William W. Fleming

Airway epithelium modulates the reactivity of guinea-pig respiratory smooth muscle.

The influence of the epithelium on the reactivity of guinea-pig tracheal smooth muscle in vitro was investigated. Mechanical removal of the epithelium produced contrasting effects on methacholine-, histamine- and K+-induced contractions of tracheal strips. Epithelium removal resulted in 2.1-fold shifts to the left of histamine and methacholine concentration-response curves, and an increase in the maximum response to histamine; the maximum response to methacholine was unaffected. Epithelium removal had little effect on the sensitivity, but decreased the maximum response to K+. The results suggest that inhibitory and excitatory factor(s) are released from epithelial cells, and that these factors modulate the reactivity of the smooth muscle. Indomethacin (1 microM) produced in epithelium-containing preparations qualitatively identical effects on the sensitivity to methacholine and on the maximum response to histamine as removal of the epithelium. However, indomethacin was without effect on the sensitivity to histamine in the presence or absence of the epithelium. Alterations in the production or release of epithelial cell-derived factors may contribute to the airway hyperreactivity observed in respiratory disorders.

Epithelium modulates the reactivity of ovalbumin-sensitized guinea-pig airway smooth muscle

Mechanical removal of the airway epithelium alters the in vitro reactivity of airway smooth muscle. The modulation of reactivity may involve the release of inhibitory and excitatory factors from epithelial cells. Guinea pigs sensitized with ovalbumin have been used as an animal model of airway hyperreactivity. We evaluated the influence of the epithelium on the reactivity of in vitro tracheal smooth muscle from control and ovalbumin-sensitized guinea pigs, and the extent to which the presence of the epithelium affects the contractile response to in vitro challenge with ovalbumin. In both control and ovalbumin-sensitized tissues, epithelium removal increased the sensitivity of the preparations to histamine, methacholine and isoproterenol to a similar extent, i.e., 2- to 2.5-fold. Epithelium removal resulted in an 8.1-fold increase in sensitivity to ovalbumin in sensitized tissues. The epithelium appears not only to modulate the reactivity of the tissues to bronchoactive agents, but it also influences the magnitude of the contractile response following antigen challenge.

Sensitivity of caudal arteries and the mesenteric vascular bed to norepinephrine in DOCA-salt hypertension.

This study was undertaken to determine what factors might contribute to arterial supersensitivity to norepinephrine associated with deoxycorticosterone acetate (DOCA)-sait hypertension in the rat. Experimental groups of male rats were uninephrectomized and 1 week later began receiving twice weekly Injections of DOCA (20 mg/kg s.c. in sesame oil) plus 1% NaCl and 0.2% KC1 in their drinking water. For each experimental group, a group of age-matched male rats underwent a sham operation and received injections of sesame oil and the NaCl-KCl drinking water. Perfused caudal arteries from 3- week-hypertensive rats were supersensitive to intraluminal and extraluminal norepinephrine administration. However, this difference hi sensitivity between hypertensive and control caudal arteries was demonstrable at low rates of perfuslon, 0.5 to 1.0 ml/min, but not at rates of 2.0 to 2.6 ml/min. The supersensitivity was not due to differences hi neuronal uptake or to inhibition of extraneuronal uptake by DOCA. The perfused mesenteric vascular bed from 3- or 6-week-hypertensive rats was also supersensitive to intraluminal norepinephrine. However, the demonstration of supersensitivity hi the mesenteric vasculature was independent of perfusion rate (2.3–6.8 ml/min) and perfusion pressure in the range of 30 to 60 mm Hg. There was little or no supersensitivity to transmural nerve stimulation hi either the caudal artery or the mesenteric vasculature, a finding consistent with the observed decrease hi endogenous norepinephrine content. Microelectrodes were used to determine resting membrane potential hi the smooth muscle cells. No differences hi resting membrane potential were detected between caudal or mesenteric arteries from hypertensive compared with control rats 2, 3, or 6 weeks after initiation of the DOCA-salt regimen. It is concluded that 1) the perfusion rate is a critical factor hi designing experiments to test the sensitivity of caudal arteries to drugs, 2) the perfused mesenteric vascular bed is a useful preparation for studying sensitivity of blood vessels hi hypertension, 3) the supersensitivity of blood vessels hi the DOCA-salt model may be of greater importance relative to circulating catecholamines than to sympathetic innervation, and 4) the supersensitivity of blood vessels to norepinephrine in the DOCA-salt model is not due to changes in neuronal uptake, extraneuronal uptake, or membrane potential of the vascular smooth muscle cells.

