Inhibition of N-type calcium channels: the only mechanism by which presynaptic alpha 2-autoreceptors control sympathetic transmitter release

Alpha 2-Adrenoceptors are known to inhibit voltage-dependent Ca2+ channels located at neuronal cell bodies; the present study investigated whether this or alternative mechanisms, possibly downstream of Ca2+ entry, underlie the presynaptic alpha 2-adrenergic modulation of transmitter release from chick sympathetic neurons. Using chick sympathetic neurons, overflow of previously incorporated [3H]noradrenaline was elicited in the presence of extracellular Ca2+ by electrical pulses, 25 mM K+ or 10 microM nicotine, or by adding Ca2+ to otherwise Ca(2+)-free medium when cells had been made permeable by the calcium ionophore A23187 or by alpha-latrotoxin. Pretreatment of neurons with the N-type Ca2+ channel blocker omega-conotoxin GVIA and application of the alpha 2-adrenergic agonist UK 14304 reduced the overflow elicited by electrical pulses, K+ or nicotine, but not the overflow caused by Ca2+ after permeabilization with alpha-latrotoxin or A23187. In contrast, the L-type Ca2+ channel blocker nitrendipine reduced the overflow due to K+ and nicotine, but not the overflow following electrical stimulation or alpha-latrotoxin- and A23187-permeabilization. The inhibition of electrically evoked overflow by UK 14304 persisted in the presence of nitrendipine and the L-type Ca2+ channel agonist BayK 8644, which per se enhanced overflow. In omega-conotoxin GVIA-treated cultures, electrically evoked overflow was also enhanced by BayK 8644 and almost reached the value obtained in untreated neurons. However, UK 14304 lost its effect under these conditions. Whole-cell recordings of voltage-activated Ca2+ currents corroborated these results: UK 14304 inhibited Ca2+ currents by 33%, nitrendipine caused a 7% reduction, and BayK 8644 increased the currents by 30%. Moreover, the dihydropyridines failed to abolish the inhibition by UK 14304, but pretreatment with omega-conotoxin GVIA, which reduced mean amplitude from 0.95 to 0.23 nA, entirely prevented alpha 2-adrenergic effects. Our results indicate that the alpha 2-autoreceptor-mediated modulation of noradrenaline release from chick sympathetic neurons relies exclusively on the inhibition of omega-conotoxin GVIA-sensitive N-type Ca2+ channels. Mechanisms downstream of these channels and voltage-sensitive Ca2+ channels other than N-type appear not to be important.     

Effects of imidazoline derivatives on cholinergic motility in guinea-pig ileum: involvement of presynaptic alpha2-adrenoceptors or imidazoline receptors?

The present study investigates the possibility that imidazoline receptors mediate modulation of cholinergic motor functions of the guinea-pig ileum. For this purpose, the effects of a series of compounds with known affinity for alpha2-adrenoceptors and/or imidazoline recognition sites were examined on the cholinergic twitch contractions evoked by electrical field stimulation (0.1 Hz) of longitudinal muscle-myenteric plexus preparations. Additional experiments were carried out on ileal strips preincubated with [3H]choline, superfused with physiological salt solution containing hemicholinium-3, and subjected to electrical field stimulation (1 Hz). The stimulation-induced outflow of radioactivity was taken as an index of endogenous acetylcholine release. Alpha-methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline and xylazine caused a concentration-dependent inhibition of twitch responses (IC50 from 0.13 to 1.05 microM; Emax from 85.9 to 92.5%). Rilmenidine and agmatine were less potent in reducing the twitch activity, and the latter compound acted also with low intrinsic activity (IC50=44.9 microM; Emax=35.5%). In interaction experiments, the inhibitory action of clonidine on twitch responses was competitively antagonized by RX 821002 (2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline), idazoxan, rauwolscine, yohimbine and BRL 44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)-methyl] -4,5-dihydroimidazoline), whereas prazosin (10 microM), ARC 239 (2-(2,4-(O-methoxy-phenyl)-piperazin-1-yl)-ethyl-4,4-dimethyl- 1,3-(2H,4H)-isoquinolindione; 10 microM) and BRL 41992 (1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo[c,f]imidazol[1,5-a]a zepine; 10 microM) were without effect. Rauwolscine antagonized the inhibitory effects of various agonists on ileal twitch activity in a competitive manner and with similar potency. Agmatine and idazoxan did not significantly modify the twitch contractions when tested in the presence of alpha2-adrenoceptor blockade by rauwolscine (3 microM) or RX 821002 (1 microM). Linear regression analysis showed that the affinity values of antagonists correlated with their affinity at the alpha2A and alpha2D binding sites as well as at previously classified alpha2A/D adrenoceptor subtypes, whereas no significant correlation was obtained when comparing the potency estimates of agonists and antagonists with the affinity at I1 or I2 binding sites. When tested on the electrically induced outflow of tritium, alpha-methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline, xylazine and rilmenidine yielded inhibitory concentration-response curves which were shifted rightward to a similar extent in the presence of rauwolscine (3 microM). In the absence of further drugs, agmatine significantly reduced the evoked tritium outflow at the highest concentrations tested (10 and 100 microM), whereas idazoxan (up to 100 microM) was without effect. When RX 821002 (1 microM) was added to the superfusion medium, neither agmatine nor idazoxan modified the evoked outflow of radioactivity. The results argue against modulation by imidazoline receptors of acetylcholine release from myenteric plexus nerve terminals. They provide evidence that compounds endowed with imidazoline-like structures affect the cholinergic motor activity of the guinea-pig ileum by interacting with presynaptic alpha2-adrenoceptors belonging to the alpha2D subtype.     

