Carotid body denervation effect on cytochrome oxidase activity in pre-Botzinger complex of developing rats.

Previously, we found that the rat pre-B?tzinger complex (PBC) exhibited reduced cytochrome oxidase (CO) activity on postnatal days (P) 3-4 and especially on P12, with a concomitant decrease in glutamate and N-methyl-d-aspartate receptor subunit 1, and an increase in GABA, GABA(B), glycine receptor, and glutamate subunit 2. We hypothesized that the PBC would be more affected by carotid body denervation (CBD) during the two critical windows than at other times. Pairs of CBD and sham animals at each postnatal day from P2 to P14 and at P21 were operated on and survived for 3 days. Brain stems were processed for CO and neurokinin-1 receptor for the identification of PBC. Results indicate that CBD caused a significant loss in body weight in all animals and a reduction in PBC somal size when the surgery was between P2 and P7. CBD also induced a significant decrease in CO activity of the PBC in most animals and a distinct delay, as well as prolongation of the maturational process, especially when induced close to P3 and P11-P13.     

Respiratory actions induced by cholecystokinin at the brainstem level

A functional differentiation of the action of cholecystokinin octapeptide (CCK-8) on the respiratory centers was accomplished by the topical application to the ventral surface of the medulla and to the dorso-rostral pontine surface in cats. In the medulla, CCK-8S at doses ranging from 0.09 nmol to 0.88 nmol, stimulated tidal volume in a dose-dependent fashion, with minimal or no changes in frequency. The antagonist proglumide (30 nmol) inhibited specifically the action on the respiratory amplitude. In the pons, CCK-8S did not modify the respiratory activity even at the dose of 8.8 nmol. The results suggest a specific involvement of CCK-8S in the mechanisms controlling respiratory amplitude, which appear mostly restricted to the medullary level. The lack of effect of the peptide in the pons is in agreement with the absence of CCK receptors in the respiration related nuclei located at that level, as evidenced by autoradiographic studies.     

Excitatory effects of bombesin receptors in urinary tract of normal and diabetic rats in vivo

Aims

Bombesin receptors (BB receptors) and bombesin related peptides are expressed in the lower urinary tract of rodents. Here we investigated whether in vivo activation of BB receptors can contract the urinary bladder and facilitate micturition in sham rats and in a diabetic rat model of voiding dysfunction.

Material and methods

In vivo cystometry experiments were performed in adult female Sprague–Dawley rats under urethane anesthesia. Diabetes was induced by streptozotocin (STZ; 65 mg/kg, i.p.) injection. Experiments were performed 9 and 20 weeks post STZ-treatment. Drugs included neuromedin B (NMB; BB1 receptor preferring agonist), and gastrin-releasing peptide (GRP; BB2 receptor preferring agonist).

Key findings

NMB and GRP (0.01–100 μg/kg in sham rats; 0.1–300 μg/kg in STZ-treated rats, i.v.) increased micturition frequency, bladder contraction amplitude and area under the curve dose dependently in both sham and STZ-treated rats. In addition, NMB (3, 10 μg/kg i.v.) triggered voiding in > 80% of STZ-treated rats when the bladder was filled to a sub-threshold voiding volume. NMB and GRP increased mean arterial pressure and heart rate at the highest doses, 100 and 300 μg/kg.

Significance

Activation of bombesin receptors facilitated neurogenic bladder contractions in vivo. Single applications of agonists enhanced or triggered voiding in sham rats as well as in the STZ-treated rat model of diabetic voiding dysfunction. These results suggest that BB receptors may be targeted for drug development for conditions associated with poor detrusor contraction such as an underactive bladder condition.     

Suppressive effects of cholecystokinin and bombesin on growth hormone and prolactin secretion in urethane-anesthetized rats

The effects of cholecystokinin octapeptide (CCK) and bombesin on rat plasma growth hormone (GH) and prolactin (PRL) levels were investigated with the animals under urethane anesthesia. Intraventricular administration of both CCK (0.3 micrograms) and bombesin (2 micrograms) completely suppressed the GH secretion induced by FK 33-824, chlorpromazine (CPZ) or prostaglandin E2(PGE2). Both peptides also completely suppressed the PRL secretion induced by FK 33-824 or PGE2, and partially that induced by CPZ, but not that induced by domperidone. The intravenous administrations of CCK and bombesin had no or lesser potency in inhibiting the stimulated GH or PRL releases. These results indicate that the CCK and bombesin act much in the same manner to inhibit GH and PRL. These peptides may suppress the GH and PRL secretions via a hypothalamus-related action.     

