A quantitative method for assessing protein abundance using enhanced chemiluminescence.
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Animals↗
Biomedical subjects
W W Fleming
Publications and source records attributed to W W Fleming.
Adaptive supersensitivity in the guinea pig vas deferens is associated with a reduction in the abundance of the alpha 2 subunit isoform of Na+/K(+)-ATPase.
Adaptive supersensitivity has been demonstrated previously in the guinea pig vas deferens after chronic treatment with reserpine, postganglionic denervation, or preganglionic denervation. The magnitude of the change in sensitivity was similar regardless of the method of induction; the underlying mechanism was identified as a partial depolarization secondary to reduced activity of the Na+/K+ pump. Experiments were conducted to quantitatively determine whether the identified losses in Na+/K(+)-ATPase activity and [3H]ouabain binding were due to reductions in the levels of specific protein subunits of the sodium pump. Electrophoretic separation and quantification of the abundance of alpha subunit isoforms were accomplished using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and slot blot analysis. Supersensitivity was induced in the guinea pig vas deferens through pretreatment with reserpine (1.0 mg/kg/day x 5 days). The abundance of the alpha 2 subunit isoform was reduced by 41% in tissue homogenates obtained from animals treated with reserpine, compared with untreated controls. In contrast, there was no significant alteration in the alpha 1 subunit isoform (a protein similar in size to that previously identified in vascular smooth muscle as a "truncated" form of the protein). These data suggest that the adaptation of the guinea pig vas deferens after a chronic reduction in net stimulus is mediated through a change in a specific cellular protein. This evidence supports the assignment of the alpha 2 subunit isoform as the specific protein responsible for the development of nonspecific adaptive supersensitivity in the guinea pig vas deferens.
Animals↗
Adenylyl cyclase and guanine nucleotide-binding proteins in supersensitive guinea pig ventricles.
Chronic treatment with reserpine (0.1 mg/kg/day x 7 days) leads to the development of adaptive supersensitivity of ventricular myocardium of guinea pigs. The compensatory increase in sensitivity is associated with a small increase in beta-adrenoreceptor number. However, sensitivity is increased to a number of agonists that do not interact with beta-adrenoceptors. An evaluation of the role of both adenylyl cyclase and guanine nucleotide-binding regulatory proteins in the development of adaptive supersensitivity was carried out using crude membrane fragments from untreated control and chronically reserpine-treated guinea pigs. Quantitative analysis of Gs alpha and Gi protein concentrations was accomplished using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Chronic treatment with reserpine reduced basal levels of adenylyl cyclase activity by nearly 60%. The reduced activity was not the result of a loss of endogenous norepinephrine, because incubation of tissues in the presence of propranolol did not alter the basal level of adenylyl cyclase activity. Incubation in the presence of guanylylimido diphosphate (10(-5) M) also significantly reduced basal adenylyl cyclase activity, by nearly 70%. Chronic treatment with reserpine failed to significantly alter the activation of adenylyl cyclase by isoproterenol, impromidine, NaF, or forskolin. These data suggest that chronic treatment with reserpine does not alter agonist-induced activation of adenylyl cyclase. Furthermore, analysis of Gs alpha and Gi indicated that chronic treatment with reserpine did not affect the levels of these regulatory proteins in ventricular myocardial membranes. The data indicate that the enhanced sensitivity of guinea pig ventricular myocardium is not the result of an alteration in adenylyl cyclase activity or in the concentration of guanine nucleotide regulatory proteins. Therefore, the enhanced responsiveness to widely diverse agonists must be due to an alteration in cellular function beyond the level of adenylyl cyclase.
Adenylyl Cyclase Inhibitors↗
Membrane potential in myenteric neurons associated with tolerance and dependence to morphine.
