Reversal of cerebral vasospasm using an intrathecally administered nitric oxide donor

A systematic approach to evaluation of suspected occupational lung disorders is presented to assist the health care professional with diagnosis, treatment, and prognosis, as well as management of the disease in the workplace.     

Differential effects of intrathecally administered delta and mu opioid receptor agonists on formalin-evoked nociception and on the expression of Fos-like immunoreactivity in the spinal cord of the rat

In this study we demonstrate that Drosophila calcium/calmodulin-dependent protein kinase II (CaMKII) is capable of complex regulation by autophosphorylation of the three threonines within its regulatory domain. Specifically, we show that autophosphorylation of threonine-287 in Drosophila CaMKII is equivalent to phosphorylation of threonine-286 in rat alpha CaMKII both in its ability to confer calcium independence on the enzyme and in the mechanistic details of how it becomes phosphorylated. Autophosphorylation of this residue occurs only within the holoenzyme structure and requires calmodulin (CaM) to be bound to the substrate subunit. Phosphorylation of threonine-306 and threonine-307 in the CaM binding domain of the Drosophila kinase occurs only in the absence of CaM, and this phosphorylation is capable of inhibiting further CaM binding. Additionally, our findings suggest that phosphorylation of threonine-306 and threonine-307 does not mimic bound CaM to alleviate the requirement for CaM binding to the substrate subunit for intermolecular threonine-287 phosphorylation. These results demonstrate that the mechanism of regulatory autophosphorylation of this kinase predates the split between invertebrates and vertebrates.     

Effect of intrathecally administered local anesthetics on protein phosphorylation in the spinal cord

To elucidate the biochemical mechanisms of spinal anesthesia, we studied the effects of procaine and tetracaine on protein phosphorylation in the mouse spinal cord. Mice were injected intrathecally with either procaine, tetracaine (67 mM/approximately 2%, 10 microL, N = 5/drug), or saline (N = 4/group). Five minutes after injection, animals were killed with a guillotine, and the spinal cord was removed. The caudal 3-cm cord segment was homogenized and centrifuged, and an aliquot of the supernatant was used for phosphorylation assays. Calcium-dependent phosphorylation was initiated by incubating the samples in buffer containing [gamma-32P]ATP at 37 degrees for 30 min. The proteins were electrophoresed using slab gel and two-dimensional electrophoresis, and phosphorylated proteins were visualized by autoradiography. The data demonstrated that spinal anesthesia changes the phosphorylation state of five endogenous substrate proteins with apparent molecular masses of 130 (protein-a), 105 (protein-b), 55 (protein-c), 47 (protein-d), and 33 (protein-e) kDa. In two-dimensional electrophoresis, protein-a resolved into two proteins (a1 and a2). Analysis of variance of the densitometric data suggested a significant effect for the treatment (F(2,16) 735, P < 0.00005). Post hoc comparisons with the saline-treated controls, using the Newman-Keuls test, indicated that local anesthetics significantly affected phosphoproteins (P < 0.05) except for protein-al in the tetracaine-treated group. Further characterization of these phosphoproteins should aid in determining their role in the signal transduction cascade affected by spinal anesthesia.     