Respiratory effects of intrathecal capsaicin in arthritic and non-arthritic rats

The study determined the effects of intrathecal injection of 50 μg of capsaicin on respiration in rats with adjuvant arthritis as well as in control animals. Whole body plethysmographic measurements of steady-state frequency, tidal volume, and minute volume of respiration were made 3 hours and for up to 11 days after intrathecal injection. Capsaicin increased minute volume within 3 hours of its intrathecal injection in control animals. Intrathecal capsaicin also reduced the respiratory response to adjuvant arthritis in the experimental animals; the latter effect was apparent 11 days after injection. This biphasic pattern of capsaicin effects is consistent with a possible role of substance P in the chronic pain which is presumably associated with adjuvant arthritis in the rat.     

Respiratory and metabolic effects of decreased osmolality in conscious rats

We investigated the respiratory and metabolic effects of decreased osmolality, and the potential roles of angiotensin II (ANG II) and the subfornical organ (SFO) in mediating these effects, in conscious Sprague-Dawley (SD) rats. Gastric water loading was induced either by oral gavage or an externalized indwelling stomach tube (20 mL x kg(-1) distilled water at body temperature). Repeated measurements after oral gavage were obtained with and without water loading and with and without ANG II receptor block (saralasin, 1.3 microg x kg(-1) x min(-1) iv). At 15 min after water loading by oral gavage, ventilation (V, 1.14+/-0.08 L x kg(-1) x min(-1)) and tidal volume (10.7+/-0.6 mL x kg(-1)) were transiently higher (P < 0.05), at a time when plasma osmolality was decreased (-8+/-1 mOsm), compared with gavage tube alone (0.95+/-0.08 L x kg(-1) min(-1) and 9.1+/-0.7 mL x kg(-1), respectively). However, water loading via stomach tube did not stimulate V; only during the 60-s period of water infusion did V increase briefly, but this was due to increased respiratory frequency. Dye indicators demonstrated that oral gavage exposes upper airway and esophageal afferents to water, presumably accounting for respiratory stimulation. Lesions of the SFO did not affect respiration or metabolism. A decrease in osmolality, associated with both water loading techniques, caused a sustained increase in oxygen consumption (Vo2 ) and a decrease in the V/Vo2 ratio. ANG II receptor block reduced the Vo2 response and prevented the decrease in V/Vo2 following water loading by oral gavage, but did not affect the transient stimulation of V. Unlike larger mammals, decreased osmolality does not stimulate respiration in the SD rat.     

Direct unfolding of RuvA-HJ complex at the single-molecule level

The repair of double-stranded DNA breaks via homologous recombination involves a four-way cross-strand intermediate known as Holliday junction (HJ), which is recognized, processed, and resolved by a specific set of proteins. RuvA, a prokaryotic HJ-binding protein, is known to stabilize the square-planar conformation of the HJ, which is otherwise a short-lived intermediate. Despite much progress being made regarding the molecular mechanism of RuvA-HJ interactions, the mechanochemical aspect of this protein-HJ complex is yet to be investigated. Here, we employed an optical-tweezers-based, single-molecule manipulation assay to detect the formation of RuvA-HJ complex and determined its mechanical and thermodynamic properties in a manner that would be impossible with traditional ensemble techniques. We found that the binding of RuvA increases the unfolding force (F_unfold) of the HJ by ～2-fold. Compared with the ΔG_unfold of the HJ alone (54 ± 13 kcal/mol), the increased free energy of the RuvA-HJ complex (101 ± 20 kcal/mol) demonstrates that the RuvA protein stabilizes HJs. Interestingly, the protein remains bound to the mechanically melted HJ, facilitating its refolding at an unusually high force when the stretched DNA molecule is relaxed. These results suggest that the RuvA protein not only stabilizes the HJs but also induces refolding of the HJs. The single-molecule platform that we employed here for studying the RuvA-HJ interaction is broadly applicable to study other HJ-binding proteins involved in the critical DNA repair process.     

