Qualitative Differences between β-Adrenergic and α-Adrenergic Inotropic Effects in Rat Heart Muscle

Abstract If β- and α-adrenergic inotropic effects are cyclic AMP dependent and cyclic AMP independent, respectively, they may be qualitatively different. The inotropic effects of β-receptor stimulation (isoprenaline) and α-receptor stimulation (phenylephrine combined with propranolol) were characterized in isolated perfused rat hearts, rat atria and rat papillary muscles. The β-effect reached its maximum before the α-effect. The α-effect followed a three-phasic time-course indicating both stimulatory and inhibitory components. The aortic pressure wave (perfused heart) indicated a shorter contraction phase after β-stimulation than after α-stimulation. The time to peak tension (atrium, papillary muscle) was relatively shorter after isoprenaline than after α-stimulation, which tended to prolong it. The contraction-relaxation cycles (atrium, papillary muscle) were examined by recording the isometric tension (T), its first (T′) and second (T″) derivatives, α- and β-stimulation both increased T_max, T′_max (maximal rate of tension rise), T′_min, (maximal rate of tension decline) and T″_min (maximal rate of transition from rise to decline of tension). Isoprenaline increased T_min, (papillary muscle) and T″_min (atrium, papillary muscle) relatively more than did α-stimulation, i.e. the relaxing processes were activated relatively more by β-stimulation. The results indicate different mechanisms for the two adrenergic inotropic effects. The relatively larger activation of relaxation by β-stimulation is assumed to be caused by cyclic AMP.     

Differences between α-Adrenergic and β-Adrenergic Inotropic Effects in Rat Heart Papillary Muscles

Abstract: α-And β-adrenergic inotropic effects have been shown to be qualitatively different. In order to further characterize these differences we compared the mechanical responses to α- and β-adrenoceptor stimulation, respectively, in electrically driven left ventricular papillary muscles from rat heart. The muscles were stimulated by either isoprenaline (β-adrenoceptor stimulation), phenylephrine in the presence of propranolol (α-adrenoceptor stimulation) or phenylephrine alone (combined α- and β-adrenoceptor stimulation). Isometric tension (T), rate of rise and decline of tension (first derivative = T′) and rate of transition from tension rise to tension decline (negative part of second derivative = T″) were recorded. These recordings disclosed qualitative differences between the α- and β-inotropic response both in dose-response and time course experiments. Maximal β-adrenoceptor stimulation caused a small increase in T_max (18%), intermediate increases in T′_max (45%) and T′_min (68%) and a considerable increase in T″_min (145%) (“β-type” effect). Maximal α-adrenoceptor stimulation increased all qualities by about the same degree (23–24%) (“α-type” effect). While β-adrenoceptor stimulation gave a dose-dependent and pronounced increase in the ratio T″_min/T′_max (relaxation-onset index), α-adrenoceptor stimulation decreased it to subcontrol values and phenylephrine alone gave a small dose-dependent increase at higher doses. The time course of the α-adrenoceptor stimulation was characterized by a transient decrease in all qualities followed by an increase which reached maximum at 4–5 min. β-Adrenoceptor stimulation gave a monophasic response which reached maximum after 1–2 min. Phenylephrine alone gave mainly an “α-type” effect although T″_min increased significantly more in the absence than in the presence of propranolol and T″_min/T′_max showed a small increase which developed slowly. Thus β-adrenoceptor stimulation activated relaxation compared to contraction by a higher degree than did α-adrenoceptor stimulation. This probably reflects different mechanisms of action. While the α-effect may rely primarily on an increased calcium influx, the β-effect probably is the final result of several subcellular effects of cyclic AMP.     

