Inhibitory transmission in guinea pig stomach mediated by distinct receptors for pituitary adenylate cyclase-activating peptide.

Previous studies have shown that inhibitory transmission in guinea pig stomach involves an interplay between vasoactive intestinal peptide (VIP) and nitric oxide (NO). The present study examined the contribution of pituitary adenylate cyclase-activating peptide (PACAP), a homologous peptide present in gastric and intestinal myenteric neurons. VIP, PACAP-27 and PACAP-38 induced concentration-dependent relaxation that was partly inhibited by the antagonists VIP10-28 and PACAP6-38 and the NO synthase inhibitor NG-nitro-L-arginine (L-NNA). Only relaxation induced by PACAP-27 and PACAP-38 was partly inhibited by apamin. Electrical field stimulation (0.25-16 Hz) induced frequency-dependent relaxation and PACAP release (maximum of 35.7 fmol/100 mg-min or 7-fold above basal levels). Electrical field stimulation-induced relaxation was partly inhibited by a combination of selective monoclonal antibodies to PACAP-27 and PACAP-38 (42 +/- 7% at 16 Hz) and by the antagonists VIP10-28 (29 +/- 9%) and PACAP6-38 (29 +/- 3%). The relaxation was also partly inhibited by L-NNA (51 +/- 12% at 16 Hz) and apamin (36 +/- 4%). The effects of a combination of apamin and L-NNA were additive, amounting to 75 +/- 3% inhibition. The effect of L-NNA reflected inhibition of NO release from nerve terminals, as well as NO generation in muscle cells by the action of VIP and PACAP; the effect of apamin reflected blockade of the action of PACAP. Thus, inhibitory transmission in guinea pig gastric fundus represents the combined actions of VIP, PACAP and NO released from nerve terminals and NO generated in muscle cells. The postjunctional actions of PACAP are mediated by a VIP/PACAP-II receptor and by a PACAP-specific, apamin-sensitive receptor.

Specific monoclonal antibodies neutralize the action of PACAP 1-27 or PACAP 1-38 on intestinal muscle strips in vitro.

The gut-brain neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) is a novel highly conserved member of the secretin-glucagon-VIP peptide family comprising 38 or 27 amino acid residues. In this study, we investigate the actions of PACAP 1-27 or PACAP 1-38 on jejunal and caecal muscle strips from pig or guinea pig and demonstrate the neutralizing effect of two PACAP-specific monoclonal antibodies of the IgG1 subtype, RSP27II and RSP38. These antibodies were used to set up assay systems specific for PACAP 1-27 or PACAP 1-38. Monoclonal antibody RSP27II recognizes exclusively PACAP 1-27, whereas RSP38 binds only PACAP 1-38. PACAP 1-27 and PACAP 1-38 relax taenia caeci dose-dependently in the presence of guanethidine and scopolamine. Both peptides inhibit the spontaneous contractions of porcine jejunal muscle strips equipotently. Monoclonal antibodies RSP27II and RSP38 specifically neutralize the actions of either exogenously applied or endogenously released PACAP. Thus, they represent processing-specific tools to examine the physiological role of both molecular forms of PACAP in the gastrointestinal tract.

Method comparison of EMIT 700 and EMIT II with RIA for drug screening.

The EMIT 700 and EMIT II assays for marijuana, cocaine, opiates, barbiturates, and phencyclidine, as performed on a Hitachi 717 Analyzer, were compared with Roche Abuscreen RIA tests for high-volume drug abuse screening. The EMIT II kits offer some advantages over the EMIT 700 kits for testing large work loads. The EMIT II marijuana test in particular exhibits a calibration curve that promises improved ability to separate negative from positive samples due to the increased absorbance rate separation between the negative and cutoff calibrators. Both EMIT formulations are preferable in terms of speed and ease of analysis in comparison with our laboratory's current RIA procedures. Over 50,000 urine samples were screened by EMIT 700 and RIA and by EMIT II and RIA. The performance of both EMIT assays was approximately equivalent to RIA in terms of their ability to detect urine samples that confirmed positive for cocaine and opiates. Both EMIT assays detected approximately 90% of the urine samples screened positive by RIA and confirmed positive for marijuana. Both EMIT assays performed better than RIA in detecting confirmed-positive barbiturate samples.

Cyclosporine A inhibits protein-kinase-C-mediated amylase release from isolated rat pancreatic acini.