Respiratory effects of pressor and depressor agents in conscious rats

We hypothesized that the respiratory baroreflex in conscious rats is either more transient, or has a higher pressure threshold than in other species. To characterize the effect of arterial pressure changes on respiration in conscious rats, ventilation (V) was measured by the plethysmographic technique during injections, or infusions, of pressor and depressor agents. Bolus injections of angiotensin II (Ang II) or arginine vasopressin (AVP), transiently increased mean arterial pressure (MAP; mean +/- SE) 43+/-6 and 28+/-5 mm Hg (1 mm Hg = 133.3 Pa), respectively, and immediately reduced tidal volume (Vt) and, in the case of AVP, V. In contrast, by 10 min of a sustained elevation of MAP (40+/-3 mm Hg) with infusion of Ang II, Vt, f, and V were not different from control levels. Bolus injection of sodium nitroprusside (SNP) to lower MAP (-28+/-3 mm Hg) immediately increased breathing frequency (f) and V, whereas sustained infusion of SNP to lower MAP (-21+/-3 mm Hg) did not change for V at 10 and 20 min. In conscious rats, both injection and infusion of the pressor agent PE (+40 to 50 mm Hg) stimulated f and V; this contrasted with anesthetized rats where PE inhibited f and V, as reported by others. In conscious rats, respiratory responses associated with baroreflexes adapt rapidly and, in the case of PE, can be overridden by some other mechanism.     

Cooperative effects of bombesin, substance P and methacholine on the release of intestinal neurotensin in rats.

The secretion of ileal neurotensin (NT) results from events occurring at the apical and basal side of the N-cells. The hypothesis of a functional relationship between cholinergic and peptidergic neurones with the N-cell was investigated in the present study utilizing the isolated vascularly perfused rat ileum. Intraarterial methacholine (MC, 10(-4) M) evoked a prompt and well sustained release of NT in the portal effluent (plateau value at 500% of basal). This effect was dose-dependent over the range of 10(-6) M to 10(-4) M. Bombesin (B) provoked a dose-dependent peak secretion of NT (800% of basal at 10(-7) M) followed by a rapid return to almost basal levels. The B-induced NT release remained unaltered upon 10(-6) M tetrodotoxin (TTX) or 10(-5) M atropine infusion. Substance P (SP) potently stimulated the release of NT. The maximal response, consisting of a sustained secretion, was observed at a concentration of 10(-7) M (350% of basal) while 10(-6) M SP induced a transient release. TTX or atropine did not reduce significantly the SP-induced secretion of NT. Neurokinin A and B did not increase NT concentrations in the portal effluent. B synergistically increased the secretion of NT induced by SP. Atropine or TTX did not modify the effect of combined SP and B infusion. MC potentiated the release of NT induced by B but not that evoked by SP. Combined infusion of SP, B and MC produced the largest output of NT. In conclusion, B, SP and MC are strong stimulants of NT release in rats. In addition, the cooperative effects of these transmitters argue in favor of a complex functional relationship between the intramural nervous network and the intestinal N-cells in rats.     

Influence of respiratory network drive on phrenic motor output evoked by activation of cat pre-Botzinger complex

We have previously demonstrated that microinjection of dl-homocysteic acid (DLH), a glutamate analog, into the pre-B?tzinger complex (pre-B?tC) can produce either phasic or tonic excitation of phrenic nerve discharge during hyperoxic normocapnia. Breathing, however, is influenced by input from both central and peripheral chemoreceptor activation. This influence of increased respiratory network drive on pre-B?tC-induced modulation of phrenic motor output is unclear. Therefore, these experiments were designed to examine the effects of chemical stimulation of neurons (DLH; 10 mM; 10-20 nl) in the pre-B?tC during hyperoxic modulation of CO2 (i.e., hypercapnia and hypocapnia) and during normocapnic hypoxia in chloralose-anesthetized, vagotomized, mechanically ventilated cats. For these experiments, sites were selected in which unilateral microinjection of DLH into the pre-B?tC during baseline conditions of hyperoxic normocapnia [arterial PCO2 (PaCO2) = 37-43 mmHg; n = 22] produced a tonic (nonphasic) excitation of phrenic nerve discharge. During hypercapnia (PaCO2 = 59.7 +/- 2.8 mmHg; n = 17), similar microinjection produced excitation in which phasic respiratory bursts were superimposed on varying levels of tonic discharge. These DLH-induced phasic respiratory bursts had an increased frequency compared with the preinjection baseline frequency (P < 0.01). In contrast, during hypocapnia (PaCO2 = 29.4 +/- 1.5 mmHg; n = 11), microinjection of DLH produced nonphasic tonic excitation of phrenic nerve discharge that was less robust than the initial (normocapnic) response (i.e., decreased amplitude). During normocapnic hypoxia (PaCO2 = 38.5 +/- 3.7; arterial Po2 = 38.4 +/- 4.4; n = 8) microinjection of DLH produced phrenic excitation similar to that seen during hypercapnia (i.e., increased frequency of phasic respiratory bursts superimposed on tonic discharge). These findings demonstrate that phrenic motor activity evoked by chemical stimulation of the pre-B?tC is influenced by and integrates with modulation of respiratory network drive mediated by input from central and peripheral chemoreceptors.     