Chronic treatment of guinea pigs with morphine produces subsensitivity (tolerance) of the longitudinal smooth muscle-myenteric plexus preparation to a variety of inhibitory agonists (e.g., mu opioid, alpha adrenoceptor and adenosine receptor agonists) and supersensitivity (dependence) to a variety of excitatory agonists (e.g., nicotine, 5-hydroxytryptamine and potassium ions). The present investigation was to determine if these changes in sensitivity could be related to changes in electrical properties of the S and AH neurons in the myenteric plexus. S neurons from morphine-implanted animals were significantly depolarized (7 mV) relative to those from placebo-implanted animals, whereas the membrane potential of AH neurons was unchanged. Approximately 60% of S neurons were hyperpolarized by morphine. In this subset of neurons, membranes were significantly depolarized but the threshold was unchanged in morphine-implanted animals. This means that resting potentials of S neurons from tolerant preparations are closer to threshold. The hyperpolarization produced by morphine (0.1 microM) was similar in preparations from morphine- and placebo-implanted animals. Thus, the partially depolarized state of S neurons in the myenteric plexus is the cause of the subsensitivity and supersensitivity to agonists and can explain both tolerance and dependence. Changes in opioid receptors or their coupling to potassium channels do not appear to contribute to tolerance in the longitudinal smooth muscle-myenteric plexus.
Action Potentials↗
Mesenteric vascular responses of young spontaneously hypertensive rats.
Frequency-responses curves for nerve stimulation and dose-response curve for norepinephrine, 5-hydroxytryptamine potassium chloride, vasopressin and acetylcholine (ACh) were determined in isolated, perfused mesenteric vascular beds from young (approximately 5 weeks) spontanelouly hypertensive (SHR) and Wistar Kyoto rats. Although mean systolic blood pressure (measured by tail cuff plethysmography) was slightly higher in the SHR, this difference was not significant. Slopes and maximum responses were increased significantly for nerve stimulation and all agonists. The basal perfusion pressure was also significantly elevated in the SHR. These differences are consistent with existing evidence that structural changes occur in blood vessels of SHR at an early stage and probably precede development of hypertension. Such structural changes could therefore contribute to development of the hypertension. Cocaine (1 microM) markedly increased responses to nerve stimulation and bolus injections of norepinephrine in preparations from SHR with little or no effect on such responses in Wistar Kyoto preparations, a result consistent with the known greater density of noradrenergic nerves in SHR vasculature. In the presence of cocaine, there was unmasked a selective super-sensitivity (significantly lower ED50) to norepinephrine in the SHR. Thus SHR mesenteric vessels may possess an alteration in adrenoreceptors or their coupling to other cellular mechanisms. Responses to ACh revealed no indication of a deficient endothelial mediated relaxation. An altered media:lumen ratio of small arteries, hypernoradrenergic innervation and supersensitivity to the transmitter may contribute to development of hypertension.
Animals↗
Cyclic AMP and adaptive supersensitivity in guinea pig atria.
Supersensitivity was induced by injection of guinea pigs with reserpine, 0.1 mg/kg/day for 7 days. This treatment induced chronotropic supersensitivity of isolated right atria to isoproterenol but not to forskolin. The pretreatment induced supersensitivity to the cyclic AMP-generating effects of isoproterenol or forskolin in left, but not right, atria. These results are discussed in reference to the extensive literature describing supersensitivity in the guinea-pig heart, including the possible role of the adenylyl cyclase system.
Animals↗
Catecholamine and neuropeptide Y levels in tissues from young Dahl rats following 5 days low- or high-salt diet.
