Degradation rates of acetylcholine receptors can be modified in the postjunctional plasma membrane of the vertebrate neuromuscular junction

Denervation of vertebrate muscle causes an acceleration of acetylcholine receptor turnover at the neuromuscular junction. This acceleration reflects the composite behavior of two populations of receptors: "original receptors" present at the junction at the time of denervation, and "new receptors" inserted into the denervated junction to replace the original receptors as they are degraded (Levitt, T. A., and M. M. Salpeter, 1981, Nature (Lond.), 291:239-241). The present study examined the degradation rate of original receptors to determine whether reinnervation could reverse the effect of denervation. Sternomastoid muscles in adult mice were denervated by either cutting or crushing the nerve, and the nerves either allowed to regenerate or ligated to prevent regeneration. The original receptors were labeled with 125I-alpha-bungarotoxin at the time of denervation, and their degradation rate followed by gamma counting. We found that when the nerve was not allowed to regenerate, the degradation decreased from a t1/2 of approximately 8-10 d to one of approximately 3 d (as reported earlier for denervated original receptors) and remained at that half-life throughout the experiment (approximately 36 d). If the axons were allowed to regenerate (which occurred asynchronously between day 14 and day 30 after nerve cut and between day 7 and 13 after nerve crush), the accelerated degradation rate of the original receptors reverted to a t1/2 of approximately 8 d. Our data lead us to conclude that the effect of denervation on the degradation rate of original receptors can be reversed by reinnervating. The nerve can thus slow the degradation rate of receptors previously inserted into the postsynaptic membrane.     

Adrenergic agonists induce heterologous sensitization of adenylate cyclase in NS20Y-D(2L) cells

Adenylate cyclase activity in NS20Y cells expressing D2L dopamine receptors was examined following chronic treatment with norepinephrine and epinephrine. Initial acute experiments revealed that both norepinephrine and epinephrine inhibited forskolin-stimulated cyclic AMP accumulation via D2 receptors. Furthermore, chronic 18 h activation of D2 dopamine receptors by norepinephrine or epinephrine induced a marked increase (>10-fold) in subsequent forskolin-stimulated cyclic AMP accumulation. This heterologous sensitization of adenylate cyclase activity was blocked by D2 dopamine receptor antagonists and by pertussis toxin pretreatment. In contrast, concurrent activation of Galpha(s) or adenylate cyclase did not appear to alter noradrenergic agonist-induced sensitization.     

Inhibitory effect of ouabain on epinephrine-induced stimulation of adrenal corticosterone secretion in rats.

Chronic administration of ouabain (3 mg/Kg body weight, subcutaneously, once daily for consecutive 15 days) definitely inhibited epinephrine-induced increase of adrenal corticosterone secretion. The inhibition rate increased along with frequency of ouabain administration. Increase in adrenal corticosterone synthesis and secretion by ACTH (20-50 mU/rat) administration was partially suppressed by pretreatment with chronic ouabain administration. A slight but significant increase of adrenal corticosterone secretion caused by epinephrine administration in hypophysectomized rats was also inhibited by pretreatment with ouabain administration. Chronic administration of neither phentolamine (1 mg/rat, intraperitoneally, once daily for consecutive 15 days) nor propranolol (3 mg/Kg body weight, subcutaneously, once daily for consecutive 15 days) caused significant changes in adrenal corticosterone secretion in response to ACTH as well as to epinephrine. Chronic administration of ouabain in rats causes not only elevated secretion of ACTH from anterior pituitary but also functional change in adrenals leading to suppression of corticosterone secretion in response to ACTH or epinephrine administration.     

Depletion of epinephrine in rat hypothalamus by a dopamine agonist,pergolide

The i.p. injection of pergolide mesylate, a dopamine agonist, at doses of 0.3–0.6 mg/kg led to a decrease in epinephrine concentration in rat hypothalamus. After a 0.6 mg/kg dose of pergolide mesylate, epinephrine concentration in hypothalamus decreased within 2 hr, reached a minimum concentration at about 8 hrs, and then returned toward control values. Norepinephrine N-methyltransferase activity was not decreased after pergolide injection in vivo nor was it inhibited by pergolide added in vitro at concentrations as high as 10^–3 M. Higher i.p. doses of less potent dopamine agonists, apomorphine (10 mg/kg) and lergotrile (3 mg/kg), also decreased epinephrine concentration in hypothalamus. The pergolideinduced decrease in hypothalamic epinephrine concentration was prevented by pretreatment with haloperidol or spiperone., antagonists of dopamine receptors. Activation of dopamine receptors appears to result in a decrease in epinephrine concentration in rat brain, possibly due to, enhanced release of epinephrine.     