Decreased behavioral effects of daily intracerebroventricular bombesin

Intracerebroventricular (ICV) bombesin increases grooming and decreases food intake in rats. We examined tolerance to these effects by administering a daily injection of either saline or 25 ng bombesin to rats for 8 days via lateral ventricular cannulas. Food intake and grooming were monitored. After 8 days bombesin no longer increased grooming or decreased food intake in bombesin-treated rats, but did increase grooming and decrease food intake in saline-treated rats. This development of behavioral tolerance conflicts with previous reports using larger doses and demonstrates that repeated small doses of ICV bombesin produce different effects from larger doses.     

Inhibition of itch-related responses at spinal level in rats

The goal was to develop a rat model for determination of the effects of intrathecally administered drugs on the peripherally induced pruritic behaviors. After chronic intrathecal catheterization, a serotonin derivative (5-methoxytryptamine: MeOT, 200 μg on both sides) was injected into the lower leg skin. After the first period (phase 0: 0-30 min) MeOT injection was repeated and opioid antagonist naltrexone (10 μg), NMDA receptor antagonists ketamine (10-100 μg), kynurenic acid (1-10 μg) or their combinations were injected intrathecally. The second observational period lasted for 60 min (phases I and II, 30-60 and 60-90 min, respectively). MeOT produced pruritic behavior with high degree of interindividual differences. The second MeOT injection caused an enhanced pruritic behavior in Phase I. Naltrexone decreased the pruritic activity, while neither doses of ketamine influenced the effects of MeOT. The higher doses of kynurenic acid resulted in notable decreases in the pruritic behavior. The combinations of naltrexone with ketamine or kynurenic acid produced a prolonged antipruritic effect. Our data suggest an important direction for the development of a new itch model in rats that focuses on the spinal mechanism of itching. Besides, the results revealed the role of the spinal opioid and NMDA receptors in this process.     

Ionotropic excitatory amino acid receptors in pre-Botzinger complex play a modulatory role in hypoxia-induced gasping in vivo.

Activation of ionotropic excitatory amino acid (EAA) receptors in pre-B?tzinger complex (pre-B?tC) not only influences the eupneic pattern of phrenic motor output but also modifies hypoxia-induced gasping in vivo by increasing gasp frequency. Although ionotropic EAA receptor activation in this region appears to be required for the generation of eupneic breathing, it remains to be determined whether similar activation is necessary for the production and/or expression of hypoxia-induced gasping. Therefore, we examined the effects of severe brain hypoxia before and after blockade of ionotropic EAA receptors in the pre-B?tC in eight chloralose-anesthetized, deafferented, mechanically ventilated cats. In each experiment, before blockade of ionotropic EAA receptors in the pre-B?tC, severe brain hypoxia (6% O2 in a balance of N2 for 3-6 min) produced gasping. Although bilateral microinjection of the broad-spectrum ionotropic EAA receptor antagonist kynurenic acid (20-100 mM; 40 nl) into the pre-B?tC eliminated basal phrenic nerve discharge, severe brain hypoxia still produced gasping. Under these conditions, however, the onset latency to gasping was increased (P < 0.05), the number of gasps was reduced for the same duration of hypoxic gas exposure (P < 0.05), the duration of gasps was prolonged (P < 0.05), and the duration between gasps was increased (P < 0.05). These findings demonstrate that hypoxia-induced gasping in vivo does not require activation of ionotropic EAA receptors in the pre-B?tC, but ionotropic EAA receptor activation in this region may modify the expression of the hypoxia-induced response. The present findings also provide additional support for the pre-B?tC as the primary locus of respiratory rhythm generation.     

