Pharmacological Denervation Research Articles

Induction of Dystrophin-Associated Proteins Together with Nicotinic Acetylcholine Receptors by Denervation in the Absence of Dystrophin in Skeletal Muscles ofmdxMice

We purified the nicotinic acetylcholine receptor from digitonin-solubilized rabbit skeletal muscle by affinity chromatography and detected many proteins linked to AChR, including dystrophin, adhalin, β-dystroglycan, utrophin, rapsyn, and actin. To determine whether or not AChR links to dystrophin-associated proteins (DAPs) without dystrophin, we studied the effects of denervation on AChR and DAPs in the skeletal muscle of amdxmouse. Following surgical denervation, the levels of adhalin and β-dystroglycan dramatically increased at the extrajunctional sarcolemma with AChR, suggesting that their association is independent of dystrophin. Furthermore, the diffuse extrajunctional appearance of adhalin, β-dystroglycan, and AChR was observed after pharmacological denervation through the subcutaneous administration of succinylcholine. Since the depletion of DAPs and the subsequent disruption of sarcolemmal linkage are believed to be a primary cause of muscle cell necrosis in dystrophinopathies, pharmacological denervation may have some beneficial effect on these diseases.

Testicular injection of 5,6-dihydroxytryptamine or vasectomy interferes with the local stimulatory effect of oxytocin on testicular steroidogenesis in immature rats.

Previous studies indicated that in immature rats testicular administration of oxytocin stimulates testicular steroidogenesis. In the present study, testicular treatment with oxytocin (50 ng) was combined with pharmacological or surgical denervation of the testis in hemigonadectomized immature rats. For denervation 5,6-dihydroxytryptamine (160 micrograms/testis), a substance that destroys serotoninergic neuronal elements, was injected intratesticularly or vasectomy was performed, which also includes transection of the inferior testicular nerve. In 9-day-old animals both vasectomy and pretreatment of the testis with 5,6-dihydroxytryptamine prevented the oxytocin-induced rise in serum testosterone concentration. In addition, intratesticular injection of oxytocin combined with vasectomy resulted in a significant increase in in vitro basal testosterone secretion of the testis. A similar effect was not observed in the 5,6-dihydroxytryptamine-pretreated group receiving oxytocin. The results indicate that testicular innervation is involved in the control of local peptide effects, and data further suggest a differential role of these neural elements in intratesticular regulatory processes.

1009-48 Radiofrequency-induced Lesions Around the Coronary Sinus Ostium Assisted Solely by Endoscopy: Their Effects on Atrioventricular Nodal Properties

Although two anatomically distinct inputs from the areas around the coronary sinus ostium (CSos) to the atrioventricular node (AVN) were reported, their electrophysiologic effects on AVN properties are not well known. We have recently shown in the dog that radiofrequency catheter ablation can be assisted by means of balloon-tipped fiberoptic endoscopy. Thus, we examined the feasibility of this technique in creating lesions around CSos by radiofrequency energy and assessed the effects of such lesions on AVN properties in 11 anesthetized dogs. After pharmacological denervation with intravenous propranolol 1 mg/kg and atropine 0.2 mg/kg, basic intervals and Wenckebach cycle length (WCl) were obtained. AVN conduction curves were constructed at multiple cycle lengths using atrial extrastimulus testing. We then ablated areas between CSos and the tricuspid valve in 2 dogs (Group 1) and those above CSos in 6 (Group 2). In 4 dogs in Group 2, the same areas in Group 1 were then ablated. The remaining 3 dogs served as controls (Group 3). Measurements were repeated after each ablation procedure using the same doses of the drugs. At the end of each experiment, pathological examination was performed. Radiofrequency ablation was possible in all dogs under direct vision without fluoroscopy. Basic intervals and WCL showed no changes. Group 1 showed shifting of the AV nodal curve downward (facilitation) in 1 dog and development of 2: 1 AVN block in another after ablation. In Group 2, block in slow pathway was noted in 1 dog, facilitation of the AVN curve in 3, and no change in 2. Further ablation in the area in Group 1 resulted in AVN block in 1 and showed no change in 3. No changes were demonstrated in Group 3. Pathologic study confirmed transferal lesions in the expected areas. This technique can be used to safely assist catheter ablation in dogs. Ablation in the tested areas modified AVN properties but the changes were not consistent. These observations may suggest that these anatomical inputs do not necesarily have specific electrophysiologic characteristics but overall effects on the AVN are due to their interactions.

