New Site Of Action Research Articles

Substance P presynaptically depresses the transmission of sensory input to bronchopulmonary neurons in the guinea pig nucleus tractus solitarii.

Substance P modulates the reflex regulation of respiratory function by its actions both peripherally and in the CNS, particularly in the nucleus tractus solitarii (NTS), the first central site for synaptic contact of the lung and airway afferent fibres. There is considerable evidence that the actions of substance P in the NTS augment respiratory reflex output, but the precise effects on synaptic transmission have not yet been determined. Therefore, we determined the effects of substance P on synaptic transmission at the first central synapses by using whole-cell voltage clamping in an NTS slice preparation. Studies were performed on second-order neurons in the slice anatomically identified as receiving monosynaptic input from sensory nerves in the lungs and airways. This was done by the fluorescent labelling of terminal boutons after 1,1'-dioctadecyl-3,3,3',3'-tetra-methylindocarbo-cyanine perchlorate (DiI) was applied via tracheal instillation. Substance P (1.0, 0.3 and 0.1 microM) significantly decreased the amplitude of excitatory postsynaptic currents (eEPSCs) evoked by stimulation of the tractus solitarius, in a concentration-dependent manner. The decrease was accompanied by an increase in the paired-pulse ratio of two consecutive eEPSCs, and a decrease in the frequency, but not the amplitude, of spontaneous EPSCs and miniature EPSCs, findings consistent with a presynaptic site of action. The effects were consistently and significantly attenuated by a neurokinin-1 (NK1) receptor antagonist (SR140333, 3 muM). The data suggest a new site of action for substance P in the NTS (NK1 receptors on the central terminals of sensory fibres) and a new mechanism (depression of synaptic transmission) for regulating respiratory reflex function.

Substance P presynaptically depresses the transmission of sensory input to bronchopulmonary neurons in the guinea pig nucleus tractus solitarii

Substance P modulates the reflex regulation of respiratory function by its actions both peripherally and in the CNS, particularly in the nucleus tractus solitarii (NTS), the first central site for synaptic contact of the lung and airway afferent fibres. There is considerable evidence that the actions of substance P in the NTS augment respiratory reflex output, but the precise effects on synaptic transmission have not yet been determined. Therefore, we determined the effects of substance P on synaptic transmission at the first central synapses by using whole-cell voltage clamping in an NTS slice preparation. Studies were performed on second-order neurons in the slice anatomically identified as receiving monosynaptic input from sensory nerves in the lungs and airways. This was done by the fluorescent labelling of terminal boutons after 1,1′-dioctadecyl-3,3,3′,3′-tetra-methylindocarbo-cyanine perchlorate (DiI) was applied via tracheal instillation. Substance P (1.0, 0.3 and 0.1 μM) significantly decreased the amplitude of excitatory postsynaptic currents (eEPSCs) evoked by stimulation of the tractus solitarius, in a concentration-dependent manner. The decrease was accompanied by an increase in the paired-pulse ratio of two consecutive eEPSCs, and a decrease in the frequency, but not the amplitude, of spontaneous EPSCs and miniature EPSCs, findings consistent with a presynaptic site of action. The effects were consistently and significantly attenuated by a neurokinin-1 (NK1) receptor antagonist (SR140333, 3 μM). The data suggest a new site of action for substance P in the NTS (NK1 receptors on the central terminals of sensory fibres) and a new mechanism (depression of synaptic transmission) for regulating respiratory reflex function.

Hypothalamic insulin signaling is required for inhibition of glucose production.