Cardiovascular responses to cholinergic agonists in sinoaortic denervated rats

1. The cardiovascular effect of systemic nicotinic and muscarinic cholinergic stimulation were studied in conscious sham operated and sinoaortic denervated (SAD) rats, 7 days after the corresponding operation. 2. The administration of the nicotinic ganglionic agent, 1,1-dimethyl-4-phenylpiperazinium (DMPP, 50-100 micrograms.kg-1, i.v.) induced a fall of heart rate that was significantly higher in SAD rats than in sham rats. DMPP induced in sham rats an increase of arterial pressure but in SAD animals a biphasic response: an initial hypotension followed by an increase of arterial pressure. 3. Under muscarinic blockade, DMPP only showed a pressor and tachycardic action in both groups of rats without differences between them. 4. The muscarinic agonist, carbachol (0.1-10 micrograms.kg-1, i.v.) showed the same hypotensive and bradycardic action in both groups of rats. 5. Our results suggest that after 7 days of SAD, differences in the response to DMPP between sham and denervated animals could be due to the loss of baroreflex mechanisms. The increased bradycardic effect of DMPP in SAD rats could be mediated by a supersensitivity of parasympathetic ganglionic nicotinic receptors, whilst the sympathetic ganglionic nicotinic receptors remained unaltered. On the other side, the cardiovascular muscarinic responses to carbachol remain unaffected in SAD rats.     

Depression of C fiber-evoked activity by intrathecally administered reactive red 2 in rat thalamic neurons

To investigate the possible role of spinal purinoceptors in nociception, the potent P2-purinoceptor antagonist reactive red 2 was studied in rats under urethane anesthesia in which nociceptive activity was elicited by electrical stimulation of afferent C fibers in the sural nerve and recorded from single neurons in the ventrobasal complex of the thalamus. Intrathecal (i.t.) application of reactive red 2 (6-200 micrograms) caused a dose-dependent reduction of the evoked activity in thalamic neurons. The estimated ED50 was 30 micrograms, and the maximum depression of nociceptive activity amounted to about 70% of the control activity at a dose of 100 micrograms. Morphine, administered i.t. at a maximally effective dose (80 micrograms), inhibited the evoked nociceptive activity by only up to 55% of the control activity. An i.t. co-injection of reactive red 2 (100 micrograms) and morphine (80 micrograms) caused a maximum reduction of the evoked thalamic activity by up to 85% of the control activity, thus, exceeding significantly the effect elicited by either drug alone. Similarly, i.t. co-injection of almost equipotent dosages of reactive red 2 (30 micrograms) and morphine (30 micrograms) caused a maximum reduction of the evoked activity by up to 72% of the control activity, which again exceeded significantly the effect of either drug alone. The results suggest that in rats reactive red 2 exerts antinociception by blockade of P2-purinoceptors in the spinal cord and, hence, support the idea that ATP may play an important role in spinal transmission of nociceptive signals. An activation of the spinal opioid system does not seem to contribute to the effect of reactive red 2 but might act additive or even synergistically with its antinociceptive action.     

Plasma glucose and insulin responses to orally administered simple and complex carbohydrates

We have studied the effects of glucose, sucrose, and various starches on postprandial plasma glucose and insulin responses in 19 subjects. All carbohydrate loads were calculated to contain 50 gm. of glucose, and the response to each carbohydrate was tested twice: when given alone in a drink or when given in combination with other nutrients as a meal. The data demonstrate: (1) Glucose and sucrose elicited similar plasma glucose response curves, but sucrose elicited a somewhat greater (20 per cent) plasma insulin response. (2) Raw starch ingestion resulted in a 44 per cent lower glucose response and a 35-65 per cent lower insulin response than did either glucose or sucrose ingestion. (3) When carbohydrate was given as a meal the plasma glucose responses were 40-60 per cent lower than when the same carbohydrate was given as a drink, while the insulin responses were generally similar, and (4) when different cooked starches were compared, the plasma glucose and insulin responses to rice were significantly lower (50 per cent) than to potato. In conclusion, the size of the carbohydrate molecule appears to influence the postprandial glucose and insulin responses such that more complex carbohydrates (starches) elicit lower responses. This effect may be related to differences in digestion rather than to differences in absorption.     

