Mechanical Response of Rat Myocardium to Dibutyryl Cyclic AMP in Relation to Effects of α- and β-Adrenoceptor Stimulators

Abstract: Dibutyryl cyclic AMP, and α- and β-adrenoceptor stimulators are all able to elicit inotropic effects, α- and β-Adrenoceptor stimulation are known to change each myocardial contraction-relaxation cycle differently. In order to elucidate the myocardial function of cyclic AMP the effects of dibutyryl cyclic AMP on the contraction-relaxation cycle of isolated rat heart papillary muscle were examined and compared to the effects of α- and β-adrenoceptor stimulation, respectively. Dibutyryl cyclic AMP (in the presence of propranolol) increased developed tension (T_max) by 18%, rate of tension rise (T′_max) by 46%, rate of tension fall (T′_min) by 62% and onset-rate of relaxation (T″_min) by 136%. These changes in the contraction-relaxation cycle were strikingly similar to those produced by isoprenaline (β-adrenoceptor stimulation). The response to dibutyryl cyclic AMP, however, developed much slowlier than did the response to isoprenaline. The latter effect was associated with cyclic AMP elevation in a way indicating a trigger function for cyclic AMP. The α-adrenoceptor stimulation (by phenylephrine combined with propranolol), however, increased measures both for contraction and for relaxation by about the same degree, and the effects occurred without changes of cyclic AMP contents. Phenylephrine alone (combined α- and β-adrenoceptor stimulation) elicited a substantial cyclic AMP elevation but gave mechanical effects only slightly different from the pure α-adrenergic response. Thus cyclic AMP effects did not seem to be fully expressed in this case. As a whole, the results indicate that the effects of both dibutyryl cyclic AMP and of isoprenaline are mediated by the cyclic AMP-system while α-adrenoceptor stimulation involves other mechanisms.     

Myocardial Calcium Uptake in Streptozotocin Diabetic and Control Rats after Dibutyryl-cAMP; α-Adrenergic Stimulation and Calcium Deprivation

Previous studies have shown a decreased myocardial calcium uptake after β-adrenergic stimulation with isoproterenol in isolated perfused hearts from streptozotocin diabetic rats. Abnormalities in the β-receptor-adenylate cyclase system could explain this but in order to circumvene the receptor we studied the effect of the permeable cAMP analogue, dibutyryl-cAMP on this calcium uptake. A marked increase was seen in control hearts while no increase could be registered in diabetic hearts. Defects in the protein kinase phosphorylation system or in the protein kinase substrate in the sarcolemma are suggested. α-Adrenergic stimulation with phenylephrine, being a cAMP independent positive inotropic agent, was also tested but no increase in calcium uptake was seen in either control or diabetic hearts. This could be due to a different effect on calcium currents during action potential after α-stimulation compared to the β-adrenergic effect. Reexposure to calcium after calcium deprivation leads to excessive myocardial calcium uptake (calcium paradox), but the increase was significantly smaller in diabetic hearts, suggesting a differential responsiveness to the damage induced by this procedure. Early biochemical abnormalities in the basement membrane or in the composition and calcium binding ability of the sarcolemma could possibly constitute a common final site for the defect myocardial calcium uptake after isoproterenol, db-cAMP and calcium deprivation in streptozotocin diabetic rats.     

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.     

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.     

Qualitative Differences between the Inotropic Responses in Rat Papillary Muscles to α-Adrenoceptor and β-Adrenoceptor Stimulation by both Noradrenaline and Adrenaline

Abstract: Mammalian heart has obviously both α- and β-adrenergic inotropic mechanisms, and stimulation of these two mechanisms by appropriate agonists lead to qualitatively different inotropic responses. This difference can serve to recognize the two adrenergic inotropic effects. In order to explore if the endogenous catecholamines, noradrenaline and adrenaline, could activate both these mechanisms, and if so, to characterize them qualitatively and quantitatively, the mechanical responses in electrically driven rat left ventricular papillary muscles were examined in the absence or presence of appropriate receptor blockade. 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. α-Adrenergic and β-adrenergic inotropic effects were demonstrated both for noradrenaline and adrenaline. Maximal β-adrenoceptor stimulation (agonist in the presence of an α-adrenoceptor blocker) caused a small increase in T_max, intermediate increases in T'_max and T'_min, and a considerable increase in T_min (β-type effect). Maximal α-adrenoceptor stimulation (agonist in the presence of a β-adrenoceptor blocker) increased all parts of the contraction-relaxation cycle by about the same degree (T_minT_minT'_maxT_max 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. The time course of the response to 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 inotropic response which developed in the course of 1–2 min. Both agents alone gave a monophasic response with the characteristics of a β-type effect (i.e. relative maximal increase of T_minT_minT_maxT_max), and a marked increase in the relaxation-onset index (T_min/T_max). Thus the β-adrenergic inotropic component was the dominating one when the amines were used alone. The two different response patterns probably reflect a dual mechanism of action of the endogenous catecholamines: the β-adrenergic component which is dependent upon an increase in cyclic AMP levels and the α-adrenergic component which is independent on cyclic AMP.     