Ontogeny of thirst in the rat: Effects of hypertonic saline, polyethylene glycol, and vena cava ligation.

Describes 3 experiments in which over 465 Sprague-Dawley rats of various ages either (a) were injected with hypertonic sodium chloride solutions to produce intracellular dehydration, (b) were injected with polyethylene glycol to induce hypovolemia, or (c) underwent ligation of the inferior vena cava to stimulate the renin-angiotensin system. 16-day-old Ss drank like adults after injection of polyethylene glycol. Hypertonic saline injection did not elicit adultlike drinking until 30 days of age, and vena cava ligation did not produce adult levels of water consumption until 42 days postnatally. It is concluded that treatments which have been associated with different mechanisms of thirst, therefore, first become effective at different times during ontogeny. (27 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Engraftment of human kidney tissue in rat radiation chimera: I. A new model of human kidney allograft rejection

BACKGROUND: We have recently shown that lethally irradiated normal strains of mice and rats, reconstituted with bone marrow from severe combined immune deficiency (SCID) mice, can be engrafted with human peripheral blood mononuclear cells (PBMC). METHODS: The feasibility of transplanting human renal tissue under the kidney capsule of the SCID/Lewis and SCID/nude radiation chimera and the effects of intraperitoneal infusion of allogeneic human PBMC on the human renal implants were investigated by histology, electron microscopy, immunohistochemistry, and fluorescence-activated cell sorter analysis. RESULTS: Sequential evaluation of the human renal implants from 10 days to 2 months after transplantation showed that human parenchymal elements survive in the implants up to 2 months after transplantation. The overall architecture of the transplanted kidney tissue and the normal structure of individual cells in the glomeruli and tubuli were preserved. Infusion of allogeneic human PBMC after kidney implantation resulted in patchy cellular infiltrates, composed mainly of activated human T cells, and led to prompt rejection of the human renal tissue, whereas no signs of inflammation were observed in human renal implants of chimeric rats that did not receive human PBMC. Treatment with OKT3 antibody, anti-human CD25 antibody, or CTLA4Ig fusion protein in vivo ameliorated the rejection process. CONCLUSIONS: Human adult kidney fragments transplanted into SCID-like rats transiently retain competent parenchymal structures. When these grafts are combined with allogeneic human PBMC, acute cellular rejection develops. We suggest that this chimeric model might be useful for the investigation of the effects of experimental manipulation on the kinetics of the inflammatory response during human renal allograft rejection.     

Postnatal changes of nicotinic acetylcholine receptor alpha 2, alpha 3, alpha 4, alpha 7 and beta 2 subunits genes expression in rat brain