The effect of verapamil is reduced in isolated airway smooth muscle preparations lacking the epithelium

The effect of epithelium removal on the reactivity of rabbit airway smooth muscle to bronchoactive agents and on the effect of verapamil was studied in vitro using preparations from several levels within the respiratory tree, i.e., trachea, primary (10) and secondary (20) bronchus. Methacholine contracted tissues from all three levels of airway. Histamine contracted strips from 20 bronchus, had an inconsistent action in strips from 10 bronchus and was without effect in tracheal preparations. K+ contracted tissues from the trachea and 10 bronchus, and had a mixed action in 20 bronchial strips. Removal of the epithelial cell layer variably affected the reactivity of the smooth muscle to the three agents studied. In 20 bronchus, epithelium removal potentiated responses to histamine and methacholine. In 10 bronchus, only responses to methacholine were consistently augmented. In tracheal preparations epithelium removal did not alter the reactivity of the tissue to any agent examined. Verapamil (1 microM) attenuated responses to all agents and increased in its potency from tracheal through 10 to 20 bronchial preparations. Following epithelium removal, verapamil was substantially less effective in 20 bronchi, yet its effects were unchanged in the trachea. The results indicate that the epithelial cell layer modulates airway smooth muscle reactivity; this phenomenon is apparently widespread in mammals, the modulatory effect is more prominent in the smaller airways, and the magnitude of the effect of verapamil on airway smooth muscle is, in part, related to the presence of the epithelium.

Morphine tolerance and nonspecific subsensitivity of the longitudinal muscle myenteric plexus preparation of the guinea-pig to inhibitory agonists

Summary1.The sensitivity of the longitudinal smooth muscle/myenteric plexus (LM/MP) to agonists which reduce the amplitude of neurogenic contractions was studied in preparations obtained from animals implanted with either placebo or morphine (75 mg/pellet) pellets 7 days prior.2.Tolerance or subsensitivity to morphine was observed following chronic treatment with morphine and was revealed as a rightward shift of the concentration-response curve to morphine. The degree of tolerance decayed modestly with time after removal from a morphine containing environment suggesting a time dependence for the loss of subsensitivity to morphine.3.LM/MP preparations from animals pretreated with morphine also developed subsensitivity to the inhibitory effects of the purine analogue, 2-chloroadenosine. Subsensitivity to 2-chloroadenosine was seen as a parallel rightward shift of the concentration-response curve in morphine-tolerant preparations. The magnitude of the loss in sensitivity was comparable to that observed to morphine.4.A reduction in sensitivity of the LM/MP following chronic treatment with morphine was also observed to the inhibitory effects of the alpha2 adrenoceptor agonists, clonidine and xylazine. In contrast to the results obtained with morphine and 2-chloroadenosine, the development of subsensitivity to alpha2 adrenoceptor agonists was characterized by a marked reduction in slope and a depression of the maximum response.5.These data suggest that myenteric neurons possess spare receptors for morphine and 2-chloroadenosine but not for clonidine and xylazine. Furthermore, the studies support the idea that tolerance is associated with a general cellular change or adaptation which impacts on all of these inhibitory substances in such a way as to reduce their efficacy.

The role of GABAA receptors in the subsensitivity of Purkinje neurons to GABA in genetic epilepsy prone rats

The GABA receptor subtype mediating responses of cerebellar Purkinje neurons to the neurotransmitter was evaluated and compared in GEPR-9 vs. nonepileptic, genetic control GEPR-NE rats. Quantitative analysis of responses to microiontophoretically applied GABA, muscimol and baclofen indicated that the inhibitory action of GABA on cerebellar Purkinje neurons was mediated by GABAA receptors since muscimol produced responses similar to those of GABA and baclofen was without substantial electrophysiological action. In addition, Purkinje neurons in GEPR-9 animals showed a similar reduced sensitivity to both GABA and muscimol. Radioligand binding studies using the GABAA receptor selective ligand, [3H]muscimol, and the benzodiazepine receptor selective ligand, [3H]flunitrazepam, were conducted on cerebellar and cortical homogenates from GEPR 9, GEPR-NE and Sprague-Dawley rats. No differences in the Kd or Bmax for these ligands among the three groups studied were observed. The lack of significant changes in the Kd and Bmax for these two ligands in the cerebellum suggests that the mechanism for the observed subsensitivity to GABA in the GEPR 9 rat lies beyond the level of the receptor, perhaps at the signal transduction process for GABA mediated inhibitory responses.

