David A. Taylor

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.

Opioid receptors mediating antinociception from β-endorphin and morphine in the periaqueductal gray

beta-Endorphin and morphine produce an increase in the latency of the tail-flick reflex when administered into the PAG of awake rats. The antinociceptive effect of both opioid agonists was blocked by the sequential local injection of either CTP (D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2), a selective mu opioid receptor antagonist, naltrexone, or beta-endorphin (1-27), a putative epsilon opioid receptor antagonist, with minimal selectivity. When either CTP or naltrexone was used as the antagonist, the dose-inhibition curves generated for beta-endorphin and morphine were not parallel, suggesting the involvement of separate and distinct receptors. Also, synergism occurred when a dose of morphine producing submaximum antinociception was administered simultaneously with either a submaximal or ineffective dose of beta-endorphin. Inhibition of the antinociceptive response to beta-endorphin by mu antagonists and the non-selective antagonism of both beta-endorphin and morphine by beta-endorphin (1-27) suggested that epsilon opioid receptors were not involved. Additionally, a mu/delta opioid receptor complex was not involved, since ICI 174,864 (Allyl2-Tyr-Aib-Aib-Phe-Leu-OH), a selective delta opioid receptor antagonist, did not alter the response to beta-endorphin. Thus, although additional characterization is required, beta-endorphin and morphine appear to act (at least in part) through different opioid receptors, demonstrable using selected mu opioid receptor antagonists.

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.

Nicotine increases FosB expression within a subset of reward- and memory-related brain regions during both peri- and post-adolescence

IntroductionPeriadolescent nicotine exposure is associated with increased consumption and rewarding properties of abused drugs. In the case of peri- but not post-adolescent animals, these effects are persistent and last to adulthood, suggesting that early nicotine treatment may alter postnatal CNS development in ways that contribute to long-term problems with drug abuse.Materials and methodsTo begin to identify brain regions that may be altered by developmental nicotine exposure, we have measured expression of a transcription factor, FosB, within a series of reward- and memory-related brain regions of Sprague–Dawley rats.ResultsFosB expression is known to acutely and cumulatively increase within a subset of brain regions, particularly nucleus accumbens, after exposure to many classes of abused drugs. Our results demonstrate that FosB is increased within nucleus accumbens and also the granule cell layer of hippocampal dentate gyrus after both peri- and post-adolescent nicotine exposure (0.4xa0mg kg−1 day−1 from daysxa034 to 43 and 60 to 69, respectively). In periadolescents, expression increases were detected 2xa0days after nicotine exposure, and persisted for weeks, through at least early adulthood at 80xa0days of age. In post-adolescents, expression increases persisted for at least 11xa0days to postnatal dayxa080.DiscussionThese findings demonstrate that nicotine treatment during both peri- and post-adolescence persistently alters activity of brain regions involved in reward and memory.ConclusionBecause this altered gene expression occurs after both peri- and post-adolescent treatment, it cannot be directly responsible for increased consumption and rewarding properties of abused drugs previously established to be distinctly associated with periadolescent nicotine exposure.

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.

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.

Identification of a Nonendothelial Cell Thromboxane-Like Constrictor Response and Its Interaction with the Renin-Angiotensin System in the Aorta of Spontaneously Hypertensive Rats

Aortic ring preparations from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats were treated with N omega-nitro-L-arginine (NOLA, 10(-4) M). This produced a sustained contraction in preparations from SHR but not WKY rats. A similar contraction in aortic ring preparations from the SHR was produced with methylene blue (10(-5) M) and NG-monomethyl-L-arginine (10(-5) M). The NOLA-induced contraction was reversed with indomethacin (8 x 10(-6) M), ridogrel (10(-5) M) and SQ 29548 (10(-6) M) thus confirming the involvement of thromboxane A2/prostaglandin H2 processes. Subsequent experiments demonstrated that the thromboxane-like contraction was not dependent upon the presence of endothelial cells and occurred in preparations from young, prehypertensive (5 week) and older (17 week) SHRs. The thromboxane-like contraction was markedly suppressed with chronic captopril treatment and reinstated 4 weeks after withdrawal from captopril. The addition of saralasin (10(-6) M) or captopril (10(-6) M) to aortic ring preparations did not suppress the thromboxane-like contractions. The foregoing findings support the presence of a nonendothelial cell thromboxane-like constrictor agent in the aorta of the SHR that is revealed after impairment of nitric oxide production. The activity of the thromboxane-like constrictor process is not tightly linked to prevailing blood pressure, but is reduced with chronic in vivo inhibition of the angiotensin-converting enzyme.

The effect of reserpine treatment on the chronotropic and inotropic sensitivities of the perfused guinea-pig heart to norepinephrine and calcium

Abstract The chronotropic and inotropic responses of the isolated perfused guinea-pig heart to norepinephrine and calcium were measured in preparations obtained from untreated and reserpinetreated animals. Dose-response curves were obtained in spontaneously beating as well as electrically paced perfused hearts. The dose-response curve for the chronotropic effect of both norepinephrine and calcium was shifted to the left of control in spontaneously beating preparations from animals pretreated with reserpine (0.1 mg/kg/day) for seven days. However, in these same preparations the dose-response curve for the inotropic effect of only norepinephrine was shifted to the left. This may be reflection of the enhanced sensitivity to the chronotropic action of the agonist since a changing rate is known to influence inotropic response. This conclusion is supported by the finding that, in electrically paced perfused hearts, no postjunctional supersensitivity to the inotropic effects of norepinephrine was demonstrable. Since no supersensitivity was observed to the inotropic effects of calcium, it is concuded that true postjunctional supersensitivity develops to the chronotropic but not to the inotropic effects of drugs.

Renal cortical Na+-K+-ATPase activity and abundance is decreased in normal pregnant rats.

During late pregnancy, the rat undergoes massive plasma volume expansion due to cumulative renal sodium retention. In the present study, conducted in virgin, mid- ( days 11-13), and late-pregnant ( days 18-20) rats, we measured both Na+-K+-ATPase activity (by coupled enzyme assay) and abundance of the α-subunits of the Na+-K+-ATPase (by Western and slot blot analyses) in renal cortex, medulla, and brain stem. Unexpectedly, Na+-K+-ATPase in renal cortex, in both stages of pregnancy, is reduced versus the virgin, consistent with our finding of a reduced quantity of the α1-subunit. In renal medulla, there is a small rise in activity at midterm, but there is no difference in either activity or abundance of the α1-subunit in late pregnancy, when renal Na retention is maximal. In brain stem, where only α2- and α3-subunits are evident, pregnancy has no impact on enzyme activity or abundance of either isoform. In conclusion, the outcome of these experiments was unexpected in that we did not observe increased renal Na+-K+-ATPase activity in late pregnancy in the rat. In fact, in renal cortex, Na+-K+-ATPase activity and abundance are reduced. Whatever promotes net sodium retention in pregnancy must be capable of overwhelming this and several other strong natriuretic signals.