We have previously demonstrated the development of acoustically reflective liposomes as a novel ultrasound contrast agent, that can be conjugated to antibodies for site specific acoustic enhancement of pathologically altered vascular tissue. The liposomes are echogenic due to the lipid composition, without gas entrapment, and have a size of less than one micron (Alkan-Onyuksel et al., 1996). When conjugated to anti-fibrinogen antibodies, the liposomes have the ability to attach to fibrin coated surfaces and thrombi in vitro as demonstrated by scanning electron microscopy and ultrasound imaging (Demos et al., 1997a). Anti-fibrinogen liposomes were shown to attach to fibrous atheroma and thrombi in a Yucatan miniswine model of induced atherosclerosis whereas liposomes conjugated to anti-intercellular adhesion molecule-1 (anti-ICAM-1) were demonstrated to target early stage atherosclerotic plaques (Demos et al., 1997b). The purpose of this study is to evaluate the binding characteristics of anti-fibrinogen liposomes in vitro under a variety of flow conditions in order to optimize the targeting ability of the immunoliposomes. Radiolabeled anti-fibrinogen liposomes were applied to fibrin coated filter paper and placed in a flow circuit under controlled flow conditions. Flow conditions were altered to study the effects of different shear stresses, temperature, plasma flow and pulsatile flow on the retention of liposomes to fibrin after set time periods. The retention of liposomes conjugated to polyclonal and monoclonal antibodies as well as Fab fragments made from monoclonal antibodies were compared. The binding characteristics of liposomes conjugated to different quantities of polyclonal antibodies were analyzed. At physiological shear stress of 1.5 N/m2 (15 dynes/cm2) over 70% of the liposomes remained attached to fibrin after two hours. A smaller and greater portion of the liposomes remained attached at higher and lower shear stresses respectively. Plasma components and temperature had no effect on liposomal retention whereas pulsatile flow resulted in a slight reduction in binding. Monoclonal antibodies showed a slight trend of reduced retention to fibrin over time as compared with polyclonal antibodies and Fab fragments. The quantity of antibody conjugated to the liposomes plays a role in liposome retention as demonstrated by the reduction in liposome retention caused by reducing the quantity of antibody conjugated to the liposomes. Anti-fibrinogen liposomes were retained to the fibrin surface to a large extent under all flow conditions likely to occur in vivo and therefore can provide site specific ultrasound contrast for a long enough time period to allow for imaging after injection.     

Differential distribution of alpha2A and alpha2C adrenergic receptor immunoreactivity in the rat spinal cord

alpha2-Adrenergic receptors (alpha2-ARs) mediate a number of physiological phenomena, including spinal analgesia. We have developed subtype-selective antisera against the C termini of the alpha2A-AR and alpha2C-AR to investigate the relative distribution and cellular source or sources of these receptor subtypes in the rat spinal cord. Immunoreactivity (IR) for both receptor subtypes was observed in the superficial layers of the dorsal horn of the spinal cord. Our results suggest that the primary localization of the alpha2A-AR in the rat spinal cord is on the terminals of capsaicin-sensitive, substance P (SP)-containing primary afferent fibers. In contrast, the majority of alpha2C-AR-IR was not of primary afferent origin, not strongly colocalized with SP-IR, and not sensitive to neonatal capsaicin treatment. Spinal alpha2C-AR-IR does not appear to colocalize with the neurokinin-1 receptor, nor is it localized on astrocytes, as evidenced by a lack of costaining with the glial marker GFAP. However, some colocalization was observed between alpha2C-AR-IR and enkephalin-IR, suggesting that the alpha2C-AR may be expressed by a subset of spinal interneurons. Interestingly, neither subtype was detected on descending noradrenergic terminals. These results indicate that the alpha2-AR subtypes investigated are likely expressed by different subpopulations of neurons and may therefore subserve different physiological functions in the spinal cord, with the alpha2A-AR being more likely to play a role in the modulation of nociceptive information.     

Expression of laminin alpha 1, alpha 5 and beta 2 chains during embryogenesis of the kidney and vasculature

Laminins, found predominantly in basement membranes, are large glycoproteins consisting of different subsets of alpha, beta and gamma chain subunits. To resolve conflicting data in the literature concerning coexpression of alpha 1 and beta 2 chains, expression of alpha 1 chain was studied with two different antisera against the E3 fragment of laminin alpha 1 chain. Expression of the alpha 1 chain was seen in several types of epithelial basement membranes throughout development, but its expression in rat glomerular basement membranes and some other types of epithelial basement membranes occurred only during early stages of development. By contrast, beta 2 chains were detected by immunofluorescence only during advanced stages of glomerulogenesis and vascular development. By Northern and Western blots, beta 2 chains were detected somewhat earlier, but in situ hybridization revealed that beta 2 chain was also confined to vasculature during the earlier stages. It thus seems that, in the tissues studied here, the expression of alpha 1 and beta 2 chains was mutually exclusive. To explore whether the newly described alpha 5 chain is expressed in locations lacking alpha 1 chain, expression of alpha 5 chain was studied by Northern blots and in situ hybridization. The alpha 5 chain was not uniformly expressed in all embryonic epithelial cell types but was present mainly in epithelial sheets which produce very little alpha 1 chain. There also appeared to be a developmental trend, with alpha 1 chain appearing early and alpha 5 later, in maturing epithelial sheets. The alpha 5 chain could be a major alpha chain of the adult glomerular basement membrane.     