The mesenteric vasculature of Dahl salt-sensitive (DS) rats on high-salt diet is supersensitive to nerve stimulation and to norepinephrine. The current experiments were undertaken to examine whether the enhanced sensitivity to nerve stimulation is due solely to the postsynaptic supersensitivity to norepinephrine, to increased sympathetic innervation, to altered transmitter release or to the presence of another transmitter acting as a potentiator. Catecholamine content and neuropeptide Y (NPY) presence were determined in tissues from young (approximately 5 weeks old) male Dahl rats exposed to 5 days of high (7%) or low (0.45%) salt diet. Catecholamine content from mesenteric artery, renal artery, caudal artery, right atrium, aorta, vas deferens and adrenal gland was quantified by high-pressure liquid chromatography with an electrochemical detector. A strain difference, independent of diet, between young DS and Dahl salt-resistant (DR) rats was seen only in adrenal epinephrine content. DS high-salt (+) rats displayed reduced norepinephrine content relative to DR+ in the mesenteric artery and right atrium. The release of norepinephrine from isolated mesenteric vasculature into the perfusate in response to transmural stimulation showed no significant differences between DS+ and DR+ preparations under basal, or deoxycorticosterone acetate (DOCA; 30 microM) perfusion conditions. The addition of 5 microM cocaine to the DOCA perfusion, while increasing total norepinephrine outflow in all preparations, failed to differentiate between DS+ and DR+. NPY immunofluorescence along mesenteric artery sections of DS+ and DR+ rats was not significantly different. Thus, in the tissues examined, enhanced responsiveness of vascular smooth muscle may not be explained by hypernoradrenergic innervation, elevated NPY innervation or altered release of transmitter.
Animals↗
Specific supersensitivity of the mesenteric vascular bed of Dahl salt-sensitive rats.
Dahl salt-sensitive (DS) and salt-resistant (DR) rats were maintained on a diet containing normal (0.45%) or high (7%) salt for 5 days. The DS rats had slightly higher systolic blood pressures than DR rats, although a high salt diet failed to significantly elevate pressure in either group when compared with their appropriate (low salt diet) controls. The sensitivity of the isolated, perfused mesenteric vasculature from DS rats fed a high salt diet to nerve stimulation was greater when compared with all other groups in the presence or absence of cocaine (1 microM). A similar difference in sensitivity between high salt DS rats and high salt DR rats to bolus injections of norepinephrine was observed only in the presence of cocaine. The change in sensitivity was characterized by a leftward shift of the dose-response curve without a change in maximum response. No difference in sensitivity between the high salt DS group and any other treatment group was observed in response to the pressor agents KCl, angiotensin II, 5-hydroxytryptamine or the depressor agent acetylcholine. These data indicate that DS rats on a short-term, high salt diet possess a significant and specific elevation in sensitivity to nerve stimulation and norepinephrine in the absence of an increase in blood pressure. Differences in the effectiveness of cocaine among the groups suggest that differences may exist in neuronal uptake (uptake 1).(ABSTRACT TRUNCATED AT 250 WORDS)
Acetylcholine↗
Involvement of mu, delta and kappa receptors in morphine-induced tolerance in the guinea pig myenteric plexus.
Tolerance in the longitudinal muscle-myenteric plexus (LM-MP) preparation to opioids appears to represent two separate phenomena. One phenomenon is characterized by marked subsensitivity to mu-selective opioid substances and a very short half-life. The other is characterized by moderate subsensitivity to morphine and to several nonopioid inhibitory substances and has a long half-life. The present investigation was to determine if the long half-life type of tolerance involves cross-tolerance between mu-selective opioids, such as morphine, and opioids selective for delta or kappa receptors. Tolerance was induced by s.c. implantation of morphine pellets for 7 days. Control guinea pigs received placebo pellets. Isolated LM-MP preparations were tolerant to the mu-selective agonists morphine and [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin as well as to [D-Ala,2D-Leu5]enkephalin and [D-Ser2]leucine enkephalin-Thr, agonists only slightly selective for delta receptors over mu. The delta-selective agonist, [D-Pen2,D-Pen3]enkephalin, produced a biphasic concentration-response curve. The first phase, which included only very weak effects, presumably represented delta-mediated effects. The second phase reached virtually 100% inhibition of the twitch. Significant subsensitivity (tolerance) was demonstrated only for the second phase, presumably representing mu-mediated effects. There was no significant tolerance to the kappa-selective agonist, U50,488H. Consistent with the literature, the results indicate effective twitch inhibition mediated by mu and kappa receptors, but not delta receptors. Among opioids, tolerance induced by morphine pellets in the LM-MP is limited to mu receptor-mediated inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Sensitivity of cerebellar Purkinje neurons to neurotransmitters in genetically epileptic rats.
