Effects of hyperoxia and allergic airway inflammation on cough reflex intensity in guinea pigs

Toxic influence of high oxygen concentration on pulmonary function and structures has been known for many years. However, the influence of high oxygen concentration breathing on defensive respiratory reflexes is still not clear. In our previous experiments, we found an inhibitory effect of 100 % oxygen breathing on cough reflex intensity in healthy guinea pigs. The present study was designed to detect the effects of hyperoxia on cough reflex in guinea pigs with allergic airway inflammation. In the first phase of our experiment, the animals were sensitized with ovalbumin. Thirty-two sensitized animals were used in two separate experiments according to oxygen concentration breathing: 100 % or 50 % oxygen for 60 h continuously. In each experiment, one group of animals was exposed to hyperoxia, another to ambient air. The cough reflex was induced both by aerosol of citric acid before sensitization, then in sensitized animals at 24 h and 60 h of exposition to oxygen/air in awake animals, and by mechanical stimulation of airway mucosa in anesthetized animals just after the end of the experiment. In contrast to 50 % oxygen, 100 % oxygen breathing leads to significant decrease in chemically induced cough in guinea pigs with allergic inflammation. No significant changes were present in cough induced by mechanical stimulation of airways.     

Encoding of the cough reflex in anesthetized guinea pigs

We have previously described the physiological and morphological properties of the cough receptors and their sites of termination in the airways and centrally in the nucleus tractus solitarius (nTS). In the present study, we have addressed the hypothesis that the primary central synapses of the cough receptors subserve an essential role in the encoding of cough. We found that cough requires sustained, high-frequency (≥8-Hz) afferent nerve activation. We also found evidence for processes that both facilitate (summation, sensitization) and inhibit the initiation of cough. Sensitization of cough occurs with repetitive subthreshold activation of the cough receptors or by coincident activation of C-fibers and/or nTS neurokinin receptor activation. Desensitization of cough evoked by repetitive and/or continuous afferent nerve activation has a rapid onset (<60 s) and does not differentiate between tussive stimuli, suggesting a central nervous system-dependent process. The cough reflex can also be actively inhibited upon activation of other airway afferent nerve subtypes, including slowly adapting receptors and pulmonary C-fibers. The sensitization and desensitization of cough are likely attributable to the prominent, primary, and unique role of N-methyl-d-aspartate receptor-dependent signaling at the central synapses of the cough receptors. These attributes may have direct relevance to the presentation of cough in disease and for the effectiveness of antitussive therapies.     

Antigen-nonspecific and specific suppression of lymphokine production in desensitized guinea pigs

Doubly immunized guinea pigs may be desensitized with respect to delayed hypersensitivity reactions against both antigens (anergy) by injection of large doses of either one. This anergic response therefore has both a specific and nonspecific component. The specific component of desensitization persists longer than the nonspecific one. In the present study, we have explored the mechanism of both antigen-specific and antigen-nonspecific suppression during the later stages of desensitization. Guinea pigs immunized with two antigens, DNP-KLH and DNP-EA, were desensitized with DNP-EA. The lymph node cells obtained from the animals 1 day after desensitization were unable to produce MIF in the presence of either antigen. The cells obtained 3, 5, and 7 days after desensitization were able to generate MIF when stimulated with the non-specific antigen (DNP-KLH), but not with specific antigen (DNP-EA). It was shown that both T- and non-T-cell fractions obtained 1 day after desensitization had the capacity to antigen-nonspecifically suppress MIF production. In contrast, if the cells were obtained 3 or 5 days after desensitization, T cells could inhibit only the antigen-specific production of MIF, while non-T cells were still capable of suppressing antigen-specific and nonspecific MIF production. Interestingly, when these two populations were mixed back again, it was now only suppressive to the specific antigen-induced MIF production. This latter observation indicates that nonspecific suppressor non-T cells may themselves be regulated by suppressor T cells. Furthermore, antigen-specific suppressor T cells were shown to produce soluble factor(s) which inhibited the production of MIF.     

Differential effects of airway afferent nerve subtypes on cough and respiration in anesthetized guinea pigs

The hypothesis that respiratory reflexes, such as cough, reflect the net and often opposing effects of activation of multiple afferent nerve subpopulations throughout the airways was evaluated. Laryngeal and tracheal mucosal challenge with either citric acid or mechanical probing reliably evoked coughing in anesthetized guinea pigs. No other stimulus reliably evoked coughing in these animals, regardless of route of administration and despite some profound effects on respiration. Selectively activating vagal C-fibers arising from the nodose ganglia with either adenosine or 2-methyl-5-HT evoked only tachypnea. Selectively activating vagal afferents arising from the jugular ganglia induced respiratory slowing and apnea. Nasal afferent nerve activation by capsaicin, citric acid, hypertonic saline, or histamine evoked only respiratory slowing. Histamine, which activates intrapulmonary rapidly adapting receptors but not airway or lung C-fibers or tracheal bronchial cough receptors induced bronchospasm and tachypnea, but no coughing. The results indicate that the reflexes initiated by stimuli thought to be selective for some afferent nerve subtypes will likely depend on the net and potentially opposing effects of multiple afferent nerve subpopulations throughout the airways. The data also provide further evidence that the afferent nerves regulating cough in anesthetized guinea pigs are distinct from either C-fibers or intrapulmonary rapidly adapting receptors.     