The present study was performed to characterize a direct influence of cyclosporine A (CsA) on exocrine pancreatic enzyme secretion. CsA inhibited dose-dependently amylase release from isolated rat pancreatic acini in response to carbachol or cholecystokinin octapeptide (CCK-8). A significant reduction in amylase release by 17% was observed at 0.2 mumol/l CsA (p < 0.001) when compared to controls. At 0.2 mmol/l, CsA reduced amylase release in response to both secretagogues by maximally 45%, whereas basal secretion was not affected. CsA had no influence on CCK-8-stimulated increase in intracellular Ca2+ concentrations or amylase release in response to the Ca2+ ionophore A 23187. In contrast, the dose-response curve for amylase secretion induced by the phorbol ester phorbolmyristate-13-acetate was shifted to the right without a reduction of the maximal secretory response. Unexpectedly, vasoactive-intestinal-polypeptide- and secretin-stimulated acinar secretion was not diminished by CsA. In isolated pancreatic lobules exposed to 0.1 mmol/l CsA, amylase release stimulated by cerulein or veratridine was reduced by 26.7 +/- 2 or 28.3 +/- 4%, respectively. CsA had no influence on the displacement of 125I-Bolton-Hunter-labeled CCK-8 from acinar CCK receptors. The ultrastructure of cellular organelles in isolated lobules was not altered after incubation with 0.1 mmol/l CsA for 60 min. Our data suggest that CsA interferes with protein-kinase-C-mediated signal transduction in isolated rat pancreatic acini, without affecting cAMP-dependent signalling.

Method comparison of EMIT II and online with RIA for drug screening.

The newest formulation of the EMIT assay for drugs of abuse, EMIT II, and a new immunoassay, OnLine, using the kinetic interaction of microparticles in solution methodology, were evaluated for marijuana, cocaine, opiates, barbiturates, and phencyclidine. Both types of immunoassays were performed on an Hitachi 717 analyzer. Calibration curves, the degree of separation between negative and cutoff calibrators, precision, probability of carryover from positive to negative samples, and overall ease and speed of analysis were evaluated. EMIT II and OnLine were compared with RIA tests for the five drugs to determine each assay's ability to detect samples which confirm positive by GC/MS. The RIA and OnLine marijuana tests detected > 99% of confirmed positive samples while EMIT II detected about 90%. All three immunoassays performed equivalently for cocaine and opiates, each assay detecting at least 98% of positives. Barbiturates showed the greatest disparity with OnLine detecting 96%, EMIT II 85%, and RIA 79% of confirmed positive samples. Too few phencyclidine positive samples were detected for a method comparison study. The fully automated EMIT II and OnLine assays are preferable for a variety of reasons to our laboratory's current semi-automated RIA tests for large volume urine testing. The immunoassays offer comparable performance for some drugs but not for others.

Interpretation of methamphetamine and amphetamine enantiomer data.

Interpretation of drug testing results is a challenging and complex task, particularly when the interpretation can result in establishing legitimate use of a drug or illicit use with all of its attendant complications (i.e., loss of job, criminal prosecution, etc.). One of the more challenging drugs to interpret is methamphetamine. While methamphetamine is a schedule II controlled substance, the l-enantiomer of methamphetamine is found in the Vick's Inhaler, which is a product exempted from control. For this reason, while identification of methamphetamine and amphetamine in the urine of an individual can clearly establish the use of methamphetamine, it does not prove the use of a controlled substance. Use of racemic methamphetamine can make the interpretation even more difficult because of the different metabolism and excretion of l- and d-methamphetamine. Enantiomeric characterization of methamphetamine may not give unequivocal results. Evaluation of experimentally derived and published data from urine samples containing l- and d,l-methamphetamine indicates that use of the enantiomeric distribution of amphetamine affords unambiguous interpretation. Because the l-enantiomer is the only possible finding in an individual who is using the Vick's Inhaler, detection of the d-enantiomer or a mixture of the d- and l-enantiomers clearly establishes the use of a controlled substance. Without a prescription from appropriate medical personnel, this detection would indicate the illicit use of a controlled substance.

Enzyme immunoassay, kinetic microparticle immunoassay, radioimmunoassay, and fluorescence polarization immunoassay compared for drugs-of-abuse screening.