Both α1- and β-Adrenoceptor Stimulation Determine the Time Course of the Inotropic Effect of Noradrenaline in Rabbit Heart

It has been a matter of controversy whether α₁-adrenoceptor stimulation contributes to the final inotropic and lusitropic responses in mammalian myocardium to noradrenaline during concomitant and unopposed β-adrenoceptor stimulation. In the present paper we report studies that compare time courses of the inotropic and lusitropic responses to separate and combined α- and β-adrenoceptor stimulation, respectively, in electrically driven rabbit papillary muscles by a submaximal concentration of noradrenaline. Separate α₁- or β-adrenoceptor stimulation (presence of appropriate receptor blocker) showed the characteristic slow and fast development, respectively, of the inotropic responses. Qualitatively, the respective characteristic changes were also observed: α₁-adrenoceptor stimulation caused a negative lusitropic effect giving a prolongation of the time to peak tension (TPT), while β-adrenoceptor stimulation caused a pronounced positive lusitropic effect giving a shortening of TPT. The time course of the inotropic response to combined adrenoceptor stimulation had characteristics that deviated from the respective time courses to separate α₁- or β-adrenoceptor stimulation thus indicating a contribution from both adrenoceptor populations to the final inotropic response. Combined α₁- and β-adrenoceptor stimulation gave a pronounced positive lusitropic response as might be expected due to the obviously dominating role of the β-adrenergic component. However, the maximal lusitropic effect and the shortening of TPT were both slightly less during combined adrenoceptor stimulation compared to separate β-stimulation thus indicating an influence of the α₁ -adrenoceptor mediated negative lusitropic effect. Quantitatively, the separate α₁ - and the separate β-adrenoceptor mediated inotropic effects were not additive. In accordance with other recent studies, this indicated an inhibitory interaction between the two adrenergic receptor populations in myocardium.     

Inhibition of Cyclic AMP Production by α2-Adrenoceptor Stimulation in the Guinea-Pig Spinal Cord Slices

Abstract: In spinal cord slices isolated from guinea-pig and preincubated with ³H-adenine, 0.3–30 μM forskolin induced a dose-dependent increase in the content of ³H-cAMP, the maximal increase being about 8-fold. The selective α₂-adrenergic agonist UK-14,304 (10 μM) reduced both the basal and the forskolin stimulated levels of ³H-cAMP by 18–32%. Dose response curves of the effect of UK-14,304 on cAMP production in the spinal cord slices, stimulated with 3 μM forskolin, showed an IC50 of 37 nM and a maximally inhibitory effect of 27%. A number of other α₂-adrenergic agonist (clonidine, guanfacine, B-HT 920 and B-HT 933) also inhibited the forskolin stimulated ³H-cAMP production; clonidine and guanfacine being almost equipotent with UK-14,304, but their maximal inhibitory effects being only about 6–7%. B-HT 920 and B-HT 933 were less potent and their maximal inhibitory effects about 16–21%. The dose response curve of UK-14,304 on inhibition of forskolin stimulated cAMP production was shifted almost 50-fold to the right by 0.3 μM yohimbine. Prazosin (0.3 μM) did not affect the UK-14,304 dose resonse curve. It is concluded that α₂-adrenoceptor stimulation mediates inhibition of cAMP production in the guinea-pig spinal cord.     

β-Endorphin-(1–27) inhibits the spinal beta-endorphin-induced release of Met-enkephalin

The effect of intraventricular β-endorphin-(1–27) on the spinal release of Met-enkephalin induced by intraventricular β-endorphin was studied using the intrathecal superfusion technique in urethane anesthetized rats. Intraventricular injection of β-endorphin at a dose of 15 μg released Met-enkephalin from the spinal cord. This release of Met-enkephalin induced by β-endorphin was significantly reduced by β-endorphin-(1–27), 60 μg, injected intraventricularly. Injection of β-endorphin (1–27) itself did not cause any release of Met-enkephalin. The finding is in line with the previous report that β-endorphin (1–27) inhibited the analgesia induced by β-endorphin.     