Antinociceptive responses to nicotinic acetylcholine receptor ligands after systemic and intrathecal administration in mice

The objective of this study was to determine which nicotinic receptor subtypes are involved in antinociception and their site of action. For that, the antinociceptive effects of several nicotinic receptor ligands were evaluated in the tail-flick test both after s.c. and intrathecal (i.t.) administration. Nicotine and other nicotine agonists increased tail-flick latencies in a dose-dependent manner after both routes of administration. Epibatidine enantiomers were the most potent agonists examined. Cytisine, a potent nicotinic ligand, failed to elicit antinociception when injected either i.t. or s.c. Despite some similarities in the effects of nicotinic agonists after i.t. and s.c. injections, their rank-order potency was different. In contrast to the s.c. results, the stereoselectivity of nicotine's effect after i.t. administration was minimal. When various nicotinic antagonists were compared after i.t. and s.c. administration, the results showed that mecamylamine and dihydro-beta-erythroidine differ in potency and their degree of antagonism of some of the nicotinic agonists given i.t. These data suggest that different subtypes of nicotinic receptors may exist in the spinal cord. A good correlation was found between binding affinity to [3H]-nicotine binding sites and analgesic potency after i.t. (r = 0.82), suggesting the involvement of alpha 4 beta 2 receptor subunits. In contrast, studies with MLA and alpha-BGTX suggested a minimal role for alpha-BGTX-sensitive receptors in the antinociceptive effect of nicotinic agonists.     

The potential of subtype-selective neuronal nicotinic acetylcholine receptor agonists as therapeutic agents

Neuronal nicotinic acetylcholine receptors (NAChRs) are pentameric ligand-gated ion channel receptors which exist as different functional subunit combinations which apparently subserve different physiological functions as indicated by molecular biological and pharmacological techniques. It is possible to design and synthesize novel compounds that have greater selective affinities and efficacies than nicotine for different NAChRs, which should translate into different behavioral profiles and therapeutic potentials. Examples of NAChR agonists studied are nicotine, SIB-1508Y, SIB-1553A and epibatidine. These compounds have different degrees of selectivity for human recombinant NAChRs, different neurotransmitter release profiles in vitro and in vivo and differential behavioral profiles. Preclinical studies suggest that SIB-1508Y is a candidate for the treatment of the motor and cognitive deficits of Parkinson's disease, whereas SIB-1553A appears to have potential as a candidate for the treatment of Alzheimer's disease. Epibatidine has a strong analgesic profile, however the ratio between pharmacological activity and undesirable effects is so low that it is difficult to envisage the use of this compound therapeutically. Nicotine has a broad profile of pharmacological activity, for instance demonstrating activity in models for cognition and analgesia. As for epibatidine, the adverse effects of nicotine severely limits its therapeutic use in humans. The discovery of subtype-selective NAChR agonists such as SIB-1508Y and SIB-1553A provides a new class of neuropsychopharmacological agents with better therapeutic ratios than nonspecific agents such as nicotine.     

Differential blockade of the antinociceptive effects of centrally administered cannabinoids by SR141716A

We evaluated delta-9 tetrahydrocannabinol (Delta9-THC), delta-8 tetrahydrocannabinol (Delta8-THC), CP55,940 (CP55), 1-deoxy-11-hydroxy-Delta8-THC-dimethylheptyl (deoxy-HU210, a CB2-selective cannabinoid that also binds the CB1 receptor) and the endogenous cannabinoid anandamide (ANA) via i.c.v. and/or intrathecal (i.t.) routes of administration, alone and in combination with SR141716A (SR), a CB1 antagonist, using the tail-flick test. Our studies were performed in order better to characterize potential diversity in interactions of the cannabinoids with the cannabinoid (CB1) receptor. When SR was administered i.c.v. or i.p. before Delta9-THC, Delta8-THC or CP55 (i.c.v. or i.t.), SR was a potent antagonist and the blockade was complete (AD50 相似文献   

Antinociceptive effect of intrathecally administered pituitary adenylate cyclase activating polypeptide (PACAP) on the rat formalin test

Diclofenac (0.5-2 mM) dose- and time-dependently reduces the viability of isolated hepatocytes. This effect cannot be counteracted by the calcium channel blockers diltiazem (0.05-0.1 mM) and verapamil (0.05-0.5 mM), the calmodulin antagonist calmidazolium (0.01 mM) or Quin 2-AM (0.1 mM), an intracellular calcium chelating agent. On the contrary, verapamil even accentuates the toxic effects of diclofenac. It is concluded from these results, that diclofenac causes cell damage by other mechanisms than calcium overload.     