The trophic influence of autonomic nerves on electrical properties of the cell membrane in smooth muscle.

Abstract The relationship of neurotransmission to sensitivity in a variety of effector cell types is briefly surveyed. The several cellular changes which contribute to supersensitivity in denervated skeletal muscle are listed. Subsequently evidence is reviewed regarding the role of a partial depolarization of the smooth muscle cells in the development of supersensitivity in the guinea-pig vas deferens. The depolarization appears to be the result of the decreased activity of an electrogenic Na + , K + pump. The applicability of these findings to other autonomically innervated effectors is discussed.

Na+, K+ ATPase α-subunit isoform distribution and abundance in guinea-pig longitudinal muscle/myenteric plexus after exposure to morphine

Previous work in the myenteric plexus has shown that the resting membrane potential of morphine-tolerant guinea-pig myenteric S neurons is significantly depolarized relative to placebo-implanted controls, and that this depolarization is associated with reduced electrogenic Na+, K+ pumping. Identification of the subunits of the sodium pump which are in the myenteric plexus was undertaken in order to facilitate direct qualitative and quantitative measurements of the abundance of sodium pump isoforms after morphine exposure, thereby confirming and extending the electrophysiological data to the molecular level. Seven days prior to the experiments, tolerance was induced by subcutaneous implantation of morphine pellets (one pellet, 75 mg/100 g body weight) while control guinea pigs received placebo pellets. Using immunohistochemistry and confocal microscopy, the distribution of the alpha subunit isoforms of the Na+/K+ -ATPase in placebo and morphine-tolerant guinea-pig ileum was determined. Only the alpha1 and alpha3 subunit isoforms were in sufficient abundance to be observed. The alpha1 subunit isoform was most highly concentrated in the mucosa and in neurons. In contrast, the alpha3 subunit isoform was uniquely localized to neurons. Western and slot blot analyses of longitudinal muscle/myenteric plexus homogenates identified a significant reduction of the alpha3 but not the alpha1 subunit isoform in tolerant preparations. It is concluded that the reduced electrogenic pumping in the S neurons after morphine exposure is associated with a reduction in the alpha3 subunit isoform.

Effects of denervation and reserpine on nexuses in the rat vas deferens.

Permanganate-fixed vasa deferentia from rats were examined for the presence of nexal-like contacts by electron microscopy. There was a significantly greater incidence of nexuses (2X) in chronically denervated tissues (5-7 days) but not in tissues from reserpine-pretreated animals (1.0 mg/kg/day for 5-7 days). The results suggest that an increase in nexal regions may not be a general feature of postjunctional supersensitivity but rather may contribute to other denervation-induced changes in contractile response.

Quantification of the α3 subunit of the Na+/K+-ATPase in developing rat cerebellum

Abstract Cerebellar Purkinje neurons of rats have been shown to exhibit a progressive increase in resting membrane potential as the animals develop postnatally. The magnitude of this increase was equivalent in magnitude to the increase in the depolarizing action of ouabain, consistent with a role for the Na+/K+-pump in the hyperpolarization. Ouabain binding sites in whole cerebellum also increased with age. The present study was undertaken to confirm that the increases in ouabain binding and the electrophysiological responses to ouabain were a consequence of increases in the sodium pump and to determine whether the changes seen at the whole organ level were reflective of changes taking place at the cellular level. Using antibodies directed against the α1, α2, and α3 subunits of the Na+/K+-ATPase, rats between 13 and 19 days of age exhibited a statistically significant increase in the relative amount of the α3 subunit at the level of the whole organ, as determined by Western and slot blot analyses, with no change in the levels of either the α1 or the α2 subunit. Using immunohistochemistry, the α3 subunit was shown to increase in both the Purkinje cell layer and the white matter during this postnatal time period, while the α1 subunit increased in the granular layer. These results support and extend previous work, which pointed to a role for the electrogenic sodium pump in the developmental increase in Purkinje cell membrane potential. Furthermore, the data provide a cellular mechanism underlying the increase in resting membrane potential, that is, by the specific modulation of the α3 subunit isoform.