Contraction-excitation feedback in atrial reentry: role of velocity of mechanical stretch

To determine the effects of atrial stretch on atrial flutter, we analyzed variations in atrial flutter cycle length (CL) caused by ventricular contraction in five dogs with artificially induced atrioventricular block. In contrast to absence of variations in CL during sustained ventricular asystole, atrial stretch by ventricular contraction consistently produced phasic variations in CL. The variations were not confined to any specific atrial sites, but depended on the time of ventricular contraction relative to atrial activation phase. The phase-response curve of the variations was closely related to that of the velocity of changes in the atrial pressure with no time lags, but not to that of the atrial pressure itself. Pharmacological denervation did not abolish the variations. Conventional electrophysiological study revealed greater changes in intra-atrial activation time by atrial stretch at a short pacing cycle length than at a longer one, suggestive of changes in repolarization by mechanical stretch. In conclusion, atrial stretch caused by ventricular contraction produces phasic variations in CL, the extent of which depends on the velocity of the mechanical stretch of atrial muscles.

Effect of interventions that increase cyclic AMP levels on susceptibilty to ventricular fibrillation in unanesthetized dogs

Although the autonomic nervous system has been implicated in the formation of ventricular fibrillation, the precise mechanism by which this is mediated remains undetermined. In particular, the role of second messengers, generated by β-adrenoceptor activation, has been postulated to mediate the pro-arrhythmic effects of the sympathetic nervous system. Thus, a 2 min occlusion of the left circumflex coronary artery was initiated during the last minute of exercise in canines with healed myocardial infarctions (produced by ligation of left anterior descending artery). Fifteen dogs were found to be susceptible to the formation of ventricular fibrillation while 17 animals were resistant. Nine resistant dogs were treated with the phosphodiesterase inhibitor isobutylmethyl xanthine (IBMX,1 mg/kg) in combination with an infusion of 8-bromo-cAMP (100–150 μg/kg/min beginning 45 min prior to exercise). Heart rate and left ventricular d P/d t max significantly increased, but failed to elicit, arrhythmias during the exercise and ischemia test. Nine resistant animals were also treated with the adenylate cyclase activator forskolin, (100 μg/kg), which provoked the same hemodynamic changes as the cyclic AMP infusion but also failed to induce ventricular fibrillation. Both forskolin ( n = 3) and IBMX ( n = 3) induced large increases in myocardial cAMP levels (control 5.2 ± 0.5, forskolin 8.1 ± 0.8 pmol/mg non-collagen protein; control 5.0 ± 0.8, IBMX 6.8 ± 0.3 pmol/mg non-collagen protein). Ten resistant animals were treated with the β-adrenoceptor agonist isoproterenol (1–10 μg/kg/min), which failed to cause ventricular fibrillation despite significant increases in the hemodynamic parameters described above. Finally, experiments were repeated after 8-bromo-cAMP infusion and IBMX pretreatment in 8 susceptible animals with pharmacologic denervation (atropine + propranolol + prazosin). In spite of hemodynamic increases indicative of an increase in myocardial cyclic AMP levels, arrhythmias were not re-introduced. These data suggest that changes in cAMP may not be responsible for ventricular fibrillation in this model of sudden cardiac death.

