A Highly Specific and Sensitive Radioimmunoassay of Caerulein,An Analogue of Cholecystokinin-8

Abstract

A highly specific and sensitive competitive radioimmunoassay was developed for caerulein (CLN), an analogue of cholecystokinin-8 (CCK-8), in plasma and brain. Antiserum was produced in rabbit by immunization with Nδ-[CLN-(1-6)]-ornithine amide conjugated with bovine serum albumin by the glutaraldehyde method. Nα-[CLN-(1-6)]-lysine amide was labelled with ¹²⁵I-Bolton & Hunter reagent and used as a labelled antigen after purification by high-performance liquid chromerography. This assay was highly specific for CLN, and cross reactivities for other related peptides, CCK-4, CCK-8, gastrin-I, and gastrin-(14–17), were not observed (<0.01%). The limits of determination in biological specimens after CLN administration were 11 pg/ml in human plasma and rat plasma and 80 pg/g in rat brain. This study showed that the slight structure difference between hapten and ¹²⁵I-labelled antigen is important to the assay performance.     

Atrial natriuretic polypeptides: structure-activity relationship in the central action--a comparison of their antidipsogenic actions

The structure-activity relationship of atrial natriuretic polypeptides (ANPs) in the central action was investigated by examining the suppressive effects of the intracerebroventricular (i.c.v.) administration of ANPs on water intake induced by the i.c.v. injection of angiotensin II (AII) (0.1 nmol) in rats. alpha-Rat ANP (alpha-rANP), alpha-human ANP, alpha-rANP4-28 and alpha-rANP5-28 at a dose of 1.5 nmol exerted equipotent antidipsogenic actions, while alpha-rANP7-23-amide had no inhibitory effect on water drinking at this dose.     

Nearby inverted repeats fuse to generate acentric and dicentric palindromic chromosomes by a replication template exchange mechanism

Gene amplification plays important roles in the progression of cancer and contributes to acquired drug resistance during treatment. Amplification can initiate via dicentric palindromic chromosome production and subsequent breakage–fusion–bridge cycles. Here we show that, in fission yeast, acentric and dicentric palindromic chromosomes form by homologous recombination protein-dependent fusion of nearby inverted repeats, and that these fusions occur frequently when replication forks arrest within the inverted repeats. Genetic and molecular analyses suggest that these acentric and dicentric palindromic chromosomes arise not by previously described mechanisms, but by a replication template exchange mechanism that does not involve a DNA double-strand break. We thus propose an alternative mechanism for the generation of palindromic chromosomes dependent on replication fork arrest at closely spaced inverted repeats.     

Non-NMDA and NMDA receptor agonists induced excitation and their differential effect in activation of superior salivatory nucleus neurons in anaesthetized rats

We investigated the effects of the ionophoretic application of ionotropic non-NMDA receptor agonist (AMPA) and NMDA receptor agonist (NMDA) on extracellularly recorded and antidromically identified superior salivatory nucleus (SSN) neurons. A great majority (93%) of SSN neurons was induced to fire by ionophoretic application of AMPA, and they were classified into high firing rate (more than 6 spikes/s), and low firing rate (less than 3 spikes/s) neurons. No clear differences were found between high firing rate and low firing rate neurons according their fibre type and histological locations. Of the SSN neurons that excited by AMPA, 22% (4/18) and 50% (5/9) of the neurons also were induced to fire following ionophoretic application of the NMDA receptor agonist NMDA in different concentrations, 20 mM and 100 mM, respectively. In neurons that induced firing by AMPA and by NMDA, AMPA-evoked firings were induced by the lower intensities of applied current and had higher mean firing rates than NMDA-evoked firing. Neurons that were induced firing by AMPA and by NMDA had B fibre and C fibre axons as well as those that induced firing only by AMPA. Neurons that were fired only by AMPA were found in whole SSN area, whereas neurons that were induced firing by AMPA and by NMDA were mainly found in intermediate SSN area. In conclusion, activation of ionotoropic non-NMDA receptor has a greater excitatory effect on the SSN neurons than that of ionotropic of NMDA receptor. Our data support the view that non-NMDA receptor plays a major role, whereas NMDA receptor plays a minor role, in the activation of SSN neurons.     

Ionotropic NMDA receptor evokes an excitatory response in superior salivatory nucleus neurons in anaesthetized rats