The newest formulation of the Syva EMIT assay for drugs of abuse, EMIT II, and a new immunoassay, OnLine (Roche), utilizing the kinetic interaction of microparticles in solution (KIMS) methodology, RIA tests, and TDx fluorescence polarization immunoassay (FPIA) procedures were compared for marijuana, cocaine, opiates, and barbiturates. Both EMIT II and OnLine immunoassays were performed with a Hitachi 717 analyzer. Calibration curves, the degree of separation between negative and cutoff calibrators, precision, likelihood of carryover from positive to negative samples, and overall ease and speed of analysis were evaluated. RIA and OnLine detected 99% of gas chromatography/mass spectrometry (GC/MS)-confirmed marijuana samples; TDx, 95%; and EMIT II, 88%. All four immunoassays detected approximately 99% of confirmed cocaine-positive urines. RIA, OnLine, and TDx all detected 100% of opiate-confirmed samples; EMIT II, 97%. Barbiturate assays exhibited the greatest disparity, with OnLine and TDx detecting 100% of confirmed positives; EMIT II, 88%; and RIA, 78%. For a variety of reasons, we prefer the fully automated EMIT II and OnLine assays for high-volume urine testing, in comparison with our laboratory's semiautomated RIA tests and the limited-throughput TDx system. The four immunoassays investigated delivered comparable performance in terms of detection rates for GC/MS-confirmed positives for some drugs but not for others.

PACAP and VIP stimulate enzyme secretion in rat pancreatic acini via interaction with VIP/PACAP-2 receptors: additive augmentation of CCK/carbachol-induced enzyme release.

The binding and biological effect of pituitary adenylate cyclase activating polypeptide (PACAP), a novel hypothalamic peptide with high sequence homology to vasoactive intestinal polypeptide (VIP), were studied in rat AR 4-2 J pancreatic carcinoma cells and isolated rat pancreatic acini. PACAP(1-27) and analogue PACAP(1-23, VIP-24-28), but not VIP, displaced potently and reversibly 125I-PACAP(1-27) from binding to an abundantly expressed high affinity PACAP-preferring receptor on AR 4-2 J cells, referred to as "PACAP-1 receptor." High affinity binding was dependent on N-terminal and C-terminal residues of PACAP(1-27): PACAP(1-24,Cys-25) (7.3 +/- 1.6 microM), PACAP(1-23) (8.2 +/- 1.5 microM), VIP (> 30 microM), PACAP(3-27), PACAP(1-19), PACAP(3-19), PACAP(1-12), and PACAP(18-38) (all > 50 microM) showed low or no binding potency. In contrast, high and low affinity binding of 125I-VIP to AR 4-2 J cells was displaced equipotently by PACAP(1-27) and VIP, thus defining on these cells, in addition, two scarcely expressed binding sites, designated "VIP/PACAP-2 receptor," similar or identical to the previously described high and low affinity acinar VIP receptor. Binding of 125I-PACAP(1-27) to a high and low affinity binding site on rat pancreatic acini was inhibited equipotently by PACAP(1-27) and VIP, identifying these sites as VIP/PACAP-2 receptors. PACAP(1-23) recognized both type 2 binding sites with only slightly lower affinity. PACAP(1-27), PACAP(1-38), PACAP(1-23, VIP-24-28), and PACAP(1-23) equipotently stimulated acinar lipase release and cyclic AMP production in pancreatic acini. Co-incubation of PACAP(1-27) or VIP with cholecystokinin-8 or carbachol revealed additive effects on enzyme secretion. Our results suggest the predominant expression of VIP/PACAP-2 receptors on rat pancreatic acini, whereas AR 4-2 J cells express mainly PACAP-1 receptors. PACAP is a potent ligand for both receptor types and has to be regarded as a novel VIP-like pancreatic secretagogue.

The effects of adulterating agents on FPIA analysis of urine for drugs of abuse.

A variety of chemical agents were evaluated to determine their effects on fluorescence polarization immunoassays for drugs of abuse. Sixteen different agents, at concentrations up to 10%, were tested against urine assays for cannabinoids, cocaine (metabolite), amphetamines, opiates, phencyclidine (PCP), and barbiturates. The potential to cause both false positive and false negative results was evaluated, and assays were performed one and seven days after sample adulteration to simulate different collection/testing formats. All six drug assays were susceptible to one or more adulterating agents, but the degree varied considerably between assays. The cannabinoid assay was most susceptible to adulterant-induced false negative results, and the barbiturate assay was most susceptible to false positive results. The remaining assays demonstrated relatively few, but characteristic effects, some of which were attributable to drug degradation and others to assay interference. Although the results of pH measurement on adulterated samples verified its utility in identifying some samples adulterated with interfering agents, other adulterants that cause substantial effects would not be identified by pH measurements alone.