Changes in sensitivity of the isolated guinea-pig vas deferens induced by a lyophilized Phoradendron latifolium leaf infusion

The effect of a lyophilized mistletoe infusion (LMI) was studied on isolated guinea-pig vas deferens. LMI caused a contraction which was partially blocked by phentolamine but not by atropine. LMI caused a shift to the left of the norepinephrine concentration-effect curve (CEC), an effect which appeared to be blocked by atropine and was absent in animals previously treated with reserpine and α-methyl- para-tyrosine. The increase of the norepinephrine maximal response induced by LMI was not blocked by atropine or pharmacological denervation. LMI caused a shift to the right of the acetylcholine CEC and had no effect on the acetylcholine maximal response. These results suggest that the effects seem to be due mainly to the presence of potassium ion in the LMI; however, the participation of muscarinic agonist(s) of reduced intrinsic activity or some tyramine-like substance could not be ruled out.

Dopaminergic alterations in cotreatments attenuating haloperidol-induced hypersensitivity

Chronic treatment of the laboratory rat with haloperidol results in an increased stereotypic behavioral response to subsequent dopamine agonist challenge. This behavioral hypersensitivity (BH) is thought to reflect an increase in DA receptor number following chronic pharmacologic denervation. Using a cotreatment strategy, we demonstrate here that a variety of agents can attenuate or prevent the development of BH when administered chronically with haloperidol. Cotreatment with lithium and amantadine prevented the changes in DA biochemistry as well as the proliferation of DA receptors normally associated with chronic haloperidol treatment. Cotreatment with thioridazine or scopolamine did alter the changes in DA biochemistry normally associated with haloperidol treatment, but failed to attenuate the DA receptor proliferation. Taken together, these data suggest that mechanisms in addition to DA biochemical and receptor changes participate in the development and subsequent expression of BH. DA receptor proliferation must, therefore, be considered permissive to the development of BH.

Striatal homogenates from animals chronically treated with haloperidol stimulate dopamine and GABA uptake in cultures of rostral mesencephalic tegmentum.

The effect of pharmacologic denervation of striatal tissue on the production of growth promoting factors was examined in a cell culture system. Relative to saline-treated controls, rats were rendered behaviorally hypersensitive to a subsequent apomorphine challenge by 2 months of chronic treatment with haloperidol. Four days following chronic treatment, the animals were killed and the striata and cerebella were homogenized in Hank's Balanced Salt solution. The supernatants of these crude homogenates were then added to E-13 rostral mesencephalic tegmentum cultures for 6 days. Within 24 h, the haloperidol-treated striatal supernatants induced an overt increase in culture growth relative to all other supernatants. After 6 days, cultures incubated with haloperidol-treated striatal supernatants exhibited a significant increase in dopamine and GABA uptake relative to cultures incubated with all other supernatants. This effect was observed in the presence and absence of glia. The relative degree of this increased uptake was dependent upon the amount of haloperidol-treated striatal supernatant added. Boiling the supernatant removed the growth promoting effect. These results suggest that pharmacologic denervation of striatal tissue leads to a "target-specific" increase in growth promoting activity that may play a role in the pharmacologic and behavioral effects of haloperidol.

Chronotropic and inotropic effects of kampo extracts in the canine isolated, blood-perfused heart preparations.

Cardiac effects of drugs used for circulatory disorders in traditional Japanese medicine based on ancient Chinese medicine (Kampo Medicine): Saikoka-ryukotsu-borei-to, Oren-gedoku-to, Toki-shakuyaku-san, Shimbu-to, Moku-boi-to, Ryo-kei-jutsu-kan-to, Sha-kanzo-to, Keishi-ninjin-to, Toki-to and Ryo-kan-kyo-mi-shin-ge-nin-to were investigated using canine isolated, blood-perfused sinoatrial node and papillary muscle preparations. Single injections of small doses of Oren-gedoku-to, Moku-boi-to and Ryo-kan-kyo-mi-shin-ge-nin-to (0.1 to 3 mg) dose-dependently increased the sinoatrial rate and the developed tension of papillary muscle, while other drugs showed almost no effect on these parameters. All the drugs had almost no effect on the blood flow through the nutrient arteries of each preparation. The positive chronotropic and inotropic effects induced by Oren-gedoku-to, Moku-boi-to and Ryo-kan-kyo-mi-shin-ge-nin-to did not show tachyphylaxis and were not affected after pharmacological denervation by tetrodotoxin treatment or by reserpine pretreatment, but were significantly suppressed by atenolol. These results indicate that these three drugs act as beta-adrenoceptor agonists to produce clinically useful cardiac effects.