Allosteric modulation of Torpedo nicotinic acetylcholine receptor ion channel activity by noncompetitive agonists

Similar to other neuroreceptors of the vertebrate central nervous system, the nicotinic acetylcholine receptor (nAChR) is subject to modulatory control by allosterically acting ligands. Of particular interest in this regard are allosteric ligands that enhance the sensitivity of the receptor to its natural agonist acetylcholine (ACh), as such ligands could be useful as drugs in diseases associated with impaired nicotinic neurotransmission. Here we discuss the action of a novel class of nAChR ligands which act as allosterically potentiating ligands (APL) on the nicotinic responses induced by ACh and competitive agonists. In addition, APLs also act as noncompetitive agonists of very low efficacy, and as direct blockers of ACh-activated channels. These actions are observed with nAChRs from brain, muscle and electric tissue, and they depend on the structure of the APL and the concentration range applied. We focus here on Torpedo nAChR because (i) the unusual pharmacology of these ligands was first discovered with this system, and (ii) large quantities of this receptor are readily available for biochemical studies.     

Direct recording of nicotinic responses in presynaptic nerve terminals

Nicotinic acetylcholine receptors are widely expressed in the nervous system, but their functions remain poorly understood. One attractive hypothesis is that the receptors act presynaptically to modulate synaptic transmission. We provide a direct demonstration of presynaptic nicotinic receptors in situ by using whole-cell patch-clamp techniques to record currents in large presynaptic calyces that midbrain neurons form on ciliary neurons. Bath application of nicotine induced inward currents in the calyces capable of generating action potentials that overrode the limited space clamp achievable. The inward currents reversed near 0 mV and showed inward rectification common for neuronal nicotinic receptors. Tetrodotoxin (TTX) blocked the action potentials but not the inward currents. alpha-Bungarotoxin blocked both, consistent with the presynaptic receptors containing alpha7 subunits. Recording from the postsynaptic ciliary neurons during nicotine exposure revealed EPSCs that TTX blocked, presumably by blocking presynaptic action potentials. The postsynaptic cells also displayed bimodal inward currents caused by their own nicotinic receptors; the bimodal currents were not blocked by TTX but were blocked partially by alpha-bungarotoxin and completely by D-tubocurarine. Dye-filling with Lucifer yellow from the recording pipette confirmed the identity of patched structures and showed no dye transfer between calyx and ciliary neuron. When calyces or ciliary neurons were labeled en mass with neurobiotin and biocytin through nerve roots, dye transfer was rarely observed. Thus, electrical synapses were infrequent and unlikely to influence calyx responses. Immunochemical analysis of preganglionic nerve extracts identified receptors that bind alpha-bungarotoxin and contain alpha7 subunits. The results unambiguously document the existence of functional presynaptic nicotinic receptors.     

111Indium-diethylenetriamine pentaacetic acid cerebrospinal fluid flow studies predict distribution of intrathecally administered chemotherapy and outcome in patients with leptomeningeal metastases