Nerve regeneration-induced recovery of quinine avoidance after complete gustatory deafferentation of the tongue

The concentration-dependent decrease in quinine licking by rats is substantially attenuated by combined bilateral transection of the chorda tympani (CT) and glossopharyngeal (GL) nerves, but transection of either nerve alone produces marginal impairments at most. Here we tested whether regeneration of one or both of these nerves after combined transection would result in recovery of taste avoidance. Water-restricted rats were presented with a series of brief-access (5 s) taste trials (water and 0.003-3.0 mM quinine-HCl) in a 5-day test block of 40-min sessions both before nerve transection and starting 75-77 days after transection. Licking avoidance returned to presurgical levels when both nerves were allowed to regenerate. When only the GL was allowed to regenerate, performance did not differ from that of sham-transected animals. This suggests that even after considerable gustatory deafferentation, regeneration has the capacity to restore normal taste-guided behavior. Surprisingly, when only the CT was allowed to regenerate, avoidance behavior was severely impaired and was not different from that of rats in which regeneration of both nerves was prevented. Taking into account prior findings, it appears that the absence of the GL in the presence of an intact CT is fundamentally different from the absence of the GL in the presence of a regenerated CT with respect to some taste functions. This represents the first reported instance to our knowledge in which the capacity of a regenerated nerve to maintain taste-guided behavior was distinctly different from that of an intact nerve in a rodent model.     

Roles of alpha 1- and beta-adrenergic receptors in adrenergic responsiveness of liver cells formed after partial hepatectomy

The adrenergic receptor involved in the action of epinephrine changed dramatically during the process of active proliferation which follows partial hepatectomy. In control or sham-operated animals, the stimulation of glycogenolysis, gluconeogenesis and ureogenesis by epinephrine was mediated through alpha 1-adrenergic receptors. In contrast, in hepatocytes obtained from animals partially hepatectomized 3 days before experimentation, the receptor involved in the stimulation of these metabolic pathways by epinephrine was of the beta-adrenergic type. Interestingly, the adrenergic receptor involved in the metabolic actions of epinephrine, in hepatocytes from rats partially hepatectomized 7 days before experimentation was again of the alpha 1-subtype. Thus, it appears that during the process of liver regeneration which follows partial hepatectomy there is a transition in the type of adrenergic receptor involved in the hepatic actions of catecholamines from beta in the initial stages to later alpha 1. A similar transition seems to occur as the animal ages. Cyclic AMP accumulation in response to beta-adrenergic stimulation was significantly enhanced in hepatocytes obtained from rats partially hepatectomized 3 days before the experiment, as compared to control hepatocytes or cells obtained from animals operated 7 days before experimentation. This enhanced beta-adrenergic sensitivity is probably related to the increased number of beta-adrenergic receptors observed at this stage. However, a clear dissociation between cyclic AMP levels and metabolic effects was evidenced when the different conditions were compared. The number and affinity (for epinephrine or prazosin) of alpha 1-adrenergic receptors did not change at any stage of the process, which indicates that the markedly diminished alpha 1-adrenergic sensitivity observed in hepatocytes obtained from rats partially hepatectomized 3 days before experimentation is probably due to defective generation or intracellular processing of the alpha 1-adrenergic signal, rather than to changes at the receptor level.     

