Na+, K+ ATPase alpha-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.

Cellular depolarization of neurons in the locus ceruleus region of the guinea pig associated with the development of tolerance to opioids.

These experiments were designed to test two hypotheses: 1) the tolerance induced by morphine pellet implantation in guinea pigs will result in subsensitivity of cells in the locus ceruleus (LC), not only to morphine, but to another agonist acting on a different receptor and transduction system, namely the gamma-aminobutyric acid(A) receptor agonist, muscimol; and 2) The nonspecific (heterologous) tolerance would be associated with a partial depolarization of the tolerant cells and a decrease in the contribution of electrogenic Na(+)/K(+) pumping. Extracellular recording from LC neurons in brain slices from animals implanted with either morphine or placebo pellets established that the tolerant preparations were subsensitive to both morphine and muscimol. Immunocytochemical analysis identified the alpha(3)-subunit as the primary isoform of the Na(+)/K(+) pump in the cells under investigation. Whole-cell patch clamp recording of neurons in brain slices demonstrated that, with electrodes containing 20 mM Na(+) (approximating [Na](i)), tolerant cells were significantly depolarized by a mean of 6.7 mV. Dialysis with antibody specific for the alpha(3)-isoform from patch pipettes produced depolarization of both control and tolerant cells. However, the depolarizing effect of the antibody was less in tolerant cells, suggesting a lesser degree of electrogenic Na(+) pumping. Furthermore, the presence of antibody reduced the membrane potentials of tolerant and placebo cells to equal values, suggesting that the diffusion potentials were not different. In contrast, antibody specific for the alpha(1)-subunit isoform in the pipettes had no effect on membrane potential in either control or tolerant cells. In conclusion, both hypotheses were supported.

Unifying perspectives of the mechanisms underlying the development of tolerance and physical dependence to opioids.

The cellular basis of tolerance to, and dependence upon, many types of drugs, including opioids, has long defied identification. Tolerance to opioids cannot be explained solely on the basis of modification of opioid receptors or altered metabolism or disposition of the opioid. The development of tolerance following chronic exposure to opioids presents at least three different types of change in cellular responsiveness, each of which has been suggested to represent some type of adaptive modification in cellular responsiveness. These different forms of tolerance are distinguishable on the basis of their time course and whether or not the tolerance is specific for opioid receptor agonists (homologous) or extends to agonists of other systems (heterologous). The adaptive modulation of responsiveness via regulation of cellular proteins has been proposed to be the basis for both longer-term forms of tolerance. The divergent signaling pathways activated by G-protein-coupled receptors like the mu-opioid receptor provide multiple downstream targets for both short- and long-term regulation of cell function that is associated with the development of tolerance and/or dependence. Since the magnitude of receptor activation is an important determinant of the degree to which various signaling pathways are activated, the expressed characteristics of tolerance and/or dependence may be functionally related to which of these diverse pathways are stimulated to the greatest degree. Thus, the possibility that different signaling events are activated either sequentially or concurrently offers the possibility to explain the interaction between these different forms of tolerance and/or dependence.

Alterations in neuronal gamma-aminobutyric acid(A) receptor responsiveness in genetic models of seizure susceptibility with different expression patterns.

The genetically epilepsy-prone rat (GEPR) is a unique animal model of seizure predisposition with substrains (i.e., GEPR-NE, GEPR-3, and GEPR-9) that exhibit different seizure patterns in response to the same stimulus. Among many deficits identified in these animals, reduced responses to GABA(A) receptor agonists have been described in several brain regions of the GEPR-9. However, few studies have quantitatively analyzed this difference in responsiveness or have examined and compared the responsiveness of GEPR-3 neurons with the other strains. Using intracellular recording, we determined and compared the responsiveness of Purkinje neurons from GEPR-3 animals with those of control (both Sprague-Dawley and GEPR-NE) and GEPR-9 rats at different developmental ages. In GEPR-9 animals, the EC(50) value for GABA and muscimol was shifted 3-fold to the right, with no reduction in maximum. In contrast, GEPR-3 animals showed a significant reduction in the maximum hyperpolarizing response to only GABA and muscimol with no change in the EC(50) values. Responsiveness to glutamate, aspartate, norepinephrine, and diazepam was unchanged in both strains, indicating that the change in responsiveness was highly selective for GABA(A) receptor agonists. Changes in responsiveness in animals <15 days of age suggests that deficits in GABAergic function exist before the development of seizure susceptibility. In addition, the data are the first to reveal that the GEPR-3 and GEPR-9 exhibit different changes in GABA(A) receptor function and may provide significant insight into the cellular mechanism underlying differences between these two strains.

Quantification of the alpha(3) subunit of the Na(+)/K(+)-ATPase in developing rat cerebellum.