Respiratory Arrest as Main Determinant of Toxicity due to Overdose with Different β–Blockers in Rats

Propranolol, timolol and sotalol were compared regarding their toxicological effects on the cardiovascular and respiratory system. Each drug was administered intravenously to anaesthetized spontaneously breathing and artificially ventilated rats. After the start of infusion in spontaneously breathing rats each drug induced an expected decrease in arterial blood pressure and heart rate. An increase in PQ, QRS and QT interval was observed. From 5/8 of the survival time onwards these changes were accentuated. PaO₂, pH and respiratory rate decreased and PaCO₂ increased. The rats died as a result of respiratory arrest. Artificial ventilation of rats infused with the same doses increased the survival time significantly. The total doses administered before the animals died as a result of cardiovascular failure were significantly higher for each drug. The initial decreases in arterial blood pressure and heart rate were similar to those in spontaneously breathing rats. Thereafter, significantly smaller decreases were observed. The increases in PQ, QRS and QT interval were significantly less than in spontaneously breathing rats. Blood gases remained unchanged except for a decrease in pH in case of timolol     

Novel lβ‐Methylcarbapenems Having Cyclic Sulfonamide Moieties: Synthesis and Evaluation of in‐vitro Biological Activity – Part II

The synthesis of a new series of 1β‐methylcarbapenems having cyclic sulfonamide moieties is described. Their in‐vitro antibacterial activities against both Gram‐positive and Gram‐negative bacteria were tested and the effect of a substituent on the pyrrolidine ring was investigated. One particular compound IIIe having a [1,2,5]thiadiazolidin 1,1‐dioxide moiety showed the most potent antibacterial activity.     

Synthesis and properties of β-endorphin analogs containing the dynorphin-(1–13) sequence

Two analogs of β_h-endorphin containing dynorphin-(5 - 13) or dynorphin-(6 - 13) sequence have been synthesized by the solid-phase method. Biological activities of the analogs have also been investigated. It was found that the opiate activity is about three times more than β_h-endorphin in the guinea pig ileum assay. Both analgesic potency and opiate receptor-binding activity of the analogs are lower when compared with β_h-endorphin. However, the analogs have interesting behavioral effects in mice.     

Effect of General Anaesthetics and Organic Solvents on α1–Adrenoceptors in the Myometrium

Abstract: The α₁ selective radioligand ³H–prazosin was used to assay α₁ receptors in membranes prepared from the rabbit myometrium. ³H–Prazosin was found to bind to a single high affinity site in these membranes which was the presumed α₁ receptor. A series of general anaesthetics and organic solvents were tested for their ability to inhibit ³H–prazosin binding. The order of potency of the tested agents to inhibit the binding was: chloroform = halothane = trichloroethylene > carbon tetrachloride > dichloromethane. The depression of ³H–prazosin binding seemed to be induced on the α₁ receptor since non–specific radioligand binding was not affected as revealed by a saturation experiment with ³H–prazosin where halothane was used as inhibiting agent. Computer analysis of the latter experiment also showed that halothane depressed mainly the affinity of ³H–prazosin for the α₁ receptor. The ability of the general anaesthetics and organic solvents to inhibit contractions elicited by α₁ stimulation with phenylephrine in the rabbit uterus was also investigated. In these tests the order of potency for the inhibition of the contractile response was: carbon tetrachloride ≥ halothane = chloroform > trichloroethylene > dichloromethane. The mechanism of action for α₁ receptor and myometrial depression is discussed     

The Ulcerogenic Effect on the Oesophagus of Three β-Adrenoceptor Antagonists,Investigated in a New Porcine Oesophagus Test Model

Abstract: Drug-induced oesophageal lesions have attracted increasing attention during the past few years. A test model is presented for assessing irritative or ulcerogenic effects of potential drugs on the oesophagus. In this model the pig oesophagus is used, as it is more similar to the human oesophagus than models used in other techniques that have been suggested. The β-blockers, alprenolol and propranolol, displayed the same ulcerogenic properties with this test model as reported with other test models in the literature. The β₁-selective blocker metoprolol, did not have such ulcerogenic effects. The pig model is suggested as the method of choice in evaluating the potential irritative or ulcerogenic effects of pharmaceutical formulations intended for human use.     