Epilepsy is a state of neuronal hyperactivity which may be caused by an altered relationship between inhibitory and excitatory influences on neurons. We have conducted experiments using microiontophoresis with in situ extracellular recording of anesthetized rat cerebellar Purkinje neuron activity to determine the sensitivity of these neurons to neurotransmitters in a genetic model of epilepsy. Quantitative evaluations of agonist-induced changes in activity were carried out by using poststimulus time histograms. Current-response curves were generated and linear regression analysis was performed to evaluate changes in responsiveness and sensitivity between control and genetically epilepsy-prone-9 rats. The current required to produce 50% inhibition of activity by GABA was 2.6-fold higher in the genetically epilepsy-prone-9 rats compared to control. In contrast, the amount of current required to produce 50% inhibition by norepinephrine was not significantly different between groups. There also was no significant change in cerebellar neuron sensitivity to the excitatory transmitter, glutamate. The lack of an alteration in sensitivity and responsiveness to norepinephrine or glutamate suggests that the hyperexcitability of neurons may be associated with a specific subsensitivity of the GABAergic system. Such a specific subsensitivity to gamma-aminobutyric acid would, therefore, yield a more excitable state of the neuron and may contribute to the development of the hyperactivity observed in epilepsy.
Animals↗
Effects of GABA in the morphine-tolerant longitudinal muscle, myenteric plexus preparation of the guinea pig.
The effect of chronic treatment with morphine via pellet implantation on the sensitivity of the longitudinal smooth muscle-myenteric plexus of the guinea pig ileum to the contractile effects of gamma-aminobutyric acid (GABAA)-receptor agonists was assessed. GABA and muscimol elicited concentration-dependent contractions of the longitudinal smooth muscle which were due to the release of acetylcholine because the contractile effects were markedly attenuated by atropine (10 nM). The contractile action of GABA agonists does not involve an intermediate step mediated by nicotinic receptors because the concentration-response curves for GABA were unaffected by hexamethonium (1 mM). Bicuculline (10 microM) produced nearly equivalent rightward shifts of the concentration-response curves for both GABA and muscimol, indicating mediation of the contractile effects of these agents by GABAA receptors. Chronic exposure to morphine via pellet implantation did not alter the sensitivity of this preparation to either GABA or muscimol. This is in contrast to the development of supersensitivity of the longitudinal smooth muscle-myenteric plexus to other excitatory agonists (nicotine, 5-hydroxytryptamine and potassium), which accompanies the development of tolerance to opioids. GABA induces depolarization of myenteric neurons that is observed most prominently in AH neurons and rarely in S neurons. The stimulatory effects of nicotine and of GABA were inhibited by morphine (a predominantly mu opioid agonist) and by U50,488H (a predominantly kappa opioid agonist). The results are discussed within the context that supersensitivity to neuronal stimulants of the myenteric plexus in morphine-tolerant preparations is limited to substances which depolarize S neurons.
Animals↗
Antagonism of norepinephrine by clonidine in the isolated rat mesenteric vascular bed.