Circulating insulin inhibits endogenous glucose production. Here we report that bidirectional changes in hypothalamic insulin signaling affect glucose production. The infusion of either insulin or a small-molecule insulin mimetic in the third cerebral ventricle suppressed glucose production independent of circulating levels of insulin and of other glucoregulatory hormones. Conversely, central antagonism of insulin signaling impaired the ability of circulating insulin to inhibit glucose production. Finally, third-cerebral-ventricle administration of inhibitors of ATP-sensitive potassium channels, but not of antagonists of the central melanocortin receptors, also blunted the effect of hyperinsulinemia on glucose production. These results reveal a new site of action of insulin on glucose production and suggest that hypothalamic insulin resistance can contribute to hyperglycemia in type 2 diabetes mellitus.

Inhibition of 20 S and 26 S Proteasome Activity by Lithium Chloride

Lithium affects several enzymatic activities, however, the molecular mechanisms of lithium actions are not fully understood. We previously showed that LiCl interacts synergistically with all-trans-retinoic acid to promote terminal differentiation of WEHI-3B D(+) cells, a phenomenon accompanied by the recovery of the retinoid-induced loss of retinoic acid receptor alpha protein pools. Here, we demonstrate the effects of LiCl on proteasome-dependent degradation of retinoic acid receptor alpha proteins. LiCl alone, or in combination with all-trans-retinoic acid, increased cellular levels of ubiquitinated retinoic acid receptor alpha and markedly reduced chymotryptic-like activity of WEHI-3B D(+) 20 S and 26 S proteasome enzymes. Neither KCl nor all-trans-retinoic acid affected enzyme activity, whereas NaCl produced a modest reduction at relatively high concentrations. In addition, LiCl inhibited 20 S proteasome chymotryptic-like activity from rabbits but had no effect on tryptic-like activity of the 26 S proteasome. This effect has significant consequences in stabilizing the retinoic acid receptor alpha protein levels that are necessary to promote continued differentiation of leukemia cells in response to all-trans-retinoic acid. In support of this concept, combination of proteasome inhibitors beta-clastolactacystin or benzyloxycarbonyl-Leu-Leu-Phe with all-trans-retinoic acid increased differentiation of WEHI-3B D(+) cells in a manner that was analogous to the combination of LiCl and all-trans-retinoic acid.

Akt1/PKBα Protects PC12 Cells against the Parkinsonism-Inducing Neurotoxin 1-Methyl-4-phenylpyridinium and Reduces the Levels of Oxygen-Free Radicals

The phosphatidylinositol (PI)-3 kinase-Akt/PKB survival pathway protects neurons from apoptosis caused by diverse stress stimuli. However, its protective effect against neurotoxins that produce oxidative stress and neurodegeneration has not been investigated. We analyzed the effect of this pathway on the action of the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Overexpression of a membrane-targeted, N-myristylated fusion protein of enhanced green fluorescence protein (EGFP) and mouse Akt1 attenuated the apoptotic effect of the neurotoxin in PC12 cells. This effect was not due to protection of mitochondrial complex I activity or restoration of energy charge. Following MPP+-treatment, myr-EGFP-Akt1-transfected cells exhibited an unaltered mitochondrial membrane potential and lower ROS levels than control cells. These results provide a new site of action of Akt/PKB at the level of the oxidative detoxifying cell machinery and suggest that this effect may be responsible in part for the resistance of myr-EGFP-Akt1-expressing cells to oxidative stress and MPP+-induced apoptosis.