The Effect of Nifedipine on the Sinus and Atrioventricular Node of the Dog Heart after β-Adrenergic Receptor Blockade

Abstract The effect of nifedipine (BAY 1040), a calcium-antagonistic inhibitor of the electromechanical coupling process was tested on atrioventricular conduction and refractoriness of the dog heart in situ by means of His-bundle electrography and programmed electrical stimulation. The animals were anaesthetized with sodium pentobarbital. As the basic effects of the compound can be altered by release of catecholamines from sympathetic nerves of heart and vessels, the dogs were pretreated with acebutolol, a β-adrenergic receptor blocking agent, which decreased heart rate and prolonged atrioventricular conduction and refractoriness. Nifedipine 1,6 and particularly 30 μg/kg body weight increased the heart rate and decreased atrioventricular conduction time during atrial pacing, whereas atrioventricular conduction time during sinus rhythm and atrioventricular refractoriness were only affected by nifedipine 30 μg/kg. In this respect, nifedipine differs distinctly from another calcium antagonistic compound, verapamil.     

Isolation and properties of β-endorphin-(1–27)-like peptide from bovine brains

A β-endorphin-(1–27)-like peptide was isolated from bovine cerebral hemisphere extracts by gel filtration, ion-exchange chromatography, high performance liquid chromatography and paper electrophoresis. The peptide had tyrosine as the amino-terminal residue and its amino acid composition was nearly identical to that of equine pituitary β-EP-(1–27). It had also the same mobility as equine pituitary β-EP-(1–27) in paper electrophoresis. In radioimmunoassay and opiate receptor-binding assay, the brain peptide had 50% activity when compared with human β-endorphin-(1–27). Evidence for the occurrence of NH₂-acetylated form of β-endorphin-(1–27) is also presented.     

Higher α-noradrenergic receptors in paraventricular nucleus of obese zucker rats: Decline after food deprivation

Norepinephrine (NE), acting through α₂-noradrenergic receptors in the hypothalamic paraventricular nucleus (PVN), has been implicated in the control of feeding behavior and body weight gain. To determine whether this hypothalamic receptor system is disturbed in genetically obese rats, the binding of radioligands to α₂-noradrenergic, as well as to α₁-noradrenergic, receptors was examined in seven hypothalamic nuclei of obese Zucker rats relative to their lean littermates. Receptor binding procedures, using the α₂-noradrenergic agonist [³H]p-aminoclonidine ([³H]PAC) and the α₁-noradrenergic antagonist [³H]prazosin, demonstrated that the obese rats, compared to the lean rats, had significantly greater α₂-noradrenergic and α₁-noradrenergic receptor binding, specifically in the PVN as opposed to other hypothalamic areas examined. Moreover, the obese rats, compared to the lean rats, exhibited greater responsiveness to the effects of food deprevation (48 h), which caused a significant decline in radioligand binding to both α₂ and α₁ receptors, specifically in the PVN. A decrease in α₂-receptor binding after deprivation in the obese rats was also seen in two basal hypothalamic areas, namely the supraoptic nucleus and acurate nucleus-median eminence. The possibility exists that these disturbances in hypothalamic α-receptors may be involved in the development and/or maintenance of the genetic obesity.     

Cortical β- and α2- Adrenoceptor Binding,Hypothalamic Noradrenaline and Pineal Melatonin Concentrations Measured at Different Times of the Day after Repeated Treatment of Rats with Imipramine,Zimeldine, Alaproclate and Amiflamine

Abstract: The effect of repeated treatment of rats for 21 days with the monoamine reuptake inhibitors imipramine, zimeldine, alaproclate (in each case 10 μmol/kg b.i.d.) and the reversible monoamine oxidase-A inhibitor amiflamine (3 μmol/kg b.i.d.) on brain noradrenergic mechanisms measured at different times of the day and night was investigated. Imipramine treatment produced a down-regulation of the B_max for ³H-dihydroalprenolol binding to cortical β-adrenoceptors that was not dependent upon the time of day the animals were killed. Zimeldine, on the other hand, reduced both B_max, and K_d of binding for day-time, but not night-time samples. Alaproclate and amiflamine were without effect on the binding. Twenty-four hour mean values for 1 nM ³H-p-aminoclonidine binding to α₂-adrenoceptors were lower for the zimeldine-treated rats than for the saline-treated rats. Pineal melatonin concentrations, which are regulated by β-adrenoceptors, showed a pronounced diurnal rhythm, with the highest concentrations being found at 02:00. At this time point, a lower pineal melatonin content was found after amiflamine treatment, whereas imipramine, zimeldine and alaproclate were without significant effect. The importance of the use of more than one time point and the use of more than one biochemical test for the determination of the effects of repeated antidepressant treatment on central noradrenergic systems measured ex vivo is discussed.