Mechanisms of β-Adrenergic Desensitization in Rat Myometrium

Abstract: This study was performed in order to elucidate the mechanism behind the decreased responsiveness to β-adrenergic stimulation occuring in uterine muscle after prolonged treatment with isoprenaline. Pretreatment of rats with isoprenaline, 20 nmol/kg, three times daily during four days, significantly decreased the myometrial relaxing effect of the β-agonist. There was also a significant decrease of the β-receptor binding capacity of the myometrial membranes measured by the (minus;)-(³H) DHA binding technique. In the animals pretreated with isoprenaline no significant increase of the adenylate cyclase activity could be observed after isoprenaline stimulation in vitro. The uterine cAMP level was diminished in the desensitized rats. The phosphodiesterase activity was increased. Thus both decreased production and increased degradation contribute to the lower level of uterine cAMP content. The activity of cAMP dependent protein kinase was also depressed. In this work, where low concentrations of isoprenaline have been administered in vivo, several biochemical parameters have been shown to contribute to the β-adrenergic desensitization in myometrial tissue.     

Competitive Blockade of α-Adrenergic Receptors in Rat Heart by Prazosi

Abstract: Pheiiykphriiie (PE) in presence of propranolol evokes an α-adrenergic inotroyic response in rat heart. The time course of this response is characterizcd by a transient ducrease in maximal developed tension (T_max) subcontrol levels (negative phase of the inotropic response) followed by an increase which reaches maximum after 4–5 min. (positive phase of the inotropic response). Prazosin (PRZ), a selective α₁-receptor blocker. inhibited preferentially the positive phase of the inotropic response and displaced the dose-responsecurve of PE to the right in nanomolar concentrations, indicating a competitive mechanism of inhibition. Phentolamine, a non-selrctive α-blocker. blocked both the negative and the positive phase olthe inotropicresponse toabout the same degree. PRZ appears to be a competitive α-adrenergic antagonist with high affinity in rat heart. Two populations of α-adrenergic receptors may he present: one stirnulatory (α₁) iind one inhibitory     

Is there any Cross-Antagonism between μ-Opioid and α2-Adrenergic Receptors in the Rat Spinal Cord?

Abstract: A functional connection, evidenced by synergism and cross-tolerance, seems to exist between the μ-opioid and the α₂-adrenergic receptors in the superficial dorsal horn of the rat spinal cord. In this study possible cross-antagonism between these systems was studied by intrathecal administration of morphine 2.5 μg or dexmedetomidine 1.25 μg 15 min. after subcutaneous injection of naloxone 1.0 mg/kg or atipamezole 3.0 mg/kg in the morphine group and 4.0 mg/kg or 1.0 mg/kg in the dexmedetomidine group, respectively. No cross-antagonism of the antinociceptive effect was seen either in the tail flick or in the hot plate test at 15, 30, 45 and 60 min. from the pretreatment.     

Different β-Adrenergic Receptor Density in Different Rat Skeletal Muscle Fibre Types

Abstract: The effects of adrenaline on skeletal muscle differ between fibre types. The aim of the present study was to investigate the β-adrenoceptor density, affinity and subtype in rat skeletal muscles with different fibre type composition. β-Adrenoceptors were determined in cryostat sections to avoid methodological problems with variable recovery, using the non-selective β-adrenoceptor ligand [³H]CGP-12177 and β₁ and β₂-selective cold ligands CGP 20712A and ICI 118,551. In the presence of protease inhibitors [³H]CGP-12177 binding was stable, saturable, reversible, and displaceable. Scatchard analysis of binding saturation data was compatible with a single class of specific binding sites. Binding site density (B_max) was higher (P<0.02) in adult soleus (9.38 ± 1.13 fmolxmg protein^–1) than in adult extensor digitorum longus (4.74 ± 0.39 fmolxmg protein^–1), whereas the dissociation constants (K_d), 0.37® pL 0.05 and 0.31 ± 0.04 nM for soleus and extensor digitorum longus, respectively, were not significantly different. For young rats (5–6 weeks), B_max was 11.21 ± 0.33 and 5.45 ± 0.11 fmolxmg protein^–1 (P<0.05), and K_d was 0.27±0.02 and 0.24±0.04 nM for soleus and epitrochlearis, respectively. These results correspond to a receptor density of 2 and 1 pmolxg w.wt.^–1 in muscles containing mainly type I and type II fibres, respectively. Displacement studies with CGP 20712A and ICI 118,551 were compatible with mainly β₂-adrenoceptors, but 7–10% β₁-adrenoceptors were present in both types of muscle. In conclusion, the receptor density is twice as high in muscles containing mainly type I muscle fibres compared to muscles containing mainly type II fibres, and this may explain some of the different effects of adrenaline between the two muscle fibre types.     