Localization and function of angiotensin AT 1 receptors

The distributions of angiotensin AT 1 and AT 2 receptors have been mapped by in vitro autoradiography throughout most tissues of many mammals, including humans. In addition to confirming that AT 1 receptors occur in sites known to be targets for the physiologic actions of angiotensin, such as the adrenal cortex and medulla, renal glomeruli and proximal tubules, vascular and cardiac muscle and brain circumventricular organs, many new sites of action have been demonstrated. In the kidney, AT 1 receptors occur in high density in renal medullary interstitial cells. The function of these cells, which span the interstitial space between the tubules and the vasa rectae, remains to be determined. Renal medullary interstitial cells possess receptors for a number of vasoactive hormones in addition to AT 1 receptors and this, in concert with their anatomic location, suggests they may be important for the regulation of fluid reabsorption or renal medullary blood flow. In the heart, the highest densities of AT 1 receptors occur in association with the conduction system and vagal ganglia. In the central nervous system, high AT 1 receptor densities occur in many regions behind the blood–brain barrier, supporting a role for neurally derived angiotensin as a neuromodulator. The physiologic role of angiotensin in many of these brain sites remains to be determined. The AT 2 receptor also has a characteristic distribution in several tissues including the adrenal gland, heart, and brain. The role of this receptor in physiology is being elucidated, but it appears to inhibit proliferation and to participate in development. Thus, receptor-binding studies, localizing the distribution of AT 1 and AT 2 receptors, provide many insights into novel physiologic roles of angiotensin.

Localization of Sir2p: the nucleolus as a compartment for silent information regulators.

In wild-type budding yeast strains, the proteins encoded by SIR3, SIR4 and RAP1 co-localize with telomeric DNA in a limited number of foci in interphase nuclei. Immunostaining of Sir2p shows that in addition to a punctate staining that coincides with Rap1 foci, Sir2p localizes to a subdomain of the nucleolus. The presence of Sir2p at both the spacer of the rDNA repeat and at telomeres is confirmed by formaldehyde cross-linking and immunoprecipitation with anti-Sir2p antibodies. In strains lacking Sir4p, Sir3p becomes concentrated in the nucleolus, by a pathway requiring SIR2 and UTH4, a gene that regulates life span in yeast. The unexpected nucleolar localization of Sir2p and Sir3p correlates with observed effects of sir mutations on rDNA stability and yeast longevity, defining a new site of action for silent information regulatory factors.

Pharmacological characterization of a new purinergic receptor site in rabbit aorta

1. 1. The pharmacological properties of a vasodilating purine-activated receptor that is not a P 1 or P 2-purinoceptor were investigated. 2. 2. In rabbit isolated thoracic aorta precontracted with noradrenaline, ATP induced a 50% relaxation at 0.25 mM ( EC50%); in the absence of endothelium, EC50% was 2.5 mM. 3. 3. Adenosine induced a relaxation that was not different in both the presence and absence of endothelium, being EC50% 0.48 and 0.37 mM, respectively. 4. 4. The potent and selective P 2 y -purinoceptor agonist 2-methylthio-ATP (0.03–10 μM) induced a relaxation only in the presence of endothelium. 5. 5. In de-endothelialized aorta, 8-phenyltheophilline (8-PT: P 1 antagonist) and 3,7-dimethyl-1-propargylxanthine (DMPX: A 2 antagonist) did not antagonize ATP- and adenosine-induced relaxation. 6. 6. The present data support the presence of a new site of action for purines in rabbit isolated thoracic aorta. 7. 7. A P 3 subtype of purinoceptor, that may be identified in the hypothesized “nucleotide” receptor, is proposed.

Arthropod toxins as leads for novel insecticides: An assessment of polyamine amides as glutamate antagonists

In the search for new toxins, preferably with new sites of action, the polyamine amides represent a new class of compounds with potential as insecticides and as pharmaceutical agents due to their antagonism of ligand-gated cation channels. In particular, they are potent antagonists of the l-glutamate receptors of insect skeletal muscle. In this paper, we report on synthetic studies to produce hybrid analogues based upon the argiotoxin spider toxins and philanthotoxin-433 which is obtained from a solitary, parasitic wasp. We speculate upon possible modes and sites of action for these antagonists and we discuss their potential as insecticides and in the possible treatment of ischaemic damage. The synthesis and characterization of 4-hydroxyphenylpropanoylspermine is reported and the locust muscle biological assay is described. Using this pharmacological screen, structure-activity relationships have been determined in our laboratories. These are reviewed in the light of the current literature. Voltage clamp studies of the synthetic analogue philanthotoxin-343 and the effects of this polyamine amide on glutamate receptors expressed in Xenopus oocytes are outlined. In conclusion, a description of our current ideas and understanding of the many sites and modes of action of the polyamine amides, based both upon our own studies and also upon those recently reported, is presented.