Chronotropic and Inotropic Effects of Kampo Extracts in the Canine Isolated, Blood-Perfused Heart Preparations

Cardiac effects of drugs used for circulatory disorders in traditional Japanese medicine based on ancient Chinese medicine (Kampo Medicine): Saiko-ka-ryukotsu-borei-to, Oren-gedoku-to, Toki-shakuyaku-san, Shimbu-to, Moku-boi-to, Ryo-kei-jutsu-kan-to, Sha-kanzo-to, Keishi-ninjin-to, Toki-to and Ryo-kan-kyo-mi-shin-ge-nin-to were investigated using canine isolated, blood-perfused sinoatrial node and papillary muscle preparations. Single injections of small doses of Oren-gedoku-to, Moku-boi-to and Ryo-kan-kyo-mi-shin-ge-nin-to (0.1 to 3 mg) dose-deoendently increased the sinoatrial rate and the developed tension of papillary muscle, while other drugs showed almost no effect on these parameters. All the drugs had almost no effect on the blood flow through the nutrient arteries of each preparation The positive chronotropic and inotropic effects induced by Oren-gedoku-to, Moku-boi-to and Ryo-kan-kyo-mi-shin-ge-nin-to did not show tachyphylaxis and were not affected after pharmacological denervation by tetrodo-toxin treatment or by reserpine pretreatment. but were significantly suppressed by atenolol. These results indicate that these three drugs act as beta-adrenoceptor agonists to produce clinically useful cardiac effects.

Histological investigations of muscle atrophy and end plates in two critically ill patients with generalized weakness

We describe pathological alterations at the light microscopical and ultrastructural level of motor end plates and muscle fibres in 2 critically ill patients with generalized muscular atrophy and weakness. Axonal degeneration of intramuscular nerve fibres was not conspicuous. The sural nerve in one patient showed only minor abnormalities. There was a marked atrophy of type 1 and especially type 2 muscle fibres. Nodal and ultraterminal nerve sprouts were seen in both biopsies. Motor end plates showed features of regeneration. They were enlarged in one patient. This patient was treated for a prolonged period with vecuronium bromide. Pharmacological denervation may explain the presence of fibrillation potentials, and, partially, of the histological abnormalities. Another factor which must be considered is inhibition of neuromuscular transmission by antibiotics. In addition to disuse atrophy and a minor degree of axonal neuropathy, the production of muscle proteolytic factors may be involved in the rapidly occurring massive loss of muscle fibre size in critically ill patients.

Diuresis from atrial receptors after hypophysectomy and local ablation with no changes in plasma vasopressin.

Stimulation of the left atrial receptors in dogs anaesthetized with chloralose results in a reflex diuresis and natriuresis. The efferent limb of this reflex is though to have at least three components: nervous, haemodynamic and humoral. The present study was designed to investigate the humoral component in dogs anaesthetized with chloralose; to determine whether a humoral agent, other than vasopressin, might be causative in this reflex diuresis. The nervous and haemodynamic components were prevented by pharmacological denervation. Any possible contribution to the diuresis by a decrease in the plasma concentration of vasopressin was prevented by the removal of the pituitary gland. In animals in which a spontaneous diuresis followed hypophysectomy, an infusion of arginine vasopressin sufficient to maintain urine flow in the normal range for these dogs anaesthetized with chloralose was given. Distension of small balloons at the pulmonary vein-atrial junctions and in the left atrial appendage discretely to stimulate the atrial receptors in eleven dogs anaesthetized with chloralose resulted in a significant diuresis. It was concluded that a blood-borne agent other than vasopressin was responsible for the observed diuresis. At the moment it is not known whether vasopressin or the diuretic agent, or both, are involved in the diuresis accompanying the stimulation of atrial receptors.