Effects of β-Adrenergic Agonists on Rat Uterine Motility and cAMP Level in Vivo

Abstract: Intravenous injection of isoprenaline and salbutamol into rats caused an inhibition of the spontaneous contractions of uterus and increased the uterine cAMP level in a dose-dependent manner. The effects of isoprenaline (0.036 nmol/kg) were diminished or completely prevented by propranolol (0.07–7 μmol/kg). Intravenous infusion of the β-adrenergic agonists during one hour increased the uterine cAMP level. The highest level of cAMP measured was at 3 min. after start of infusion, thereafter it declined first rather fast but later more slowly. This time pattern of cAMP was very similar to what was seen during incubation of uterine tissue with isoprenaline in vitro. Pretreatment with isoprenaline three times daily 4–7 days before the experiments, decreased both the degree of inhibition and the increase of cAMP in uterus caused by an intravenous injection of isoprenaline. A slight effect on the cAMP rise was detectable after one day of pretreatment, and it seemed to have reached its maximal effect after 4 days, since no further decline in the cAMP response was seen when the pretreatment was prolonged. A desensibilization to P-adrenergic agonists was thus seen in uterus after excessive stimulation of isoprenaline in vivo.     

Effects of α2-Adrenergic Drugs on the Alcohol Consumption of Alcohol-Preferring Rats

The effects of seven-day subcutaneous infusion of an α₂-adrenoceptor agonist, medetomidine, and an α₂-adrenoceptor antagonist, atipamezole, on the voluntary alcohol consumption of alcohol-preferring rats were studied. The drugs were administered by means of implanted osmotic minipumps. Sham-operated control rats had no pumps implanted. The rats had a free choice between 10% alcohol and plain water for 30 days before pump implantation and again for six days starting 24 hr after the operation. Atipamezole increased the alcohol consumption during the first day of free choice. Medetomidine had no significant effect. During the remaining period of infusion, the alcohol consumption did not differ from that preceding the pump implantation in each treatment group. Animals in the atipamezole group gained more weight during the seven-day trial than did those in the medetomidine and control groups. The amine changes in different regions of the brain were consistent with medetomidine decreasing and atipamezole increasing the noradrenaline turnover. The present results indicate that specific drugs acting on the α₂-adrenoceptors produce only minor changes in the voluntary alcohol drinking of alcohol-preferring rats.     

Does α1-Acid Glycoprotein Act as a Non-functional Receptor for α1-Adrenergic Antagonists?

Abstract— The ability of a variety of α₁-acid glycoproteins (AAG) to affect the intrinsic activity of the α₁, -adrenergic antagonist prazosin was studied in rabbit aortic strip preparations. From these studies, the activity of AAG appears to be linked to their ability to bind the antagonist. However, a capability to bind prazosin was not the only requirement for this effect. The removal of sialic acid and partial removal of the galactose and mannose residues by periodate oxidation of human AAG all but eliminated the ability of AAG to affect the intrinsic pharmacologic activity of prazosin, although the binding of prazosin was not significantly affected. The presence of bovine AAG, a protein that has a low ability to bind prazosin, reduced the effect of human AAG on prazosin activity. Based upon these results, we propose that AAG is able to bind in the vicinity of the α₁-adrenoceptors, therefore extending the binding region for antagonists in such a way as to decrease the ability of the antagonist to interact with the receptor. The carbohydrate side-chains are important for the binding of AAG in the region of the adrenoceptor.     