Abnormal CSF flow can impair the distribution of intrathecally administered drugs. We examined the relationship between 111indium-diethylenetriamine pentaacetic acid (111In-DTPA) CSF flow studies and methotrexate levels in ventricular and lumbar CSF and correlated these findings with outcome in patients with leptomeningeal metastases (LM). Seven men and 10 women with LM (10 solid tumors, 6 lymphoma, 1 leukemia) received 12 mg methotrexate and 0.5 mCi 111In-DTPA by intra-Ommaya injection; images were obtained immediately and after 4, 24, and 48 hours. Ventricular and lumbar CSF methotrexate and radioactivity levels were measured 6 hours after injection. Thirteen patients had abnormal CSF flow studies, 9 with multiple sites of obstruction. CSF flow obstruction was observed at ventricular outlets in 13 patients, cerebral convexities in 9 and in the spine in 2. With one exception, all obstructions were explicable by tumor deposits on MRIs. For all patients, ventricular and lumbar methotrexate and radioactivity levels correlated closely. Three patients with a normal CSF flow study are alive at 15+, 7.5+, and 3.9+ months from treatment. Of 12 with abnormal CSF flow studies, 11 are dead a median of 2 months from diagnosis. Two patients had diffusely delayed flow studies and both developed methotrexate leukoencephalopathy. CSF flow studies using 111In-DTPA reliably predict distribution of intrathecal methotrexate. Abnormal flow studies correlate with structural abnormalities, are an unfavorable prognostic factor, and may predict intrathecal chemotherapy toxicity.     

Tacrine inhibits nicotinic secretory and current responses in adrenal chromaffin cells

Previous studies have demonstrated an infiltration of monocytes and increased levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the asthmatic lung. To study the possible effects of this cytokine upon the differentiation and function of these newly recruited monocytes, we have developed a model in which monocytes isolated from human peripheral blood were differentiated into macrophages in serum in the presence or absence of GM-CSF. After 7 days, the macrophages increased in size and granularity, had increased phagocytic activity, and expressed various adhesion molecules, CD14 and major histocompatibility complex (MHC) class II. The effects of GM-CSF on antigen presentation by cultured macrophages on the antigen-specific proliferative response of CD4+ T cells to Dermatophagoides pteronyssinus or purified protein derivative of tuberculin and the mitogen phytohaemagglutinin was determined. CD4+ T-cell proliferation was reduced when either antigen was presented by macrophages cultured in serum alone, compared with the values obtained with freshly isolated monocytes. However, CD4+ cell proliferation was comparable to that observed with monocytes when antigen was presented by macrophages which had been pre-cultured with 50 U/ml GM-CSF. CD4+ T-cell proliferation to phytohaemagglutinin was similar when all three populations were used as accessory cells. High numbers of macrophages partially suppressed CD4+ T-cell proliferation in response to antigen presented by monocytes, but there was no significant difference between macrophages cultured in the presence or absence of GM-CSF. This data suggests that GM-CSF directs monocyte differentiation into macrophages with an antigen-presenting, rather than a suppressive, phenotype. Elevated levels of GM-CSF in the asthmatic lung may therefore maintain recently recruited monocytes in an inflammatory and T-cell activating state.     

Competitive and non-competitive NMDA antagonists block the development of antinociceptive tolerance to morphine, but not to selective mu or delta opioid agonists in mice