Effects of acute renal denervation on kidney function in deoxycorticosterone acetate-hypertensive swine.

Deoxycorticosterone acetate-induced hypertension in Yucatan miniature swine appears to involve elevated peripheral sympathetic activity. Abnormalities in renal function in these hypertensive animals are also apparent. To determine the extent to which renal nerve activity controls kidney function in animals with established deoxycorticosterone acetate hypertension, the effects of acute renal surgical denervation were assessed in five normal and 10 deoxycorticosterone acetate-treated swine. After 12 to 16 weeks of treatment, mean arterial pressure rose from the normal level of 110 to 120 to 164 +/- 4 mm Hg but was decreased to 131 +/- 4 mm Hg by anesthesia. In the normal animals, blood pressure under anesthesia was 114 +/- 9 mm Hg. Acute left kidney surgical denervation significantly decreased renal vascular resistance and increased renal blood flow, glomerular filtration rate, urine flow, and sodium excretion only in the treated animals. In an additional group of six normal and eight deoxycorticosterone acetate-treated swine, the responses to renal pharmacological denervation with intrarenal guanethidine were evaluated. Guanethidine had no significant effect on renal blood flow, vascular resistance, glomerular filtration rate, urine flow, or sodium excretion in the normal animals. In contrast, in the mineralocorticoid-hypertensive animals, guanethidine significantly decreased renal vascular resistance and caused a diuresis and natriuresis with no change in glomerular filtration rate. We conclude that, in deoxycorticosterone acetate-treated miniature swine with established hypertension, renal nerve activity appears to be elevated and important in determining renal hemodynamics and sodium and water excretion.

Tardive Dyskinesia

The basic pathogenesis of tardive dyskinesia appears to relate to chronic pharmacologic denervation of specific dopaminergic receptor sites in the striatum. The mainstay of treatment includes withdrawal of neuroleptics where feasible and the use of dopamine-depleting agents. The benefit of manipulating other neurotransmitters remains experimental. Treatment with neuroleptics should be avoided.

Sinus node function after autonomic blockade in normals and in sick sinus syndrome

Electrophysiologic studies were performed in 10 normals and 33 patients with sick sinus syndrome before and after total autonomic blockade with propranolol and atropine. In normals both corrected sinus node recovery time (SNRT) and sinoatrial conduction time (SACT) decreased significantly after autonomic blockade. In patients with sick sinus syndrome the corrected SNRT was abnormal (> 450 msec) in 16 (48.5%) cases before and 25 (76%) cases (> 285 msec) after autonomic ablation ( P < 0.02). Thirteen of 21 patients (62%) with normal intrinsic heart rate and all 12 cases with abnormally low intrinsic rate after autonomic blockade had abnormal corrected SNRT (> 285 msec). Mean SACT measured in 19 patients also shortened significantly following pharmacologic denervation. During control it was prolonged (> 226 msec) in 8 patients (44%). After autonomic blockade 2 of 13 patients with normal intrinsic heart rate and 3 of 6 with low intrinsic rate showed abnormal SACT (> 151 msec). The data suggest that the majority (76%) of patients with sick sinus syndrome have intrinsic abnormality of sinus node automaticity while in a minority (24%) disturbed autonomic regulation is the pathogenetic mechanism. Patients with normal intrinsic heart rate usually have normal intrinsic SACT, while a significant proportion of those with low intrinsic rate have abnormal perinodal conduction. Subjects with abnormal intrinsic heart rate have more severe disturbances of sinus node function than those with normal intrinsic rate.