The effect of cold and heat pretreatments on anther culture response of Avena sativa and A. sterilis

The effect of stress pretreatments on embryo induction in anther cultures of selected genotypes of Avena sativa and A. sterilis was tested. A heat pretreatment of isolated anthers at +32°C for 5 days was best for the A. sativa line WW 18019 and for A. sterilis line CAV 2648. Genotype dependency may exist since in ‘Stout’ heat pretreatment did not increase embryo production. For A. sterilis 13 green and three albino regenerants were produced, of which five plants (haploids) survived transfer to the greenhouse. For A. sativa, 30 various differentiation media/treatment combinations were used in an attempt to regenerate plants from embryos, with no success. Seven day cold treatment of cut tillers increased slightly the response level in ‘Stout’ and was routinely used in subsequent experiments. Maltose proved to be better then sucrose as a carbon source for the genotypes tested. Fourteen percent maltose promoted the highest induction in A. sterilis, but the quality of embryos was improved in the presence of 10% maltose for both species. Sub-optimal carbohydrate levels did not enhance embryo induction in oats. This revised version was published online in June 2006 with corrections to the Cover Date.     

OBSERVATIONS ON SUPERNUMERARY HEAD FORMATION INDUCED BY LITHIUM CHLORIDE TREATMENT IN THE REGENERATING HYDRA, PELMATOHYDRA ROBUSTA

Lithium chloride treatment of hydras cut just proximal to the tentacle circle and just distal to the budding region induces a supernumerary head at the proximal cut surface. Such a supernumerary head does not appear in the normal course of regeneration. The bipolar hydra thus formed persists for several weeks and later separates to form two normal individuals. The supernumerary head is not formed at the proximal cut surface when the hydra is transected just distal to the budding zone and the distal portion is allowed to regenerate in the Li-containing medium. LiCl has a slight inhibitory effect on the regeneration of hypostomes or tentacles when the animal is cut at the base of the hypostome.     

Regulation of atrial natriuretic factor receptors by angiotensin II in rat vascular smooth muscle cells

Atrial natriuretic factor (ANF) is actively involved in the control of blood pressure and fluid homeostasis as a physiological antagonist of the renin-angiotensin system. To evaluate a possible interaction between ANF and angiotensin II (Ang-II) receptors, we investigated the effect of long term pretreatment (18 h) of rat cultured vascular smooth muscle cells with Ang-II. Binding of 125I-labeled ANF and cyclic GMP production induced by ANF were measured. After preincubation of the cells with Ang-II (1, 10, and 100 nM), the number of ANF binding sites (Bmax) was decreased by 30, 59, and 71%, respectively, with a slight decrease of the Kd values. Sar1-Ile8-Ang-II (100 nM), a specific Ang-II receptor antagonist, totally inhibited the down-regulation induced by Ang-II (10 nM). Moreover, the regulatory effect of Ang-II on ANF receptors appeared more slowly as compared to ANF homologous receptor regulation. Ang-II pretreatment did not desensitize but increased cyclic GMP production elicited by ANF, implying that only the number of non-guanylate cyclase-coupled receptors was affected. These findings, which were not observed with 100 nM of epinephrine, norepinephrine, histamine, serotonin, and Arg-vasopressin, demonstrate a specific and functional link between ANF and Ang-II receptors. This study also shows that the regulation of ANF receptors is heterogeneous, providing new evidence of multiple classes of ANF receptors.     

Inhibition of regeneration in the planarian Dugesia polychroa (Schmidt) by treatment with magnesium chloride: a morphological study of wound closure

At a concentration of 0.2% (21 m M) in culture water, magnesium chloride impaired muscle contraction and completely inhibited head regeneration in specimens of Dugesia polychroa cut prepharyngeally. The wound stayed open for nine days, with neoblasts accumulating beneath the wound without any signs of differentiation. Extremely delayed wound closure occurred by spreading epithelial cells, and was completed after 30 days in the magnesium chloride solution. Histological examination confirmed the absence of any regenerated head structures. Interestingly, the inhibitory effect was removed when such headless fragments were cut once more and kept in normal culture water: complete head regeneration then occurred at a normal rate. Among several possible explanations for the failure to regenerate, the following hypothesis is an attractive alternative: direct contact between parenchyma and epithelial cells during the period following injury seems to be an essential stimulus for the start of cell differentiation within the blastema, and the lack of such contact as a result of the drug action prevents normal regeneration. When the wound has eventually closed, a continuous basement membrane separates epithelium from parenchyma. Thus a direct contact between these tissues is never established. This revised version was published online in July 2006 with corrections to the Cover Date.     