Characterization of alpha 2-adrenoceptors mediating contraction of dog saphenous vein: identity with the human alpha 2A subtype

1. In the dog saphenous vein alpha 1- and alpha 2-adrenoceptors mediate noradrenaline-induced contractions in vitro. In order to study the alpha 2-adrenoceptor in isolation, alpha 1-adrenoceptors were inactivated by treatment of tissues with the alkylating agent phenoxybenzamine (3.0 microM for 30 min) in the presence of rauwolscine (1 microM) to protect alpha 2-adrenoceptors. 2. Noradrenaline-induced contractions of tissues treated with phenoxybenzamine were antagonized competitively by the selective alpha 2-adrenoceptor antagonist rauwolscine, pKB = 8.63 +/- 0.07 (means +/- s.e. mean; n = 3), consistent with an interaction at alpha 2-adrenoceptors. 3. Noradrenaline was a full agonist at alpha 2-adrenoceptors in dog saphenous vein. By use of the method of partial receptor alkylation and analysis of concentration-effect curve data by direct, operational model fitting methods, the affinity (pKA) and efficacy (tau) were 5.74 +/- 0.07 and 7.50 +/- 1.05, respectively (n = 6). Nine other agonists which were examined each had affinities higher than noradrenaline, but with the exception of the imidazoline, A-54741 (5,6-dihydroxy-1,2,3,4-tetrahydro-1-naphthyl-imidazoline) had relatively lower efficacies. 4. To compare the alpha 2-adrenoceptor in dog saphenous vein to the human recombinant subtypes, the affinities of twenty-one compounds were estimated in functional studies in the dog saphenous vein and in radioligand binding studies for the human alpha 2A, alpha 2B and alpha 2C receptor subtypes expressed in Chinese hamster lung (CHL) cells. 5. Of twenty-one compounds examined in ligand binding studies, only nine had greater than ten fold selectivity for one human receptor subtype over either of the other two. These compounds were A-54741, oxymetazoline, guanfacine, guanabenz, prazosin, spiroxatrine, tolazoline, WB 4101 and idazoxan. In dog saphenous vein, their affinities (pKA and pKB for agonists and antagonists respectively) were: A-54741 (pKA = 8.03 +/- 0.05), oxymetazoline (pKA = 7.67 +/- 0.09), guanfacine (pKA = 6.79 +/- 0.03); guanabenz (pKA = 7.02 +/- 0.13); prazosin (pKB = 5.19 +/- 0.08), spiroxatrine (pKB = 6.59 +/- 0.04), tolazoline (pKB = 6.21 +/- 0.07), WB 4101 (pKB = 7.42 +/- 0.09) and idazoxan (pKB = 7.11 +/- 0.08). 6. Comparisons of affinity estimates for these nine compounds at the receptor in dog saphenous vein and at the human recombinant subtypes suggest that the vascular receptor is most similar to the h alpha 2A subtype; correlation coefficients (r) were 0.82 (h alpha 2A), 0.24 (h alpha 2B) and 0.04 (h alpha 2C).     

A comparison of sensory nerve function in human, guinea-pig, rabbit and marmoset airways

We have investigated the role of sensory nerves in regulating airway smooth muscle function in the guinea-pig, marmoset, rabbit and man. Tissue levels of the sensory neuropeptides CGRP and substance P in the airways of the guinea-pig were significantly greater compared with the rabbit and marmoset. The relative order of tissue content was guinea-pig > rabbit = marmoset. Marmoset bronchial and tracheal preparations responded weakly to exogenously administered substance P and neurokinin A but contracted to methacholine and demonstrated atropine-sensitive cholinergic responses. In marmoset, rabbit and human airway preparations, capsaicin mediated weak contractile responses to exogenously administered capsaicin. However, high concentrations of capsaicin elicited a relaxation response that was epithelium-independent, cyclo-oxygenase-insensitive, not involving nitric oxide and not dependent on the activation of capsaicin-sensitive afferents. These results suggest that rabbit and marmoset airways respond functionally in a similar way to human airway preparations and maybe more relevant than guinea-pig airways with regard to understanding the role of sensory neuropeptides in airways.     