Rat Spinal Cord α2-Adrenoceptors are of the α2A-Subtype: Comparison with α2A- and α2B-Adrenoceptors in Rat Spleen,Cerebral Cortex and Kidney Using 3H-RX821002 Ligand Binding

Abstract: Binding of the α₂-adrenoceptor antagonist radioligand ³H-RX821002 was investigated in membranes from rat spinal cord, spleen, cerebral cortex and kidney. The ligand was found to bind to saturable binding sites with apparent uniform affinities within each tissue. Seven compounds, some of which have previously been reported to be selective for either α_2A- or α_2B-adrenoceptors, were used in competition with ³H-RX821002. By using computer modelling, competition curves generated for three of these compounds (ARC 239, prazosin and oxymetazoline) could be resolved into two site fits in the kidney, K_d's of the drugs being compatible with the notion that these sites corresponded to α_2A- and α_2B-adrenoceptors. Moreover, rauwolscine and yohimbine were found to be about 14 and 9-fold selective for α_2B-adrenoceptors in the kidney. In all other tissues studied drug competition curves were uniphasic and computer modelled into one site fits, drug K_d's being well correlated to those for the α_2A-adrenoceptor. In rat spinal cord 26 further drugs, which showed wide variation in structure, were evaluated in competition with ³H-RX821002. Of these compounds, competion curves of the agonists UK-14,304, (—) and (+) adrenaline were modelled into two site fits whereas those of the remaining compounds could be modelled only into one site fits. Since the high affinity site for UK-14,304, (—) and (+) adrenaline was eliminated when EDTA, Gpp(NH)p and 140 mM NaCl was present in the assay the heterogeniety observed in spinal cord was considered to be due to formation of high and low affinity conformations of the α₂-adrenoceptor for agonists. It is concluded that ³H-RX821002 is useful to label both α_2A- and α_2B-adrenoceptors in the rat. Moreover, the binding sites labelled by ³H-RX821002 in the spinal cord appear to consist of a single population of α₂-adrenoceptors of the α_2A-type.     

β-Adrenergic Responsiveness of the Gastrointestinal Tract in Diabetic Rats

Abstract: Recently, decreased gastrointestinal β-adrenergic responses in experimental diabetes have been demonstrated. Gastrointestinal responses to β-adrenoceptor agonists are impaired in both insulin-dependent and non-insulin-dependent diabetic rat. Insulin treatment improves the impaired gastrointestinal β-adrenergic responsiveness of diabetic rats. The improvement seen with insulin treatment on β-adrenergic responsiveness is closely related to protein biosynthesis. The decreased β-adrenergic responses in diabetic rat gastrointestinal tract seem to result from a decrease in the number of β-adrenoceptors. It is most likely that the decreased gastrointestinal β-adrenergic responsiveness is related to an impairment in the turnover of β-adrenoceptors as a consequence of diabetes and that insulin has a beneficial effect on the impaired receptor turnover.     

Effects of Trifluoperazine on β-Adrenergic Responses of Rat Papillary Muscle: Related to Calmodulin?

Abstract: The β-adrenergic stimulation of cardiac contraction and relaxation is related to an augmented Ca⁺⁺ oscillation mediated by cAMP. This Ca⁺⁺ mobilization may secondarily involve calmodulin in a way modulating the mechanical responses. We tested this possibility by studying interferences of trifluoperazine (which is able to block Ca⁺⁺-calmodulin) with β-adrenergic responses in rat heart papillary muscles. Trifluoperazine up to 10^?5 mol/l did not change the basal function. 10^?5 mol/l trifluoperazine augmented the contractile response to isoprenaline above 10^?7 mol/l. The inotropic effects of isoprenaline below 10^?7 mol/l and of the partial β-agonist prenalterol were not influenced by trifluoperazine. 10^?5 mol/l trifluoperazine attenuated the stimulation of initial relaxation by isoprenaline in the entire concentration range. Thus this β-adrenergic response was more sensitive to trifluoperazine than the contractile response. But trifluoperazine only slightly and non-significantly attenuated the stimulation of initial relaxation by prenalterol. From experiments on broken cell preparations the present results can be explained in terms of calmodulin blockade and thus inhibition of Ca⁺⁺ efflux across the sarcolemma and of Ca⁺⁺ uptake by the sarcoplasmic reticulum. Trifluoperazine effects unrelated to calmodulin can hardly account for the results. Thus a full β-agonist can apparently mobilize enough Ca⁺⁺ to activate calmodulin systems important for the final effects on the contraction-relaxation cycle.     