Roles of alpha1- and beta-adrenergic receptors in adrenergic responsiveness of liver cells formed after partial hepatectomy

The adrenergic receptor involved in the action of epinephrine changed dramatically during the process of active proliferation which follows partial hepatectomy. In control or sham-operated animals, the stimulation of glycogenolysis, gluconeogenesis and ureogenesis by epinephrine was mediated through alpha₁-adrenergic receptors. In contrast, in hepatocytes obtained from animals partially hepatectomized 3 days before experimentation, the receptor involved in the stimulation of these metabolic pathways by epinephrine was of the beta-adrenergic type. Interestingly, the adrenergic receptor involved in the metabolic actions of epinephrine, in hepatocytes from rats partially hepatectomized 7 days before experimentation was again of the α₁-subtype. Thus, it appears that during the process of liver regeneration which follows partial hepatectomy there is a transition in the type of adrenergic receptor involved in the hepatic actions of catecholamines from β in the initial stages to later α₁. A similar transition seems to occur as the animal ages. Cyclic AMP accumulation in response to β-adrenergic stimulation was significantly enhanced in hepatocytes obtained from rats partially hepatectomized 3 days before the experiment, as compared to control hepatocytes or cells obtained from animals operated 7 days before experimentation. This enhanced β-adrenergic sensitivity is probably related to the increased number of β-adrenergic receptors observed at this stage. However, a clear dissociation between cyclic AMP levels and metabolic effects was evidenced when the different conditions were compared. The number and affinity (for epinephrine or prazosin) of α₁-adrenergic receptors did not change at any stage of the process, which indicates that the markedly diminished α₁-adrenergic sensitivity observed in hepatocytes obtained from rats partially hepatectomized 3 days before experimentation is probably due to defective generation or intracellular processing of the α₁-adrenergic signal, rather than to changes at the receptor level.     

The effect of epinephrine in local anesthesia on the survival of full- and split-thickness skin grafts: an experimental study

The effect of local anesthesia containing epinephrine on the survival of split- and full-thickness skin grafts remains unclear. In this blinded study, Xylocaine with or without epinephrine was injected subdermally prior to harvesting of split-thickness and full-thickness skin grafts on the dorsum of rabbits. After procurement, the grafts were placed back into their original donor sites. Statistical analysis of graft survival 7 days postoperatively revealed a significant decrease in survival for the full-thickness skin grafts treated with Xylocaine with epinephrine as compared with similar grafts without epinephrine (p less than 0.0005). No significant difference was noted for split-thickness skin-graft survival in grafts treated with Xylocaine with and without epinephrine (p greater than 0.1).     

A simple "soaking method" for RNA interference in the planarian<Emphasis Type="Italic"> Dugesia japonica</Emphasis>

A simple method was developed for RNA interference (RNAi) in the planarian Dugesia japonica. The DjIFb ( Dugesia japonica intermediate filament b) gene was used to evaluate the effect of RNAi because both the cDNA and an antiserum against the gene product were available. After transverse cutting at the pre- and post-pharyngeal regions, the middle part of the body fragment was soaked in water containing double-stranded RNA (dsRNA) for about 5 h and then allowed to regenerate in water. On the 5th day of regeneration, little DjIFb protein was detected in the new tissues. When the worms were cut after soaking in dsRNA water, no RNAi effect was observed, suggesting that the dsRNA was introduced through the cut surface. A high concentration of dsRNA or repeated "cutting and soaking" resulted in more effective RNAi. This simple soaking method in combination with expressed sequence tag analysis should be very useful for high-throughput analyses of gene functions in planarian regeneration.     