Biochemical characterization of human osteoclast integrins. Osteoclasts express alpha v beta 3, alpha 2 beta 1, and alpha v beta 1 integrins

The study of osteoclast integrins has been previously hampered by the lack of a source of large numbers of purified osteoclasts. Osteoclastoma, a human giant cell tumor of bone, supplied a rich source of osteoclasts within a tissue containing many diverse cell types. Osteoclastoma integrin immunostaining confirmed the presence of the integrin alpha v beta 3 complex and the alpha 2 and beta 1 integrin subunits on osteoclasts. However, weak integrin expression, for example with alpha v beta 5, was difficult to interpret. Purification with magnetic beads coated with vitronectin receptor monoclonal antibody (13C2) enabled osteoclast membranes to be isolated with high purity and yield (57%) from osteoclastoma tissue. Positively (osteoclast-enriched) selected membranes were biochemically assessed for integrin expression by immunoprecipitation and visualization by non-radioactive enhanced chemiluminescence. alpha 1, alpha 4, alpha 6, alpha 8, alpha M, alpha X, gpIIb, beta 4, beta 6, and beta 8 integrin chains were undetectable at a sensitivity of 1 ng. alpha 3, alpha 5, alpha L, beta 2, and alpha v beta 5 were found in the negatively selected osteoclastoma tissue but not in the positively purified osteoclast membranes. The presence of alpha v beta 1 and alpha 2 beta 1 dimers was demonstrated biochemically on the immunoisolated osteoclast membranes. Osteoclast alpha v beta 3 isolation by Arg-Gly-Asp (RGD) affinity chromatography for NH2-terminal amino acid sequencing confirmed that the osteoclast vitronectin receptor was identical to that previously characterized on other cell types. In situ hybridization using human alpha v riboprobes in osteoclasts from human and rodent bone further demonstrated the high level and specificity of expression of alpha v vitronectin receptor in osteoclasts.     

Oxalate binding protein from the kidney of rat and human mitochondria: studies on properties

The present paper reviews current knowledge of the development and plasticity of the inhibitory gamma-aminobutyric acid (GABA) containing circuitry of the cerebral neocortex, in particular, the rat somatosensory barrel field cortex. Recent studies reveal a delayed and protracted maturation of the inhibitory compared with the excitatory cortical system, both at the neuronal and synaptic levels. This characteristic developmental pattern leaves a longer time window during which behaviourally relevant activity coming from the periphery can influence the organization of the GABA system. Indeed, sensory deprivation experiments confirm the involvement of the GABA system in phenomena of experience-dependent cortical plasticity. Changing the pattern and level of afferent activity of in the rat somatosensory system during development by removing vibrissae results in a significant decrease in the number of GABA neurons and synapses in the thalamocortical recipient layer IV. Particularly affected are GABA synapses contacting dendritic spines, the number of which decreases by almost two-thirds. The involvement of the GABA system in events of experience-dependent plasticity contributes to the adequate functioning of the cerebral cortex in the conditions of constantly changing environment and varying individual experience.     

Ontogenic development of the adenylyl cyclase enzyme and the alpha s, alpha i1 and alpha i2 G-protein regulatory subunits from rat prostate

STUDY OBJECTIVE: To compare the prophylactic administration of ondansetron with droperidol or placebo to determine its effectiveness in reducing postoperative nausea and vomiting after middle ear procedures. DESIGN: Prospective, randomized, double-blind study. SETTING: Inpatient otolaryngology service at a university medical center. PATIENTS: 120 ASA physical status I and II patients presenting for elective middle ear surgical procedures. INTERVENTIONS: Patients were randomly assigned to receive either placebo (Group 1), ondansetron 4 mg intravenously (IV) (Group 2), or droperidol 25 mcg/kg i.v. (Group 3) 10 minutes before induction of general anesthesia using thiopental 5 mg/kg i.v. with fentanyl 2 mcg/kg i.v. and maintenance anesthesia with isoflurane 1% to 2% end-tidal in a 50% air/oxygen mixture. MEASUREMENTS AND MAIN RESULTS: Total surgical, anesthesia, extubation, and postanesthesia care unit (PACU) occupancy times were recorded along with anesthesia recovery scores. The incidence and severity of nausea, vomiting, and pain along with rescue antiemetic administration, also were recorded. Similar assessments were made over the next 24 hours. Intergroup demographic data were similar except that the male to female ratio was higher in the ondansetron group. Stewart scores, reflecting emergence from anesthesia, were higher with ondansetron compared with droperidol. The incidence of nausea was similar between the groups but the severity was less after ondansetron therapy. More patients vomited after placebo than when given either droperidol or ondansetron. No intergroup differences were noted in the use of rescue antiemetics. Twenty-four hours later, more patients who received the placebo drug had nausea or vomited compared with either ondansetron or droperidol. CONCLUSIONS: Ondansetron 4 mg i.v. is as effective as droperidol and better than placebo in preventing nausea and vomiting in patients undergoing middle ear surgery. No cost advantage as determined by lower use of rescue antiemetics or shorter PACU times was noted after the prophylactic administration of ondansetron.     