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 alpha(1), alpha(2), and alpha(3) 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 alpha(3) 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 alpha(1) or the alpha(2) subunit. Using immunohistochemistry, the alpha(3) subunit was shown to increase in both the Purkinje cell layer and the white matter during this postnatal time period, while the alpha(1) 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 alpha(3) subunit isoform.

Cellular adaptation: journey from smooth muscle cells to neurons.

In the 1960s, it became clear that the adaptation of smooth muscle to denervation was different from that of skeletal muscle. The supersensitivity of denervated smooth muscle extended to agonists unrelated to the lost neurotransmitter and developed on a tissue-dependent time course of several days to several weeks. Several procedures, in addition to denervation, that interrupted excitatory transmission, elicited the phenomenon. The supersensitivity occurred without changes in density or affinity of receptors but correlated with a partial depolarization of the smooth muscle cells. The phenomenon could be mimicked by procedures that acutely depolarized the cells. Electrophysiological, biochemical, and molecular data established that the depolarization was due to reduced electrogenic pumping and reduced density of the Na(+),K(+) pump. The triggering event for the development of such supersensitivity is not interruption of contact of neurotransmitter with its receptor, but rather the decreased activity of the adapting cells. This is clear from the fact that the inhibitory action of opioids produces similar sensitivity changes in several different populations of guinea pig neurons, including S-type neurons of the myenteric plexus. Subcutaneous implantation of morphine pellets in guinea pigs induces adaptation of S neurons expressed as nonspecific subsensitivity to inhibitory agonists (opioids, alpha(2)-adrenoceptor agonists, 2-chloroadenosine) and supersensitivity to excitatory agonists (nicotine, 5-hydroxytryptamine, K(+)). These changes are accompanied by a partial depolarization of the S neurons and decreased electrogenic Na(+),K(+) pumping. Chronic implantation of morphine pellets also produces similar nonspecific changes in sensitivity in neurons of the nucleus tractus solitarius and locus ceruleus. It is suggested that depressed activity of these neurons leads to an electrophysiological adaptation, presumably due to reduced density of Na(+),K(+) pump proteins, as demonstrated in smooth muscle.

The role of the sodium pump in the developmental regulation of membrane electrical properties of cerebellar Purkinje neurons of the rat.

The postnatal development of the rat cerebellum has been well described morphologically and functionally. However, information regarding the electrical characteristics of Purkinje neurons during development is sparse. Using standard intracellular recording, the basic electrical properties of Purkinje neurons in cerebellar slices were compared at different postnatal ages. There was a significant, progressive increase in the resting membrane potential (RMP) of Purkinje neurons with age as well as a small but significant decrease in the cellular input resistance (Rin). The cardiac glycoside, ouabain (1 mM), an inhibitor of the sodium pump, depolarized Purkinje neurons significantly more with age. The magnitude of the increase in depolarizing activity of ouabain was equivalent to the magnitude of the increase in membrane potential. The number of ouabain binding sites was also found to increase with age suggesting an age related increase in the number of sodium pump sites. These results suggest that the predominant cellular mechanism which underlies the increase in membrane potential of Purkinje neurons during development is an increase in the density of Na+, K+ pump sites and in the contribution of the electrogenic sodium pump.

Preparation and application of an isolated superior mesenteric arterial vascular preparation.

Our laboratory developed an isolated perfused superior mesenteric arterial vascular bed preparation to study and correlate vascular smooth-muscle mechanics with associated biochemical events. This preparation provides consistent dose-dependent contractile responses, contains most of the superior mesenteric artery as well as first-, second-, and third-generation arterioles, and has been used for concurrent functional and biochemical analysis of vascular smooth muscle. Preparations isolated from Sprague-Dawley rats produced rapid, dose-related vasoconstrictor responses to norepinephrine (NE) and KCl, while appearing to be unresponsive to periarterial nerve stimulation. Endothelial relaxations to bolus doses of acetylcholine (ACh) in the presence of a constant infusion of NE (10 microM) were limited, producing reductions of perfusion pressures of <25%. Receptor-binding studies conducted to evaluate alpha1-adrenoceptor subtypes revealed high- and low-affinity binding sites composing 91 and 9% of the overall population, respectively. A 60-s time course for contractile response and inositol 1,4,5-triphosphate (IP3) production revealed a significant but transient increase of IP3 that paralleled the contractile response generated by using bolus injections of NE (30 microg). This preparation offers the capacity to conduct perfusion studies investigating vasoconstrictor responses, as well as biochemical studies including receptor-binding and second-messenger assays in the same tissue.

Examination by radioligand binding of the alpha1 adrenoceptors in the mesenteric arterial vasculature during the development of salt-sensitive hypertension.