A role for Gi in control of thrombin receptor-phospholipase C coupling in human platelets

Stimulation of washed human platelets with alpha-thrombin was accompanied by aggregation, formation of inositol phosphates and phosphatidic acid, liberation of arachidonic acid, mobilization of intracellular Ca2+ stores, and influx of Ca2+ from the extracellular medium. Each of these responses was potentiated by a short pretreatment with epinephrine, although alone this agent was ineffective. A prolonged (5 min) stimulation with alpha-thrombin desensitized both phospholipase C and Ca2+ mobilization to a further thrombin challenge. Epinephrine added following thrombin desensitization restored both the ability of thrombin to release Ca2+ stores and stimulate inositol phospholipid hydrolysis. Resensitization was mediated by alpha 2-adrenergic receptors and lasted about 3 min, after which the Ca2+ levels returned again to basal levels. Pretreatment of platelets with phorbol dibutyrate at concentrations which specifically activate protein kinase C increased the rate of desensitization of the thrombin-induced release of Ca2+ stores and abolished the ability of epinephrine to restore the thrombin response. The protein kinase C inhibitor, staurosporine, blocked the inhibitory effect of phorbol ester and also reduced the rate of desensitization of thrombin and subsequent epinephrine action. These results suggest that thrombin activation of protein kinase C phosphorylates and inactivates a signaling protein which is common to both thrombin and alpha 2-adrenergic receptors. This protein is involved in thrombin stimulation of phospholipase C but is not directly stimulatory since epinephrine alone does not activate this enzyme. We searched for a known second messenger protein common to both thrombin and alpha 2-adrenergic receptors which was phosphorylated in intact platelets by protein kinase C in parallel with thrombin-induced desensitization. The alpha subunit of the inhibitory GTP-binding protein, Gi, was the only candidate which fulfilled all of these criteria as shown by immunoprecipitation. Therefore, we suggest that alpha i maintains the thrombin receptor in a state which can couple to phospholipase C when activated with thrombin. This permissive state of alpha i is blocked by phosphorylation by thrombin-activated protein kinase C.     

Long-term phorbol ester treatment down-regulates protein kinase C and sensitizes the phosphoinositide signaling pathway to hormone and growth factor stimulation. Evidence for a role of protein kinase C in agonist-induced desensitization

Exposure of a nontransformed, continuous line of epithelial cells derived from rat liver (WB cells) to epidermal growth factor, angiotensin II, [Arg8]vasopressin, and epinephrine resulted in rapid accumulation of the inositol phosphates (InsP) InsP1, InsP2, and InsP3. Although short-term (5-60 min) pretreatment of WB cells with the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) markedly attenuated InsP accumulation in response to all agonists, the inhibitory effects on the InsP response were lost after 2 h incubation with PMA; and, with extended (6-24 h) preincubation, a time-dependent potentiation of the InsP response to angiotensin II, epidermal growth factor and [Arg8]vasopressin was observed. The InsP response during a 15-min challenge with angiotensin II in cells pretreated for 18 h with 600 nM and 10 microM PMA was increased by 2-3-fold and 4-6-fold, respectively. Long-term (18 h) treatment with 600 nM and 10 microM PMA caused a similar 90-100% loss of measurable Ca2+/phospholipid-dependent enzyme (protein kinase C) activity in cytosolic and soluble particulate fractions. The effects of long-term PMA pretreatment do not represent a general enhancement of hormone responsiveness since the InsP response to epinephrine was not affected. In control cells, the InsP response to angiotensin II and epinephrine desensitized very rapidly. Long-term pretreatment with PMA greatly reduced the contribution of agonist-induced desensitization to the angiotensin II response; in contrast, the extent of desensitization occurring during incubation of WB cells with epinephrine was unaltered by long-term treatment with PMA suggesting that an additional mechanism may be involved in alpha 1-adrenergic receptor desensitization. No PMA-induced change in resting levels of [3H]phosphoinositides or the metabolism of exogenous [3H]inositol 1,4,5-trisphosphate by WB homogenates occurred. Stimulation of InsP formation in intact cells by NaF and activation of phospholipase C by GTP gamma S in membranes both were unaltered by short-term or long-term PMA pretreatment. These data are consistent with the idea that following long-term treatment of WB cells with PMA, the occurrence of agonist-induced desensitization of receptors linked to the phosphoinositide/Ca2+ signaling system is reduced, apparently at least in part due to the loss of contribution of a negative feedback regulatory role of protein kinase C.     