Na,K-ATPase subunit isoforms in human reticulocytes: evidence from reverse transcription-PCR for the presence of alpha1, alpha3, beta2, beta3, and gamma

The objective of this study has been to determine which Na,K-ATPase isoforms are expressed in red blood cells and whether kinetic differences in the uncoupled sodium efflux mode between the human red blood cell Na,K-ATPase and other preparations can be explained by differences in the underlying subunit composition. To this end, human reticulocyte RNA was isolated, reverse transcribed, amplified by PCR and appropriate primers, and sequenced. Primers from highly conserved areas as well as isoform-specific primers were used. The alpha1 and alpha3 isoforms of the alpha subunit, and the beta2 and beta3 isoforms of the beta subunit were found. The complete coding regions of the cDNAs for the reticulocyte subunits were sequenced from overlapping PCR fragments. No difference was found between the reticulocyte isoforms and the ones already known. The fact that we found beta2 but not beta1 in reticulocyte single-stranded cDNA, and beta1 but not beta2 in a leukocyte library indicates that leukocyte contamination of our reticulocyte preparation was negligible. Analysis of a human bone marrow library showed that alpha1, alpha2, and alpha3 as well as all three beta isoforms were present. The extent to which the kinetic properties of uncoupled sodium efflux might depend on different isoform combinations is not yet known.     

Distribution of alpha 1A, alpha 1B and alpha 1E voltage-dependent calcium channel subunits in the human hippocampus and parahippocampal gyrus

The distribution of voltage-dependent calcium channel subunits in the central nervous system may provide information about the function of these channels. The present study examined the distribution of three alpha-1 subunits, alpha 1A, alpha 1B and alpha 1E, in the normal human hippocampal formation and parahippocampal gyrus using the techniques of in situ hybridization and immunocytochemistry. All three subunit mRNAs appeared to be similarly localized, with high levels of expression in the dentate granule and CA pyramidal layer. At the protein level, alpha 1A, alpha 1B and alpha 1E subunits were differentially localized. In general, alpha 1A-immunoreactivity was most intense in cell bodies and dendritic processes, including dentate granule cells, CA3 pyramidal cells and entorhinal cortex pre-alpha and pri-alpha cells. The alpha 1B antibody exhibited relatively weak staining of cell bodies but stronger staining of neuropil, especially in certain regions of high synaptic density such as the polymorphic layer of the dentate gyrus and the stratum lucidum and radiatum of the CA regions. The alpha 1E staining pattern shared features in common with both alpha 1A and alpha 1B, with strong immunoreactivity in dentate granule, CA3 pyramidal and entorhinal cortex pri-alpha cells, as well as staining of the CA3 stratum lucidum. These findings suggest regions in which particular subunits may be involved in synaptic communication. For example, comparison of alpha 1B and alpha 1E staining in the CA3 stratum lucidum with calbindin-immuno-reactivity suggested that these two calcium channels subunits may be localized presynaptically in mossy fibre terminals and therefore may be involved in neurotransmitter release from these terminals.     

Basal forebrain cholinergic immunolesion by 192IgG-saporin: evidence for a presynaptic location of subpopulations of alpha 2- and beta-adrenergic as well as 5-HT2A receptors on cortical cholinergic terminals

To study whether the changes in cortical noradrenergic and serotonergic mechanisms observed in patients with Alzheimer's disease are the consequence of reduced cortical cholinergic activity, a novel colinergic immunotoxin (conjugate of the monoclonal antibody 192IgG against the lower affinity nerve growth factor receptor with the cytotoxic protein saporin, 192IgG-saporin) was used to produce a specific and selective loss of cholinergic cells in rat basal forebrain nuclei. To correlate the responses to cholinergic immunolesion in cholinoceptive cortical target regions with cholinergic hypoactivity, quantitative receptor autoradiography to measure adrenoceptors and 5-hydroxytryptamine (5-HT) receptor subtypes, and histochemistry to estimate acetylcholinesterase activity, were performed in adjacent brain sections. alpha 1-adrenoceptor and 5-HT1A receptor binding were not affected by cholinergic immunolesion in any of the cortical and hippocampal regions studied. However, cholinergic immunolesion resulted in significantly reduced alpha 2- and beta-adrenoceptor as well as 5-HT2A receptor binding in a number of cortical and hippocampal regions displaying a reduced activity of acetylcholinesterase, already detectable seven days after a single injection of 192IgG-saporin and persisting up to three months post lesion without any significant recovery. The data suggest that at least a subpopulation of alpha 2- and beta-adrenoceptor as well 5-HT2A receptor subtype is present on cortical and hippocampal cholinergic terminals originating in the basal forebrain. The lesion-induced receptor changes suggest that the alterations in cortical 5-HT2 receptor binding observed in patients with Alzheimer's disease might be secondary to cholinergic deficits.     

