of rat left atria.

We studied the effects of alprenolol and bromoacetylalprenololmenthane (BAAM) on rat left atria. Alprenolol and BAAM at 10(-7), 3 x 10(-7), and 10(-6) M inhibited the cardiac stimulation response slightly, which is indicative of membrane-stabilizing activity independent of beta-adrenoceptor blockade. This membrane-stabilizing activity was readily reversible. Alprenolol and BAAM also caused surmountable antagonism of isoprenaline responses, and this beta 1-adrenoceptor antagonism was slowly reversible. Inhibition of the isoprenaline responses with alprenolol and BAAM at 10(-6) M was at equilibrium after 60 min, which is indicative of reversible antagonism. We conclude that alprenolol and BAAM are competitive slowly reversible beta 1-adrenoceptor antagonists on rat left atria.

Chronic ethanol treatment of rats and the myocardial beta-adrenoceptors.

We examined the effect of chronic treatment with ethanol on the dynamics of beta-adrenoceptor binding in left ventricular myocardium of rats. After treatment with BAAM (20 mg/kg i.p.), an irreversible inhibitor of beta-adrenoceptors, the inhibition of beta-adrenoceptor binding was less, and the recovery of receptor binding was faster in chronically ethanol-treated rats compared to the control animals given equicaloric dextrin maltose treatment. When intracellular beta-adrenoceptor recycling was inhibited with colchicine, cytoplasmic left ventricular beta-adrenoceptor binding was greater in ethanol-treated compared to dextrin maltose-treated animals. We conclude that the previously reported decreased functional activity of the beta-adrenoceptor-mediated system probably reflects the contribution of ethanol-mediated effects not entirely restricted to the receptor-binding mechanisms.

Partial agonistic activity of two irreversible beta-adrenergic receptor ligands, bromoacetylated derivatives of alprenolol and pindolol.

Our data demonstrate that the irreversible beta-adrenergic receptor probes BAAM and BIM are partial agonists. They should be used with caution until the impact of this finding on estimates of agonist affinity, non-linear receptor-effector coupling and receptor metabolism can be more precisely defined.

Improved delivery through biological membranes. LVI. Pharmacological evaluation of alprenoxime--a new potential antiglaucoma agent.

A new site-specific chemical delivery system (CDS) for alprenolol was designed and investigated as a potential novel antiglaucoma agent. The effect of this compound, alprenoxime (AO), on the intraocular pressure (IOP) of rabbits was evaluated after its uni- and bilateral administration. AO produced significant reduction of the IOP starting at 30 min and lasting for more than 6 hr after its topical administration. Both in rats and in rabbits the i.v. bolus injection of AO (6 mg/kg) led to insignificant transient bradycardia, while no activity was found after oral or topical administration. Alprenolol (ALP) in a similar dose produced a sustained and significant bradycardia for more than 30 min. When the beta-adrenergic blocking activity was assessed against isoprenaline-tachycardia, the same results were obtained, i.e., AO led to a transient brief activity, whereas ALP produced a significant long-lasting beta blockade. These results support the potent ocular hypotensive action and the weak systemic beta-adrenergic blocking and cardiovascular activity of AO: a significant improvement in the therapeutic index. This finding recommends alprenoxime as a potent site-specific antiglaucoma agent with minimal systemic side effects.

Desensitization and functional antagonism by beta-adrenoceptor and muscarinic receptor agonists, respectively: a comparison with receptor alkylation for calculation of apparent agonist affinity.

1. Apparent affinity constants (KD) for prenalterol, an agonist of low intrinsic efficacy at beta 1-adrenoceptors in rat left atria, have been determined by use of receptor desensitization and functional antagonism induced by isoprenaline and carbachol, respectively. The values obtained have been compared to those values estimated with the irreversible beta-adrenoceptor antagonist, bromoacetylalprenololmenthane (BAAM). 2. The -log KD values for prenalterol estimated by desensitization or irreversible antagonism ranged from 6.8-7.1 and 6.2-7.1, respectively. 3. Carbachol produced functional antagonism of the response to prenalterol even though it was removed before addition of prenalterol. This effect was mediated by M2-muscarinic receptors. Pretreatment of animals with pertussis toxin did not affect the functional antagonism elicited by carbachol. The apparent KD value obtained after pre-exposure to carbachol (6.8) was similar to those estimated by use of either alkylation with BAAM or desensitization with isoprenaline (see above). 4. It is concluded that acute desensitization or functional antagonism of responses to agonists of low intrinsic efficacy provides a means to estimate apparent KD constants. This approach could be useful to characterize receptors for which an irreversible antagonist may not be available.

[Synthesis and identification of bromoacetylalprenololmenthane (BAlpM)].

A highly potent beta-adrenergic irreversible antagonist--Bromoacetylalprenololmenthane (BAlpM) was synthesized by a six step method with phenol and allychloride as the starting materials. Some improvement on purification of the product was described. The final product is identified by melting point, elemental analysis, UV and IR spectral analysis and mass spectrometry as well as beta-adrenergic receptor binding assays. [125I] +/- IODOPINDOLOL binding assay of mouse lung cell membrane preparations treated with BAlpM in vitro or in vivo showed that there was a dose-dependent decrease in the density of specific binding sites with no change in the Kd values. This result confirms that BAlpM is a beta-adrenergic irreversible antagonist.