The Hyperglycaemic Activity of Some Catecholamines and the Effect of α- and β-Adrenergic Blocking Compounds in the Mouse

In order to classify the receptors involved in the hyperglycaemic response to catecholamines in the mouse, the relative potency of noradrenaline, adrenaline and isoprenaline in the induction of hyperglycaemia has been tested by giving increasing doses from 10 μg/kg to 1 mg/kg of one of the amines intraperitoneally. Following catecholamine administration the maximal concentration of glucose in the blood occurred between 10 and 30 min. and amounted to about 300 mg%. The relative potency was: adrenaline > noradrenaline while isoprenaline was inactive. The antagonistic effect on adrenaline induced hyperglycaemia of the α-adrenergic blocking compounds: phenoxybenzamine HCl (dibenzyline®) and phentolamine (regitin®) and the β-adrenergic blocking compounds: 1-(isopropylamino)-3-(o-phenoxyphenoxy)-2-propanol (Ph QA 33) and propranolol were tested by giving 1 and 10 mg/kg intraperitoneally 30 min. before the administration of adrenaline. Only phentolamine at the highest dose level was capable of preventing the hyperglycaemic response to adrenaline. Phenoxybenzamine, Ph QA 33 and propranolol proved to be inactive. It is concluded that the mechanism of adrenaline induced hyperglycaemia in mice cannot be explained simply by an α- and/or β-receptor activation.     

Selectivity of ABT-089 for α4β2* and α6β2* nicotinic acetylcholine receptors in brain

Numerous pharmaceutical efforts have targeted neuronal nicotinic receptors (nAChRs) for amelioration of cognitive deficits. While α4β2 and α7 are the more prominent nAChR in brain, other heteromeric nAChR can have important impact on agonist pharmacology. ABT-089 is a pioneer nAChR agonist found to enhance cognitive function with an exceptionally low incidence of adverse effects. To further investigate the mechanism of action of ABT-089, we evaluated its function in mouse brain preparations in which we have characterized the subunit composition of native nAChR. Among α4β2*-nAChR, ABT-089 had partial agonist activity (7–23% of nicotine) and high selectivity for α4α5β2 nAChR as evidenced by loss of activity in thalamus of α5^−/− mice. ABT-089 stimulated [³H]-dopamine release (57%) exceeded the activity at α4β2* nAChR, that could be explained by the activity at α6β2* nAChR. The concentration–response relationship for ABT-089 stimulation of α6β2* nAChR was biphasic. EC₅₀ and efficacy values for ABT-089, respectively, were 28 μM and 98% at the less sensitive α6β2* nAChR and 0.11 μM and 36% at the more sensitive subtype (the most sensitive target for ABT-089 identified to date). ABT-089 had essentially no agonist or antagonist activity at concentrations ≤300 μM at α3β4-nAChR measured by [³H]-acetylcholine release from interpeduncular nucleus. Thus, ABT-089 is a β2* nAChR ligand with demonstrable agonist activity at α4β2* and α6β2* receptors. As one form of α6β2* nAChR is sensitive to sub-μM concentrations, we propose that this receptor in particular may contribute to the enhanced cognitive performance following low doses of ABT-089.     