Multiple alpha 2 adrenergic receptor subtypes. I. Comparison of [3H]RX821002-labeled rat R alpha-2A adrenergic receptors in cerebral cortex to human H alpha2A adrenergic receptor and other populations of alpha-2 adrenergic subtypes

In the present study, we examined the binding of the alpha-2 adrenergic receptor (AR) antagonist [3H]-(2-(2-methoxy-1,4-benzodioxan- 2yl)-2-imidazoline ([3H]RX821002) to alpha-2 AR in rat cerebral cortex (CC) and compared the properties of these sites to those of rat alpha-2A (R alpha-2A) AR in submaxillary gland (SMG), human alpha-2A (H alpha-2A) AR in human platelets and alpha-2B AR in neonatal rat lung. In the presence of guanidinium phosphate, [3H]RX821002 bound with high affinity to a large and homogeneous population of sites in CC (Kd = 0.30 +/- 0.03 nM and Bmax = 271 +/- 7 fmol/mg of protein), SMG (Kd = 0.7 and Bmax = 274), human platelets (Kd = 0.6 nM and Bmx = 189) and neonatal rat lung (kd = 0.9 and Bmax = 161). A total of 34 chemically diverse AR ligands monophasically inhibited the binding of [3H]RX821002 from each site with, for the CC, the most potent ligand being atipamezole (Ki = 0.2 nM). For all ligands, and at each site, Hill coefficients did not differ significantly from unity. Although the profiles of inhibition of [3H]RX821002 were virtually identical in rat CC and SMG, these populations revealed several marked differences to human platelets; the alkaloids, rauwolscine and yohimbine, as well as the benzodioxane, [2-(2,6- dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane] (WB 4101), displayed about 10-fold lower affinity for R alpha-2A as compared to H alpha-2A sites, whereas the benzopyrrolidines, fluparoxan and des-fluorofluparoxan, showed about 10-fold greater affinity for R alpha-2A sites. Further, whereas the calculation of potency ratios for selected pairs of ligands, as well as of correlation coefficients, revealed virtual identity between R alpha-2A AR in CC and SMG, these analyses revealed that each of these populations of R alpha-2A AR clearly differed to H alpha-2A AR in human platelets. In addition, both R alpha-2A AR in rat CC and SMG as well as H alpha-2A AR in human platelets markedly differed to alpha-2B AR in neonatal rat lung; thus, they showed 20-fold higher affinity for [2-(2H-(1-methyl-1,3-dihydroisoindole)methyl)-4,5- dihydroimidazoline] (BRL 44408), oxymetazoline, guanfacine and guanabenz yet 10- to 100-fold lower affinity for [2-(2-4-o- methoxyphenyl)piperazine-1-yl)-ethyl)-4,4-dimethyl-1,3-(2H,4H)- isoquinolinedione] (ARC 239) prazosin, chlorpromazine and corynanthine. Similar differences in R alpha-2A and H alpha-2A sites to alpha-2C sites were apparent upon analysis of literature data.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bronchodilator and anti-inflammatory activities of SCA40: studies in human isolated bronchus, human eosinophils, and in the guinea-pig in vivo