Fluorescence polarization immunoassay detection of amphetamine, methamphetamine, and illicit amphetamine analogues.

The Abbott Diagnostics Amphetamine/Methamphetamine II and Amphetamine Class reagents were evaluated on the Abbott TDx for cross-reactivity to amphetamine and methamphetamine stereoisomers, several of their metabolites, and various illicit analogues, including 2-methoxyamphetamine, 4-hydroxymethamphetamine, 2,5-dimethoxy-amphetamine (DMA), 4-bromo-2,5-dimethoxyamphetamine (DOB), 4-bromo-2,5-dimethoxy-beta-phenethylamine (BDMPEA), 3,4,5-trimethoxyamphetamine (TMA), 3,4-methylenedioxy-amphetamine (MDA) N,N-dimethyl-3,4-methylenedioxy-amphetamine, N-hydroxy-3,4-methylenedioxyamphetamine (N-OH MDA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine (MDEA), 2,5-dimethoxy-4-ethylamphetamine (DOE), 2,5-dimethoxy-4-methylamphetamine (DOM), and mescaline in concentrations ranging from 100 to 100,000 ng/mL. Results demonstrate the utility of this assay for detection of several of the above compounds; unfortunately many are still not detectable. Significant differences were observed between the Amphetamine/Methamphetamine II and Amphetamine Class reagents, particularly regarding their cross-reactivity to over-the-counter medications. Detection of the drugs amphetamine, methamphetamine, and the illicit analogues is not enhanced with the Amphetamine Class reagents, and unless detection of the over-the-counter compounds is of interest, these reagents are a poor choice compared to the Amphetamine/Methamphetamine II reagents. Cross-reactivity of some of the illicit analogues is such that the assay can reliably be used for the routine screening of these compounds.

Characterization of a guanosine-nucleotide-binding-protein-coupled receptor for pituitary adenylate-cyclase-activating polypeptide on plasma membranes from rat brain.

Pituitary adenylate-cyclase-activating polypeptide (PACAP), a novel brain-gut hormone, was isolated from ovine hypothalami and represents the latest mammalian member of the secretin-glucagon peptide family. PACAP exists in two C-terminally amidated molecular forms, PACAP(1-27) and PACAP(1-38), comprising 27 or 38 amino acid residues, respectively. In order to identify a specific receptor for PACAP, we studied binding of 125I-labelled PACAP(1-27) to plasma membranes from rat brain. We identified a single high-affinity binding site (Kd, 340 pM and Bmax, 3.34 pmol/mg), specific for synthetic PACAP(1-38) and PACAP(1-27). Hormone binding was reversible and time, protein and temperature dependent. In contrast, neither the analogues PACAP(1-23), PACAP(18-38) and PACAP(3-25), nor vasoactive intestinal peptide (VIP), secretin and growth-hormone-releasing factor (GRF) revealed significant binding at concentrations up to 1 microM. A specific receptor protein, with an apparent molecular mass of 60 kDa, was identified by means of affinity cross-linking with disuccinimidyl suberate (DSS) and ethylene glycol disuccinimidyl suberate (EGS). PACAP receptors are associated with a GTP-binding protein as determined by the influence of different nucleotides on PACAP binding. PACAP-binding activity was solubilized with the detergents 3-[(3-cholamidopropyl)dimethylammonio]2-hydroxy-1-propane sulfonate (Chapso) or Triton X-100 and was characterized as a high-molecular-mass receptor complex (400 kDa) by non-reducing size-exclusion chromatography on Sepharose CL-6B. These data imply the following: high-affinity PACAP receptors are expressed abundantly on rat-brain plasma membranes; PACAP receptors are specific for PACAP and show no affinity for VIP, secretin and GRF; the PACAP receptor molecule has an apparent molecular mass of 60 kDa; the PACAP receptor complex is associated with a GTP-binding protein.

Galanin: structural requirements for binding and signal transduction in RINm5F insulinoma cells.