Presynaptic α 2-adrenergic stimulation leads to growth hormone release in the dog

In previous studies we have shown that the α 2-adrenergic receptor agonist clonidine (CLON) releases growth hormone (GH) in conscious dogs, an effect abolished by the selective α 2-receptor antagonist yohimbine (YOH) and by reserpine, but not by the α 1-receptor antagonist prazosin (1). In the present work intravenous (iv) administration of CLON in concious dogs evoked a dose-related rise in plasma GH at doses of 2–8 ug/Kg, but not at 16 and 32 ug/Kg. Acute pretreatment with the selective inhibitor of norepinephrine (NE) synthesis, DU-18288, or with a potent antagonist of presynaptic α 2-receptors, mianserin abolished the GH rise induced by CLON (4 ug/Kg iv). In contrast, a 10-day-pretreatment with YOH greatly enhanced the GH-releasing effect of CLON (2 ug/Kg iv). In all these data indicate that in the dog: 1) CLON induces GH release via activation of α 2-adrenergic receptors; 2) these receptors are likely located on presynaptic sites [experiments with reserpine (1), DU-18288, mianserin, dose-response curve with CLON 2–32 ug/Kg iv] ; 3) the adrenergic receptors involved in GH release exhibit supersensitivity upon (YOH-induced) chronic pharmacologic denervation. In view of the inhibitory action of presynaptic α 2-adrenergic receptors (autoreceptors) on NE function, it may be envisioned that in the dog noradrenergic activation is inhibitory and not stimulatory to GH release.

Intrinsic heart rate in the dog determined by pharmacologic denervation.

Intrinsic heart rate was measured in 19 dogs in 76 experiments after autonomic blockade, using various forms of anesthesia. Measurements were made in conscious dogs (n = 16) and in dogs in neuroleptanesthesia (n = 54) or under pentobarbital sodium (n = 6). Temperature, arterial pH, and blood gases were kept within narrow limits. Adrenergic blockade was achieved by phenoxybenzamine (2 mg X kg-1) and propranolol (2 mg X kg-1, followed by 2 mg X kg-1 X h-1). The parasympathetic system was blocked either by atropine (0.5 mg X kg-1, followed by 0.5 mg X kg-1 X h-1) and hexamethonium (20 mg X kg-1, followed by 10 mg X kg-1 X h-1) or by atropine and bilateral cervical vagotomy. Administration of hexamethonium or vagotomy was needed to block the vagal cardioacceleration unmasked by the administration of muscarinic blocking agents in conscious dogs and in dogs in neuroleptanesthesia. The mean denervated heart rate was 142.8 beats/min. This value is higher than that reported for surgically denervated hearts, the difference very likely reflecting the activity of the intact parasympathetic intrinsic cardiac innervation in surgical preparations. The estimated intraindividual and interindividual SD were 9.7 and 19.4 beats/min, respectively. The highly significant interindividual variation (P less than 0.01) contradicts the concept of an intrinsic heart rate as a practically constant species-dependent quantity.

Subsensitivity of mouse salivary gland after pharmacological denervation.

Subsensitivity of mouse salivary gland after pharmacological denervation.

Pharmacological denervation supersensitivity of mouse salivary gland induced by chronic administ-ration of amitriptyline and promethazine.

Pharmacological denervation supersensitivity of mouse salivary gland induced by chronic administ-ration of amitriptyline and promethazine.

Tardive Dyskinesia

The basic pathogenesis of TD appears to relate to chronic pharmacologic denervation of specific dopaminergic receptor sites in the striatum. The pathophysiology of the disorder relates to the resultant denervation hypersensitivity. The mainstay of treatment includes withdrawal of neuroleptics when feasible and the use of dopamine-depleting agents. Enhancement of the striatal cholinergic input offers potential ancillary benefit to the alleviation of abnormal movements. The possibility of benefit from manipulating other neurotransmitters remains experimental. Treatment of TD with neuroleptics themselves is clearly treatment with the presumed offending agent and should be avoided. This shortsighted therapy may temporarily abate the pathophysiology of the condition, but it serves to aggravate its pathogenesis.