Extracellular recordings were taken from preganglionic superior salivatory nucleus (SSN) neurons projecting to submandibular and intra-lingual ganglia, in order to study the action of SSN neurons resulting from ionophoretic application of ionotropic NMDA receptor agonist in urethane-chloralose anaesthetized rats. Single SSN neurons were identified by their antidromic spike responses following stimulation of the chorda-lingual nerve (CLN), chorda tympani branches (CTBs) and the lingual nerve (LN). About one-third (33%, 10/30) of the identified SSN neurons were induced to fire by ionophoretic application of the NMDA receptor agonists used, dl-homocysteic acid (DLH) and N-methyl-D-aspartic acid (NMDA). More than half exhibited firing at high frequencies, often exceeding 40 Hz. About one-fifth (20%; 6/30) of the identified SSN neurons exhibited orthodromic spike responses to the combination of NMDA receptor agonist application and sensory nerve (CLN or LN) stimulus. These excitatory responses evoked by application of NMDA receptor agonist were attenuated (n = 4) by ionophoretic application of DL-2-amino-5-phosphonovaleric acid (AP5; NMDA receptor antagonist). About half (47%) of the neurons did not respond to any combination of NMDA receptor agonist and sensory nerve stimuli. No differences were observed between SSN neurons with B fibre axons and those with C fibre axons in response to ionophoresis of the NMDA receptor agonists. The NMDA-sensitive neurons, which exhibited high frequency firing, were predominantly found in the rostral part of the SSN. In summary, activation of ionotropic NMDA receptors exerts an excitatory effect on about half of the SSN neurons. These data support the view that NMDA receptors are involved in information processing and transmission on SSN neurons.     

Inhibitory role of periventricular dopaminergic mechanisms in hemorrahge-induced vasopressin secretion in conscious rats

Acute blood loss (16 ml/kg b. wt.) in conscious rats caused, 5 min later, increases in plasma vasopressin (AVP) concentration accompanied by reductions in arterial pressure and hematocrit. The plasma AVP response was markedly enhanced by intracerebroventricular injection (10 μl) of a dopamine antagonist, haloperidol (0.15 μmol), which did not affect the responses of arterial pressure and hematocrit significantly. These results suggest that periventricular dopaminergic mechanisms may act to inhibit hemorrhage-induced AVP secretion.     

Role of the parvicellular reticular formation in facilitating the jaw-opening reflex in rats by stimulation of the red nucleus

In a previous study, we have shown that electrical and chemical stimulation of the red nucleus (RN) facilitates the jaw-opening reflex (JOR). The RN sends projection fibers bilaterally, with contralateral dominance, to the part of the parvicellular reticular formation (RFp) containing premotor neurons projecting to the trigeminal motor nucleus. This implies that RN-induced facilitation of the JOR might be mediated via last-order neurons in the RFp. Here, we address this issue by investigating whether microinjection of lidocaine or l-glutamate into the RFp affects RN-induced modulation of the JOR. Experiments were performed on rats anesthetized with urethane-chloralose. The JOR was evoked by electrical stimulation of the inferior alveolar (IA) nerve and was recorded as an electromyographic response from the anterior belly of the digastric muscle. Conditioning stimulation was delivered unilaterally to the RN 12 ms before the IA test stimulation. We found that local injections of 2% lidocaine (0.5 microl) into the RFp, contralateral to the RN, significantly (P < 0.05) reduce RN-induced facilitation of the JOR, whereas corresponding injections of 0.1 mM l-glutamate (0.5 microl) significantly (P < 0.05) increase it. These results suggest that the facilitatory effect of RN stimulation on the JOR is mediated partly by a relay in the RFp.     

The rostral parvicellular reticular formation neurons mediate lingual nerve input to the rostral ventrolateral medulla

In rats that had been anesthetized by urethane-chloralose, we investigated whether neurons in the rostral part of the parvicellular reticular formation (rRFp) mediate lingual nerve input to the rostral ventrolateral medulla (RVLM), which is involved in somato-visceral sensory integration and in controlling the cardiovascular system. We determined the effect of the lingual nerve stimulation on activity of the rRFp neurons that were activated antidromically by stimulation of the RVLM. Stimulation of the lingual trigeminal afferent gave rise to excitatory effects (10/26, 39%), inhibitory effects (6/26, 22%) and no effect (10/26, 39%) on the RVLM-projecting rRFp neurons. About two-thirds of RVLM-projecting rRFp neurons exhibited spontaneous activity; the remaining one-third did not. A half (13/26) of RVLM-projecting rRFp neurons exhibited a pulse-related activity, suggesting that they receive a variety of peripheral and CNS inputs involved in cardiovascular function. We conclude that the lingual trigeminal input exerts excitatory and/or inhibitory effects on a majority (61%) of the RVLM-projecting rRFp neurons, and their neuronal activity may be involved in the cardiovascular responses accompanied by the defense reaction.     

A highly specific and sensitive radioimmunoassay of caerulein, an analogue of cholecystokinin-8

A highly specific and sensitive competitive radioimmunoassay was developed for caerulein (CLN), an analogue of cholecystokinin-8 (CCK-8), in plasma and brain. Antiserum was produced in rabbit by immunization with N delta-[CLN-(1-6)]-ornithine amide conjugated with bovine serum albumin by the glutaraldehyde method. N alpha-[CLN-(1-6)]-lysine amide was labelled with 125I-Bolton & Hunter reagent and used as a labelled antigen after purification by high-performance liquid chromatography. This assay was highly specific for CLN, and cross reactivities for other related peptides, CCK-4, CCK-8, gastrin-I, and gastrin-(14-17), were not observed (less than 0.01%). The limits of determination in biological specimens after CLN administration were 11 pg/ml in human plasma and rat plasma and 80 pg/g in rat brain. This study showed that the slight structure difference between hapten and 125I-labelled antigen is important to the assay performance.