There is currently interest in the use of inhibitors of cyclic nucleotide phosphodiesterases (PDE) as potential anti-asthma agents. In this study we examined the effects of SCA40 (6-bromo-8-methylaminoimidazol-[1,2-a] pyrazine-2-carbonitrile), a preferential inhibitor of PDE 3 also endowed with PDE 4 and 5 inhibitory activities, on isolated bronchus and eosinophil functions and in an animal model of asthma. SCA40 (1 nM-0.1 mM) produced concentration-dependent inhibition of spontaneous and stimulated tone of human isolated bronchus and reached a maximal relaxation similar to that of theophylline (3 mM). The potency (-log EC50 values) of SCA40 against spontaneous tone (6.52 +/- 0.10) was greater than against tone raised by equieffective concentrations (approximately 70%) of histamine (5.76 +/- 0.06), leukotriene C4 (5.44 +/- 0.11), and acetylcholine (4.98 +/- 0.09). In the presence of cytochalasin B, the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 0.5 microM) induced leukotriene C4 production in human eosinophils isolated in discontinuous metrizamide gradients. The production of leukotriene C4 was inhibited by SCA40 in a concentration-related fashion (-log IC50 = 6.04 +/- 0.20; n = 6). Rolipram, a selective PDE 4 inhibitor, was also effective (-log IC50 = 7.29 +/- 0.32) but the selective PDE 3 inhibitor SKF94120 was scarcely effective (< 10% inhibition for 10 microM). In ovalbumin sensitized guinea-pigs, SCA40 (1 mg kg(-1), i.p.) given 30 min before antigen challenge significantly inhibited the acute bronchoconstriction produced by aerosol antigen (5 mg ml(-1), 30 s) (antigen response was 185 +/- 13 and 91 +/- 21 cmH2O l(-1) s(-1) in control and SCA40-treated animals, respectively, P < 0.05). Pretreatment with SCA40 (1 mg kg(-1), i.p., 30 min pre- and 3 h post-antigen exposure) prevented airway hyperreactivity to histamine which developed 24 h after exposure of conscious guinea-pigs to aerosol antigen. Eosinophil lung accumulation that accompanied airway hyperreactivity was also inhibited by SCA40 (from 6.15 +/- 0.86 in control to 1.27 +/- 0.27 in treated animals; expressed as eosinophils x 10(6); P < 0.05). SCA40 (1 mg kg(-1), i.p.) also inhibited the microvascular leakage produced after inhaled antigen (5 mg ml(-1), 30 s) at all airway levels. The haemodynamic effects of SCA40 (1 mg kg(-1), i.p.) consisted of a rapid decrease (peak at 5 min) in mean arterial blood pressure (-39.4 +/- 2.4%) and tracheal mucosal blood flow (-13.5 +/- 2.0%) that slowly recovered with time. These data support previous work showing that PDE inhibition results in antispasmogenic and anti-inflammatory effects. SCA40 was effective in vitro and in vivo and these effects are probably related to its activity as a mixed PDE inhibitor.     

Role of PKC in the effects of alpha1-adrenergic stimulation on Ca2+ transients, contraction and Ca2+ current in guinea-pig ventricular myocytes

The effects of alpha1-adrenoceptor stimulation on intracellular Ca2+ transients, contractility and L-type Ca2+ current (ICa,L) were studied in single cells isolated from ventricles of guinea-pig hearts. The aim of our study was to elucidate the mechanisms of the positive inotropic effect of alpha1-adrenergic stimulation by focussing on the role of protein kinase C (PKC). Phenylephrine, an alpha1-adrenergic agonist, at concentrations of 50-100 microM elicited a biphasic inotropic response: a transient negative inotropic response (22.9+/-6.0% of control) followed by a sustained positive inotropic response (61.0+/-8.4%, mean+/-SE, n=12). The Ca2+ transient decreased by 10.2+/-3.9% during the negative inotropic phase, while it increased by 67.7+/-10% (n=12) during the positive inotropic phase. These effects were inhibited by prazosin (1 microM), a alpha1-adrenergic antagonist. Phenylephrine increased the ICa,L by 60.8+/-21% (n=5) during the positive inotropic phase. To determine whether activation of PKC is responsible for the increases in Ca2+ transients, contractile amplitude and ICa,L during alpha1-adrenoceptor stimulation, we tested the effects of 4beta-phorbol 12-myristate 13-acetate (PMA), a PKC activator, and of bisindolylmaleimide I (GF109203X) and staurosporine, both of which are PKC inhibitors. PMA mimicked phenylephrine's effects on Ca2+ transients, contractile amplitude and ICa,L. PMA (100 nM) increased the Ca2+ transient, contractile amplitude and ICa,L by 131+/-17%, 137+/-25% (n=8), and 81.1+/-26% (n=5), respectively. Prior exposure to GF109203X (1 microM) or staurosporine (10 nM) prevented the phenylephrine-induced increases in Ca2+ transients, contractile amplitude and ICa,L. Our study suggests that during alpha1-adrenoceptor stimulation increase in ICa,L via PKC causes an increase in Ca2+ transients and thereby in the contractile force of the ventricular myocytes.