Vascular involvement in resting muscle thermogenesis: a new site of action for thermogenic drugs

Vascular involvement in resting muscle thermogenesis: a new site of action for thermogenic drugs

Cocaine inhibits baroreflex control of blood pressure by actions at arterial baroreceptors

Blood pressure and heart rate often increase during cocaine intoxication, but the mechanisms of these cardiovascular responses are poorly understood. The most often suggested theories are central nervous system mechanisms involving the blockade of neuronal transmitter uptake. Cocaine also has potent local anesthetic properties, and in this study we tested the possible role of peripheral actions of cocaine at baroreceptor afferents. Single fiber baroreceptors were recorded using an in vitro preparation of the rat aortic arch. Diameter, pressure, and baroreceptor discharge were recorded. Cocaine perfused through the lumen of the aortic arch at a suprathreshold pressure reduced baroreceptor discharge within 90 s of entering the lumen of the aorta. Slow ramps of pressure elicited complete pressure- and diameter-discharge curves every 5 min. Beginning at about 1 microM, cocaine inhibited baroreceptor function; threshold increased, the maximum discharge decreased, and at 100 microM cocaine, all discharge ceased. The vasodilator nitroprusside or the alpha 1-adrenoreceptor antagonist prazosin did not affect baroreceptor responses to cocaine. In in vivo tests in rabbits, cocaine that perfused through a vascularly isolated carotid sinus reduced the slope of the baroreflex relationship between carotid sinus pressure and systemic mean arterial pressure. Significant depression of baroreceptor function was found at concentrations similar to the plasma cocaine levels measured in clinical studies. The local anesthetic properties of cocaine may be involved in baroreceptor effects. Our studies suggest a possible contributing role of a new site of action of cocaine outside the central nervous system. Compromise of baroreceptor reflexes could facilitate the development of serious cardiovascular complications associated with cocaine abuse.

Nuclear epidermal growth factor (EGF) receptor as a new site of action for EGF

Nuclear epidermal growth factor (EGF) receptor as a new site of action for EGF

The distribution of opioid binding subtypes in the bovine adrenal medulla

Autoradiography has been used to examine the distribution of opioid binding subtypes in the bovine adrenal gland. Specific opioid binding sites were restricted to the adrenal medulla. Kappa sites, labelled with [ 3H]bremazocine (in the presence of excess unlabelled mu and delta ligands), were highly concentrated over nerve tracts. These nerve tract associated binding sites were sensitive to competition by the endogenous opioid, dynorphin (1–13). Specific [ 3H]bremazocine binding sites were also found over the adrenal medullary chromaffin tissue. These binding sites were concentrated over the peripheral, adrenaline-containing region of the medulla and were sensitive to competition by diprenorphine but not dynorphin (1–13). Delta opioid sites, labelled with [ 3H][ d-Ala 2, d-Leu 5] enkephalin (in the presence of excess unlabelled mu ligand) were selectively localized to the central, noradrenaline-containing region of the adrenal medulla. Mu opioid sites, labelled with [ 3H][ d-Ala 2, NMePhe 4,Gly-ol 5]enkephalin, were low in number and distributed throughout the adrenal medulla. These studies demonstrate that mu, delta and two distinct kappa opioid binding sites are differently distributed within the bovine adrenal medulla and suggest possible new sites of action for the adrenal medullary opioid peptides.