Effects of airway inflammation on cough response in the guinea pig

We have developed a guinea pig model for coughrelated to allergic airway inflammation. Unanesthetized animals wereexposed to capsaicin aerosols for 10 min, and cough frequency wascounted during this period. The cough evaluation was performed by the following three methods: visual observation, acoustic analysis, andmonitoring of pressure changes in the body chamber. These analysesclearly differentiated a cough from a sneeze. To elucidate therelationship between cough response and airway inflammation, animalswere immunosensitized and multiple challenged. Sensitized guinea pigspresented no specific changes microscopically, but multiple-challengedanimals showed an increased infiltration of inflammatory cells into theairway. Cough number in response to capsaicin increased significantlyfrom 4.7 ± 1.4 coughs/10 min in normal animals to 10.6 ± 2.0 coughs/10 min in sensitized animals and further to 22.8 ± 1.3 coughs/10 min in multiple-challenged animals. This augmentedcough frequency was significantly inhibited by the inhalation oftachykinin-receptor antagonists and by oral ingestion, but notinhalation, of codeine phosphate. The results suggest that airwayinflammation potentiates an elevation of cough sensitivity in this model.

     

Protease-activated receptor-2 activation exaggerates TRPV1-mediated cough in guinea pigs.

A lowered threshold to the cough response frequently accompanies chronic airway inflammatory conditions. However, the mechanism(s) that from chronic inflammation results in a lowered cough threshold is poorly understood. Irritant agents, including capsaicin, resiniferatoxin, and citric acid, elicit cough in humans and in experimental animals through the activation of the transient receptor potential vanilloid 1 (TRPV1). Protease-activated receptor-2 (PAR2) activation plays a role in inflammation and sensitizes TRPV1 in cultured sensory neurons by a PKC-dependent pathway. Here, we have investigated whether PAR2 activation exaggerates TRPV1-dependent cough in guinea pigs and whether protein kinases are involved in the PAR2-induced cough modulation. Aerosolized PAR2 agonists (PAR2-activating peptide and trypsin) did not produce any cough per se. However, they potentiated citric acid- and resiniferatoxin-induced cough, an effect that was completely prevented by the TRPV1 receptor antagonist capsazepine. In contrast, cough induced by hypertonic saline, a stimulus that provokes cough in a TRPV1-independent manner, was not modified by aerosolized PAR2 agonists. The PKC inhibitor GF-109203X, the PKA inhibitor H-89, and the cyclooxygenase inhibitor indomethacin did not affect cough induced by TRPV1 agonists, but abated the exaggeration of this response produced by PAR2 agonists. In conclusion, PAR2 stimulation exaggerates TRPV1-dependent cough by activation of diverse mechanism(s), including PKC, PKA, and prostanoid release. PAR2 activation, by sensitizing TRPV1 in primary sensory neurons, may play a role in the exaggerated cough observed in certain airways inflammatory diseases such as asthma and chronic obstructive pulmonary disease.     

Effects of methylprednisolone pulses on renal function of guinea pigs

Methylprednisolone produced a dose dependant significant increase in glomerular filtration rate, a significant decrease in sodium excretion, and a significant increase in urinary alkaline phosphatase activity in guinea pigs. The renal histology in groups with 4, 5 and 6 doses revealed mild degenerative changes in the tubular epithelial cells. The results suggest the beneficial effects of methylprednisolone pulse therapy on renal function mainly by increasing glomerular filtration rate with only minimal side effects.     

Possible mechanisms of dose-dependent cough suppressive effect of Althaea officinalis rhamnogalacturonan in guinea pigs test system

The rhamnogalacturonan, isolated from the roots of medicinal plant Althaea officinalis L., showed various biological effects on the citric acid-induced cough reflex and reactivity of airways smooth muscle in vitro and in vivo conditions. It possessed dose-dependent cough suppression effect comparable with opioid agonist codeine. However, reactivity of the airways smooth muscle, measured in vitro as well as in vivo conditions was not significantly affected by rhamnogalacturonan and thus bronchodilatory activity did not participate in the cough suppression effect of polysaccharide tested. Moreover, the cough suppression effect of the polymer was not significantly modified by pretreatment of K⁺_ATP ion channels with selective antagonist and therefore activation of this type of ion channels is not involved in the mechanism of rhamnogalacturonan cough suppressive ability. On the contrary, pretreatment of animals with selective 5-HT₂ receptors antagonist significantly decreased rhamnogalacturonan antitussive efficacy. From this point of view it seems that the cough suppression effect of the polymer is associated with the serotonergic 5-HT₂ receptor's function.     

Effects of antiprogesterones on myometrial cell-to-cell coupling in pregnant guinea pigs.

We used intracellular microelectrodes to investigate the effects of the antiprogesterone (AP) compounds RU 486 and ZK 299 on cell-to-cell coupling in the guinea pig myometrium during pregnancy. The input resistance (Ro) of myometrial cells was high in nonpregnant tissues (44.6 +/- 6.39 M omega), but decreased by midgestation (Day 44 or 45 of gestation; 22.9 +/- 3.17 M omega), and was lowest at term (between 17.7 +/- 2.90 m omega and 13.1 +/- 4.34 M omega on Days 59-69). Treatment with the AP RU 486 or ZK 299 in three groups of midgestational animals reduced Ro to a similar level within 24 h. Lucifer Yellow (LY) was injected into smooth muscle cells as a direct but qualitative measure of metabolic coupling. In term and AP-treated animals, LY spread rapidly to neighboring cells within 60 sec, but little spread occurred in midgestational control tissues and no spread was seen even after 10 min in nonpregnant tissues. This correlation of decreased Ro (implying increased electrical coupling) with the development of extensive spread of LY indicates increased electrical and metabolical coupling between myometrial cells during labor. These data show that myometrial smooth muscle cells of guinea pigs are moderately well coupled before the onset of labor, and the coupling increases further, just prior to spontaneous delivery or due to treatment with APs. These events may be required for synchronizing and coordinating the electrical, metabolic, and contractile activity of labor.