The action of the collagen-Fe2+ complex at tissular level

The aim of this paper is to know the iron dynamics and action of the collagen-Fe2+ complex at tissular level. The results showed that the collagen-Fe2+ complex is biocompatible at tissular level and the ferrous iron entered the animal organism on the well-known metabolic pathways. When a high dose of the complex was administered, an overloading with hemosiderin of macrophages and hepatocytes was noticed.     

Healing-promoting effect of bombesin treatment on chronic gastric ulcer in rats

To evaluate whether bombesin treatment has a facilitatory effect on the healing of chronic gastric ulcer, following the induction of ulcer by serosal application of acetic acid, rats were given bombesin (30 microg/kg/day; subcutaneously) or vehicle three times a day for 7, 14 or 21 days until they were decapitated. Neither food intake nor gastric emptying rate in either vehicle-treated or bombesin-treated groups was not statistically different from control rats. Similarly, ulcer indices and gastric myeloperoxidase (MPO) activities at the first and second weeks of injury were not different among the groups. However, in the 3-week ulcer group, bombesin treatment reduced tissue MPO level significantly back to control levels. Moreover, the analysis of the surface epithelium by scanning electron and light microscopy demonstrated a significant reduction in the severity of ulcers by bombesin treatment. Pretreatment with CCK antagonists (L-364,718 or L365,260; 25 micromol/kg/day) before bombesin treatment showed that neither of the CCK antagonists had a significant effect on the bombesin-mediated healing process, suggesting that CCK receptors are not involved in the action of bombesin. In accordance with the previous studies that show its acute gastroprotective effects, bombesin is also effective in promoting the healing process of chronic gastric ulcer in rats.     

Prevention by bombesin of cold-restraint stress induced hemorrhagic lesions in rats

Several neuropeptides, injected intraventricularly (ivt), were assessed for their effects on cold-restraint-induced hypothermia and hemorrhagic gastric lesions in 24 hr fasted rats. Bombesin (5-1 μg) further enhanced the drop in body temperature following stress and markedly prevented the gastric erosions in a dose-dependent fashion (5-0.1 μg). β-endorphin exerted a similar effect, but only at the 5 μg dose level. Other peptides (neurotensin, substance P, somatostatin and TRH: 5 μg) did not influence susceptibility to the gastric mucosal damage. Somatostatin and TRH reduced the hypothermic effect of stress. Bombesin is 250 times less potent when injected systemically than ivt and its actions are not reversed by nalaxone. The prevention of gastric erosions by bombesin could initially involve a central mechanism of action, independent of opiate receptors and possibly related to the sustained and marked hyperglycemia observed in bombesin treated rats exposed to stress.     

Gamma integrase complementation at the level of DNA binding and complex formation

Site-specific recombinases of the gamma Int family carry out two single-strand exchanges by binding as head-to-head dimers on inverted core-type DNA sites. Each protomer may cleave its own site as a monomer in cis (as for Cre recombinase), or it may recruit the tyrosine from its partner in trans to form a composite active site (as for Flp recombinase). The crystal structure of the gamma Int catalytic domain is compatible with both cleavage mechanisms, but two previous biochemical studies on gamma integrase (Int) generated data that were not in agreement. Support for cis and trans cleavage came from assays with bispecific DNA substrates for gamma and HK022 Ints and from functional complementation between recombination-deficient mutants, respectively. The data presented here do not provide new evidence for cis cleavage, but they strongly suggest that the previously described complementation results cannot be used in support of a trans-cleavage mechanism. We show here that IntR212Q retains some residual catalytic function but is impaired in binding to core-type DNA on linear substrates and in forming higher-order attL intasome structures. The binding-proficient mutant IntY342F can stabilize IntR212Q binding to core-type DNA through protein-protein interactions. Similarly, the formation of higher-order Int complexes with arm- and core-type DNA is boosted with both mutants present. This complementation precedes cleavage and thus precludes any conclusions about the mechanism of catalysis. Cross-core stimulation of wild-type HK022-Int cleavage on its cognate site (in cis) by mutant gamma Ints on bispecific core DNA suicide substrates is shown to be independent of the catalytic tyrosine but appears to be proportional to the respective core-binding affinities of the gamma Int mutants.