Receptors for galanin, a neuropeptide inhibiting insulin release, have been described on RINm5F insulinoma cells. To characterize structural requirements for binding and biological activity of galanin, we studied binding and inhibition of hormone stimulated intracellular cAMP-production of N-terminal galanin fragments and -analogues in RINm5F cells. Half-maximal binding and potency were the same for all peptides used. Active peptides had the following rank of potency: galanin = galanin(1-22(23)Cys) greater than galanin(1-29(4)NLe) greater than galanin(1-18) greater than galanin(1-29(7)DAla) greater than galanin(1-29(2)DTrp4NLe7DAla) greater than galanin(1-29(2)DTrp). Galanin(3-29) was inactive. Therefore the first two amino acids of the galanin molecule with the indole side chain of the tryptophane residue in the right steric position are crucial for receptor binding.

Impact of adulterants on RIA analysis of urine for drugs of abuse.

The effect of various adulterants on radioimmunoassay (RIA) tests for amphetamine, cannabinoids, cocaine (metabolite), phencyclidine (PCP), barbiturate, and morphine was evaluated. A total of 16 readily available agents, at concentrations ranging from 1 to 25%, were tested with negative and positive urine samples. The results demonstrate that both false positive and false negative RIA results can be produced by easily achievable levels of different agents in urine specimens. Of the RIA tests evaluated, the one for cannabinoids was most sensitive to the presence of adulterants, whereas the PCP assay was most resistant. Although the presence of some adulterants could be detected by measuring the pH or specific gravity of the urine, other agents, at concentrations sufficient to alter the RIA results, could not be as easily detected. Gas chromatography/mass spectrometry (GC/MS) analysis of adulterated samples with false positive RIA results showed no detectable drug, indicating that the adulterants interfered with the RIA test itself. In contrast, GC/MS analysis of samples with false negative RIA results confirmed that at least some adulterants acted by reducing the drug concentrations in the specimen.

Hybridization of Candida albicans through fusion of protoplasts.

Protoplasts of complementing auxotrophs of Candida albicans can fuse in the presence of polyethylene glycol and generate prototrophic cells. The yields of prototrophs from fusion mixtures depend greatly on the particular combinations of auxotrophies involved but not on other features of the strain backgrounds of protoplasts. The initial cellular products of fusions isolated on selective media are heterokaryons which replicate slowly but also segregate single parental nuclei into blastospores in high frequency. Karyogamy within heterokaryons produces hybrid nuclei which, on segregation, give rise to rapidly growing, uninucleate substrains. Analyses of the substrains show that hybrid nuclei either stabilize as diploid or undergo random loss of chromosomes to stabilize at various levels of aneuploidy prior to segregation. Chromosome losses and radiation induced mitotic crossing-over can effect recombination for parental auxotrophic markers in hybrids; patterns of recombination for ader and arg markers provide the first documented example of chromosomal linkage in C. albicans. Thus, protoplast fusions offer opportunities otherwise unavailable for applying the incisive tools of genetic recombination to analysis of this important, asexual yeast.

Growth inhibition of Proteus mirabilis by cyclic adenosine 3'-5'-monophosphate.

Growth of Proteus mirabilis on a synthetic agar medium containing either glycerol, galactose, or trehalose as the sole source is inhibited by 5 mM cyclic adenosine 3',5'-monophosphate (cAMP). Inhibition on an agar medium is evident as loss of viability, but in broth cAMP only slightly inhibits growth rate. Inhibition is associated with the accumulation of methylglyoxal in the medium. A nonswarming mutant of P. mirabilis is not inhibited by cAMP on either of the three carbon sources, but it is sensitive to exogenous methylglyoxal.

Evidence against the involvement of chemotaxis in swarming of Proteus mirabilis.

Nonswarming and nonchemotactic mutants of Proteus mirabilis were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine or ultraviolet light. These mutants were used in experiments to determine if chemotaxis is involved in the swarming of P. mirabilis. Nonchemotactic mutants failed to form chemotactic bands in a semisolid casein hydrolysate medium, yet they swarmed on the same medium containing 1.5% agar. Nonswarming mutants were attracted towards individual amino acids and components of tryptose. In cross-feeding experiments, no evidence was obtained to indicate the production of a diffusable chemical repellent. In studies with the wild-type P. mirabilis, no clear-cut negative chemotaxis was seen even though three different assays were used and numerous chemicals were tested. Additional evidence against the involvement of chemotaxis in swarming comes from finding that dialysis does not interfere with swarming; swarm cells will swarm immediately when transferred to fresh media, and swarm cells will swarm on an agar-water medium supplemented with a surfactant. These data indicate that chemotaxis is not involved in the swarming of P. mirabilis.