Some examples of the use of thin layer spectroelectrochemistry in the study of electron transfer between metals and enzymes

Some examples of the use of optically transparent thin layer electrodes to study electron transfer between platinum or carbon and enzymes are given. With respect to the biomolecule, thin layer spectroelectrochemistry has been used to choose the best electrochemical pretreatment necessary to promote the heterogeneous electron transfer (in the case of lactate dehydrogenase and glucose oxidase), to visualize intermediates in electrochemical reactions (in the case of glucose oxidase and horseradish peroxidase), to determine the electron transfer rate constant between oxidized, intermediate and reduced forms, and to prepare intermediates compounds (in the case of compound III of horseradish peroxidase), or to regenerate by electrochemical means enzymatic cofactors (in the case of hydrogenase).     

Contractile responses to epinephrine, serotonin, and potassium in arterial smooth muscle.

Effects of epinephrine, serotonin, and KCl on development of isometric tension were studied in strips of rat femoral and tail artery smooth muscle. Strkining differences were detected between the preparations. Tension development in tail artery smooth muscle was greatest with epinephrine, intermediate with serotonin, and least with K+. In femoral preparations tension developed was greatest with serotonin. Smaller but comparable contractions were elicited with epinephrine and K+. Theoretical and experimental dose-response curves for epinephrine and serotonin agreed closely, whereas curves for K+ differed markedly. The relationship between fractional response and fractional stimulus was hyperbolic for epinephrine and serotonin but sigmoid for K+. The Hill coefficient for serotonin was about 1, slightly smaller than 1 for epinephrine (0.5 to 0.8) and significantly greater for K+ (5 to 5). These findings show that responses elicited with either epinephrine or serotonin are mechanistically consistent with receptor occupancy theory. They suggest that specific receptors for K+ exist and that they may involve positive cooperative interactions similar those described for receptor mechanisms in electroplax.     

Topical epinephrine causes a decrease in density of beta-adrenergic receptors and cathecholamine-stimulated chloride transport in the rabbit cornea.

A single administration, or twice daily administration for 4.5 days, of topical 2% epinephrine to the rabbit eye in vivo causes a 30-40% decrease in the density of beta-adrenergic receptors on membranes prepared from the cornea. Such treatment also causes complete loss of the ability of excised corneas to respond to epinephrine in vitro with enhanced active chloride transport. These findings indicate that stimulation with a high concentration of catecholamine depresses the entire pathway from receptor to physiological response.     

Formation of pattern in regenerating tissue pieces of Hydra attenuata: I. Head-body proportion regulation

The precision with which an almost uniform sheet of hydra cells develops into a complete animal was measured quantitatively. Pieces of tissue of varying dimensions were cut from the body column of an adult hydra and allowed to regenerate. The regenerated animals were assayed for number of heads (hypostomes plus tentacle rings), head attempts (body tentacles), and basal discs. To ascertain whether the head and body were reformed in normal proportions, the average number of epithelial cells in the heads and bodies was measured. Pieces of tissue, from $\text{1}\text{2}$ to $\text{1}\text{20}$ an adult in size, formed heads that were a constant fraction of the regenerate. Thus, over a 10-fold size range, a proportioning mechanism was operating to divide the tissue into head area and body area quite precisely, but appeared to reach limits at the extremes of the range. However, the regenerates were not all normal miniatures with one hypostome and one basal disc. As the width-length ratio of the cut piece was increased beyond the circumference-length ratio of the intact body column, the incidence of extra hypostomes in the “head” and body tentacles and extra basal discs in the “body” rose dramatically. A proportioning mechanism based on the Gierer-Meinhardt model for pattern formation is presented to explain the results.     

Topical epinephrine causes a decrease in density of β-adrenergic receptors and catecholamine-stimulated chloride transport in the rabbit cornea

A single administration, or twice daily administration for 4.5 days, of topical 2% epinephrine to the rabbit eye in vivo causes a 30–40% decrease in the density of β-adrenergic receptors on membranes prepared from the cornea. Such treatment also causes complete loss of the ability of excised corneas to respond to epinephrine in vitro with enhanced active chloride transport. These findings indicate that stimulation with a high concentration of catecholamine depresses the entire pathway from receptor to physiological response.