Experiments were done in isolated, perfused mesenteric vascular beds from Sprague-Dawley rats. Bolus injections of norepinephrine (3-100 nmol) induced dose-dependent increases in perfusion pressure with a maximum increase greater than 100 mm Hg. In the same dose range, clonidine had no effect on perfusion pressure. In the presence of an elevated pressure caused by constant infusions of norepinephrine (6-20 microM), bolus injections of clonidine (0.1-10 nmol) or acetylcholine (0.007-7 nmol) caused dose-related decreases in perfusion pressure. Procedures which damage endothelium (brief exposure to methylene blue or reactive oxygen radicals) abolished the depressor action of acetylcholine but only moderately reduced the depressor action of clonidine. The depressor action of clonidine was not antagonized by the alpha-2 adrenoceptor antagonist, idazoxan. Acetylcholine produced depressor responses in the presence of 5-hydroxy-tryptamine or vasopressin, but clonidine did not. Dose-response curves to bolus doses of norepinephrine were shifted markedly to the right by an alpha-1 selective concentration of prazosin (1 nM) and were shifted to the right with depression of maximum by infusions of clonidine (0.3 and 1.0 microM). It is concluded that, in the mesenteric vasculature of the rat: 1) the role of alpha-2 adrenoceptors, in responses to clonidine, is minimal; 2) endothelial factors play little role, if any, in the depressor effects of clonidine and 3) clonidine has a potent ability to interfere with the alpha-1 adrenoceptor-mediated vasoconstriction induced by norepinephrine. This antagonistic action may be at the level of the receptor but could involve postreceptor steps.
Acetylcholine↗
The development and reversal of the tolerance to morphine in the longitudinal smooth muscle-myenteric plexus preparation of the guinea pig.
Chronic treatment with morphine results in a reduction in the potency of morphine in the longitudinal smooth muscle-myenteric plexus of the guinea-pig ileum. Implantation of morphine pellets leads to the development of tolerance to the inhibitory effects of morphine upon neurogenic contractions of this preparation. Tolerance develops within 24 hours, peaks between days 4 and 7 and disappears by day 14. A similar time course for the development of tolerance to the inhibitory effects of 2-chloroadenosine is also seen in these same morphine-tolerant preparations. The rate of reversal of morphine tolerance was assessed after the removal of the morphine pellets four days after implantation. In this situation, tolerance to the effects of morphine were maintained for at least 24 hours, were partially reversed at day 2 and were totally reversed by day 4. The delay in the development and reversal of the effect are consistent with the fact that chronic treatment with morphine evokes an adaptive sensitivity change.
2-Chloroadenosine↗
Mechanisms of adaptive supersensitivity in smooth muscles vs cardiac muscle: a brief review.
1. Depending upon the type of cell being studied, adaptive supersensitivity has been traced to molecular changes in receptors, the Na+/K+ pump or the G-protein/adenylate cyclase system. 2. Evidence is reviewed that indicates that adaptive supersensitivity in guinea-pig atria is the result of changes in the G-protein/adenylate cyclase system.
Animals↗
Mechanisms of cellular adaptive sensitivity changes: applications to opioid tolerance and dependence.
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Adaptation, Physiological↗
Morphine tolerance and nonspecific subsensitivity of the longitudinal muscle myenteric plexus preparation of the guinea-pig to inhibitory agonists.
1. 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.
2-Chloroadenosine↗
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)-salt 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% KCl 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 in sensitivity between hypertensive and control caudal arteries was demonstrable at low rates of perfusion, 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 in 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 in 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 in either the caudal artery or the mesenteric vasculature, a finding consistent with the observed decrease in endogenous norepinephrine content. Microelectrodes were used to determine resting membrane potential in the smooth muscle cells. No differences in 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 in 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 in hypertension, 3) the supersensitivity of blood vessels in 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.
Animals↗
Membrane potential and vascular smooth muscle sensitivity. A minireview.
A review of research on the relationship between the regulation of cellular sensitivity to drugs and transmitters and membrane potential is presented, with an emphasis on experiments with the rabbit saphenous artery. Chronic depression of the adrenergic innervation to the saphenous artery induces an adaptive and nonspecific increase in sensitivity which appears with a delay of 3 days. Coincident with the supersensitivity, the smooth muscle cells undergo a partial depolarization which brings the resting potential closer to the threshold potential for contraction. The evidence is that the depolarization is due to the loss of electrogenic Na+, K+ pumping and is the primary factor responsible for the supersensitivity.
Animals↗