N-Methyl-D-aspartate (NMDA) receptor antagonists have been shown to block the development of antinociceptive tolerance to morphine. Assessment of the effects of NMDA antagonists on development of antinociceptive tolerance to selective opioid mu (mu) and delta (delta) agonists, however, has not been reported. In these experiments, selective mu and delta receptor agonists, and morphine, were repeatedly administered to mice either supraspinally (i.c.v.) or systemically (s.c.), alone or after pretreatment with systemic NMDA antagonists. Antinociception was evaluated using a warm-water tail-flick test. Repeated i.c.v. injections of mu agonists including morphine, fentanyl, [D-Ala2, NMePhe4, Gly-ol]enkephalin (DAMGO) and Tyr-Pro-NMePhe-D-Pro-NH2 (PL017) or [D-Ala2, Glu4]deltorphin, a delta agonist, or s.c. injections of morphine or fentanyl, produced antinociceptive tolerance as shown by a significant rightward displacement of the agonist dose-response curves compared to controls. Single injections or repeated administration of MK801 (a non-competitive NMDA antagonist) or LY235959 (a competitive NMDA antagonist) at the doses employed in this study did not produce behavioral toxicity, antinociception or alter the acute antinociceptive effects of the tested opioid agonists. Consistent with previous reports, pretreatment with MK801 or LY235959 (30 min prior to agonist administration throughout the tolerance regimen) prevented the development of antinociceptive tolerance to i.c.v. or s.c. morphine. Neither NMDA antagonist, however, affected the development of antinociceptive tolerance to i.c.v. fentanyl, DAMGO, or [D-Ala2, Glu4]deltorphin. Additionally, MK801 pretreatment did not affect the development of antinociceptive tolerance to i.c.v. PL017 or to s.c. fentanyl. Further, MK801 pretreatment also did not affect the development of tolerance to the antinociception resulting from a cold-water swim-stress episode, previously shown to be a delta-opioid mediated effect. These data lead to the suggestion that the mechanisms of tolerance to receptor selective mu and delta opioids may be regulated differently from those associated with morphine. Additionally, these findings emphasize that conclusions reached with studies employing morphine cannot always be extended to 'opiates' in general.     

Human immune responses to influenza virus vaccines administered by systemic or mucosal routes

Healthy adult volunteers were immunized by parenteral or oral routes with trivalent inactivated influenza vaccine (A/Chile/1/83 (H1N1), A/Mississippi/1/85 (H3N2), and B/Ann Arbor/1/86), or intranasally with live attenuated, cold-adapted influenza type A/Texas/1/85 (H1N1) reassortant virus. In all volunteers, cells spontaneously secreting IgA, IgG or IgM antibodies specific to influenza virus were detected in peripheral blood on days 6-13 after immunization, and specific IgA, IgG and IgM antibodies to influenza vaccine were measured in sera and external secretions (saliva and nasal lavage). Following systemic immunization, a raise in specific antibodies of all isotypes was observed in sera beginning on day 13. Although small variations in IgA and IgM antibodies in saliva and nasal lavages were detected, antigen-specific IgG significantly increased between days 13 and 27. Intranasal administration of attenuated virus induced IgA and IgG antibodies in serum as well as in secretions. Serum antibodies were not substantially influenced by oral immunization, only a small increase in all isotypes was observed in volunteers' sera 21 days after ingestion of vaccine. However, in secretions, antigen-specific IgA and IgG responses were detected one week after immunization and reached a peak response on day 20. These studies show that different routes of immunization can be effective for the induction of specific antibodies, and support the concept of the common mucosal immune system in humans by demonstrating that the oral or intranasal administration of antigen-induced specific antibodies of IgA isotype in external secretions, preceded by the transient appearance in peripheral blood of specific antibody-producing cells.     

Real-time patch-cram detection of intracellular cGMP reveals long-term suppression of responses to NO and muscarinic agonists

Cyclic GMP (cGMP) is a crucial intracellular messenger in neuronal, muscle, and endocrine cells. The intracellular concentration of cGMP is regulated by various neurotransmitters, including acetylcholine (ACh) and nitric oxide (NO). While much is known about the biochemical steps leading to cGMP synthesis, little is known about cGMP kinetics in intact cells. Here, we use "patch-cramming," in which an excised, inside-out membrane patch containing cyclic nucleotide-gated ion channels is used as a biosensor, to obtain the first real-time measurements of cGMP in intact cells. Patch-cramming experiments on neuroblastoma cells show that both muscarinic agonists and NO rapidly elevate cGMP. NO elicits cGMP responses repeatedly without decrement, whereas responses to muscarinic agonists exhibit a profound and prolonged desensitization. Remarkably, muscarinic agonists also cause long-term (>30 min) suppression (LTS) of cGMP responses elicited by NO. Biochemical measurements reveal that rat sympathetic neurons also exhibit LTS of cGMP, suggesting that LTS is a widespread mechanism that may contribute to synaptic plasticity.