Analysis of γβ, βγ, γγ, ββ multiple turns in proteins

Abstract: The number of γ‐turns in a representative protein dataset selected from the current Protein Data Bank has increased almost seven times during the past decade. Eighty percent classic γ‐turns and 57% inverse γ‐turns are associated as multiple turns with either another γ‐turn or a β‐turn. We refer to these as multiple turns of the (γβ)_1,2,3 or (βγ)_1,2,3 type, depending upon whether the γ‐turn is before or after the β‐turn along the protein chain, respectively. However, for multiple turns involving only γ‐turns, we follow the nomenclature analogous to that proposed earlier for the multiple (or double) β‐turns. Fifty‐eight per cent β‐turns are associated as multiple turns with another β‐turn. We extracted multiple turns from the protein dataset and classified them on the basis of individual γ‐ or β‐turn types and the number of overlapping residues. Furthermore, we evaluated the amino acid positional potentials and determined the statistically significant amino acid preferences, hydrogen bond/side‐chain interaction preferences in the multiple turns and secondary structure preferences for residues immediately flanking these turns. The results of our analysis would be useful in the modeling, prediction or design of multiple turns in proteins. The amino acid sequence corresponding to the multiple turn, position in the protein chain, PDB Code/chain in which multiple turn is present and the individual turn types constituting the multiple turns are available from our website and this information would also be integrated in our Database of Structural Motifs in Proteins ( http://www.cdfd.org.in/dsmp.html ).     

Analysis of γβ, βγ, γγ, ββ continuous turns in proteins

Abstract: We report the observation of continuous turns in proteins which comprise individual γ‐turns or β‐turns or both that are situated immediately one after the other along the polypeptide chain. The continuous turns were identified from a representative data set of three‐dimensional protein crystal structures. The γβ/βγ, γγ and ββ continuous turns represent peptides of varying amino acid residue lengths and conformations. The continuous turns frequently observed in proteins were: γβ, between a coil and a strand; βγ, between a helix and a strand; γγ, between coils; and ββ, either between a strand and a coil or between strands or coils. We determined the statistically significant amino acid residue preferences at individual positions in the turn, calculated amino acid positional potentials and analyzed main chain hydrogen bonds and side‐chain interactions likely to stabilize the continuous turns. The data on continuous turns have been integrated in the database of structural motifs in proteins (DSMP) on our web server at ( http://www.cdfd.org.in/dsmp.html ). This is useful to make queries on sequences compatible with different continuous turns.     

Effects of Terbutaline on α-Adrenergic Responses and Ca2+ Influx in Isolated Rabbit Aorta

Effects of terbutaline applied in vivo or in vitro on α-adrenergic receptors in the rabbit aorta in normal and Ca²⁺–free solution, and on basal, high potassium-, and phenylephrine-stimulated Ca²⁺ uptake into aorta were investigated. Three day terbutaline administration (25 mg/kg, subcutaneously three times daily) to rabbits increased the pK_B for phentolamine in aorta rings (control 7.3 + 0.2, n = 9; terbutaline 7.8 +0.2, n = 15). It also depressed phenylephrine-stimulated contractions of aorta rings in Ca²⁺–free but not those in normal Krebs solution. It did not significantly depress the basal, or phenylephrine-evoked Ca²⁺ influx into aorta rings, but decreased high potassium-induced Ca²⁺–influx (control 0.58 + 0.05 umoles/g aorta; n = 3, terbutaline 0.41 +0.06 umoles/g aorta, n = 3). In vitro application of 50 μM terbutaline did not significantly alter phenylephrine-stimulated contractions of aorta rings in Ca²⁺–free Krebs solution or significantly depress basal or phenylephrine-induced Ca²⁺ influx into aortas, but did decrease high potassium-stimulated Ca²⁺–influx. Thus, 3-day terbutaline administration increased the affinity of α-adrenergic receptors for phentolamine and had a tendency to increase contractions of aorta rings to phenylephrine. It also decreased high potassium-stimulated Ca²⁺ influx, and depressed phenylephrine-induced contractions in Ca²⁺–free Krebs solution, while in vitro terbutaline application also decreased potassium-induced Ca²⁺ influx.