Role of thrombin in the proliferative response of T-47D mammary tumor cells: Mitogenic action and pleiotropic modifications induced together with epidermal growth factor and insulin

The growth of the human metastatic cell line (T-47D) in a chemically defined medium (DM) is shown to be dependent on the presence of three traditional growth factors: epidermal growth factor, insulin, and transferrin. The addition of thrombin further stimulates its growth. The mitogenic action on a human mammary tumor cell line from epithelial origin is a novel action of thrombin. Cells in the DM show striking morphological changes which are dramatically enhanced by the addition of thrombin. These observations are part of a pleiotropic response to the growth factors: the protein content of the cells increases in the defined medium; the 2DG gels of the 35S- and 32P-labeled proteins show important changes in spots, several of which are probably of cytoskeletal origin. It is also shown that cells in a semisolid growth factor-supplemented medium have growth advantages over their counterparts grown with serum. All the phenotypic changes mentioned above reveal the important role of growth factors in the growth and behavior of this mammary cell line. The results obtained with thrombin indicate a new site of action of this enzyme which may be important in the metastatic spread of human mammary tumor cells.

Anticholinergic agents prevent guinea-pig airway constriction induced by histamine, bradykinin and leukotriene C 4: Relationship to circulating TXA 2

Various doses of histamine, bradykinin and leukotriene C 4 caused bronchoconstriction in anaesthetized guinea-pigs which were breathing spontaneously or artificially ventilated; there was a simultaneous, dose-related increase of circulating TXA 2. The animals were prepared for continuous recording of extracororeal circulation in order to detect the appearance in the blood of bioassayable levels of TXA 2-like substance. Furthermore, a TXA 2 derivative, the mono-O-Me-TXB 2, was radioimmunoassayed. Atropine, oxytropium bromide and ipratropium bromide given in μmol doses prevented both the increased airway resistance and the release of TXA 2-like substance in the blood. Pirenzepine dihydrochloride was the least active of the drugs tested. The protecting activity of anticholinergics and the relationship with their ability to affect TXA 2-like substance generation suggest a new site of action for these drugs besides the blockade of muscarinic receptors.

Atropine inhibits thromboxane A2 generation in isolated lungs of the guinea-pig.

1 Histamine (0.5 to 5 micrograms) and slow reacting substance of anaphylaxis (SRS-A, 0.05 to 0.3 u), injected in the isolated, perfused lungs of normal and ovalbumin-sensitized guinea-pigs, promote formation and release of thromboxane A2(TXA2) and other arachidonate metabolites, the effect being more pronounced in sensitized lungs. 2 Carbachol injected (1 to 10 micrograms) or perfused (1 micrograms ml-1 min-1) through normal or sensitized lungs does not elicit formation of TXA2 and prostaglandins. Furthermore the increased generation of arachidonate metabolites due to histamine is not altered by carbachol. 3 Atropine and ipratropium bromide (1 microgram ml-1 min-1) reduce significantly the increased rate of production of TXA2 caused by histamine and SRS-A both in normal and sensitized lungs, whereas hexamethonium (10 to 25 micrograms ml-1 min-1) is ineffective. 4 The mechanism of action of atropine in inhibiting the increased generation of TXA2 is clearly not related to its antimuscarinic or antihistaminic properties. The drug might act at the early events, involved in the activation of arachidonic acid metabolism. The results suggest new sites of action for atropine which, besides the control of the vagal bronchomotor tone, interferes directly with the primary mediators of anaphylaxis.

Cardiovascular effects of angiotensin mediated by the central nervous system.

Cardiovascular effects of angiotensin mediated by the central nervous system.

Autoradiographic distribution studies of 3h-labeled prostaglandin e 1 in mice. prostaglandins and related factors 31

3H-labeled prostaglandin E 1 (2–7 μg) was injected intravenously into mice and the distribution of radioactive material was studied by autoradiography. Autoradiograms obtained from sagittal sections of the whole body showed high concentration of 3H in the kidney, liver and connective tissue. Lower but significant uptakes were also observed in the lungs and in the myometrium of uterus. The results were discussed in relation to current concepts of metabolism and biological effects of prosta- glandin E 1, and also in relation to possible new sites of action.