Previous experiments have suggested that the vascular smooth muscle of Dahl salt-sensitive (DS) rats may possess a difference in the alpha1-adrenoceptor population or its transduction processes compared to Dahl salt-resistant (DR) rats. The purpose of the current research is to study the role of alpha1-adrenoceptors in the specific supersensitivity to norepinephrine (NE) seen prior to and early in the development of hypertension in the DS rat. Experiments in isolated perfused superior mesenteric arterial vasculature from DS rats chronically fed a high (7%) salt diet for 5 days or 3 weeks, in the absence or presence of an elevation in systolic blood pressure, respectively, demonstrated a specific supersensitivity to NE relative to DR rats. The enhanced responsiveness was specific to NE after 5 days of high salt since no differences in sensitivity of these preparations was observed to either KCl or 5-HT. A small but significant elevation in sensitivity to KCl following 3 weeks of treatment suggests that multiple factors may contribute to tissue responsiveness at this time. Radioligand binding experiments were performed using [125I]-HEAT to study the alpha1-adrenoceptor population and its subtypes. Saturation experiments using membranes prepared from the superior mesenteric arterial vasculature or mesenteric arterial branches showed no significant differences in overall alpha1-adrenoceptor population between DS and DR rats fed a high-salt diet for 5 days or 3 weeks. Competition experiments using membranes prepared from the superior mesenteric arterial branches in the presence of the alpha1A-subtype selective antagonist 5-methylurapidil showed two binding sites (high and low affinity) in these resistance vessels but no significant differences in nature or ratio of these sites between the DS and DR groups. These results suggest that changes in the alpha1-adrenoceptor population are not responsible for the specific supersensitivity to NE, which may be an early event in the induction and development of hypertension.

Hyperpolarizing effects of morphine, clonidine and 2-chloroadenosine in myenteric neurons associated with tolerance to morphine.

Chronic treatment of guinea pigs with morphine produces non-specific subsensitivity (tolerance) of the longitudinal smooth muscle myenteric plexus (LM/MP) preparation of the guinea pig ileum to morphine, clonidine and 2-chloroadenosine correlated with a partial depolarization of myenteric S neurons. The purpose of our investigation was to gain further evidence regarding the cellular mechanism of tolerance. Either morphine or placebo pellets were implanted s.c. in guinea pigs 7 days before the experiment. Subsensitivity was confirmed by a marked decrease of the inhibitory effect of 0.1 microM morphine and 0.3 microM clonidine on neurogenically induced twitches in longitudinal smooth muscle myenteric plexus preparations from the morphine-pretreated guinea pigs. Intracellular microelectrode recording established that only myenteric S neurons that were hyperpolarized by morphine exhibited the depolarized state (difference of 7.2 mV), which occurred without a change in the threshold for initiation of action potentials. S neurons that were hyperpolarized by superfusion with solution containing morphine, 0.1 microM, were acutely hyperpolarized an equivalent amount (6-8 mV) by clonidine, 0.3 microM, or 2-chloroadenosine, 0.1 microM. Morphine and clonidine, but not 2-chloroadenosine, reduced input resistance. The hyperpolarizations and changes in conductance were not different between tolerant and control preparations for any agonist. It is concluded that 1) the receptors for the three agonists are colocalized on selected S neurons, 2) the transduction process for the hyperpolarizing effect of 2-chloroadenosine is different than that for morphine and clonidine, 3) cross-tolerance among the agonists is not a function of altered receptors or signal transduction processes and 4) the depolarized state is associated with tolerance of myenteric S neurons.

Evidence that tolerance and dependence of guinea pig myenteric neurons to opioids is a function of altered electrogenic sodium-potassium pumping.

Ouabain acutely depolarizes most types of cells through inhibition of electrogenic Na+,K+ pumping and is a useful tool with which to study conditions that affect electrogenic pumping. Intracellular recording techniques were used with neurons of the guinea pig myenteric plexus/longitudinal muscle preparation exposed to ouabain. Of 35 S neurons exposed to ouabain (1 microM), 15 were hyperpolarized by 10 +/- 2 mV, 11 were depolarized by 8 +/- 2 mV and the remaining neurons had no change in membrane potential. The nonselective potassium channel antagonist tetraethylammonium chloride (TEA; 0.5 mM) alone evoked modest (<5 mV) and inconsistent changes in the resting membrane potential of S neurons. However, in the presence of TEA, the hyperpolarizing response to 1 microM ouabain was eliminated, and the proportion of cells depolarized by ouabain increased from 31% to 83%. Glibenclamide (10 microM) and 100 nM iberiotoxin did not change the pattern of membrane potential changes induced by 1 microM ouabain. Calcium-free buffer eliminated the hyperpolarization and potentiated the depolarization induced by 1 microM ouabain. Ouabain (5 microM), in either the presence or absence of TEA, induced depolarization in all neurons tested (mean, 15-16 mV), indicating a predominant effect of inhibition of electrogenic pumping. These data suggest that ouabain may directly or indirectly activate myenteric S neuron calcium-sensitive potassium channels as well as inhibit the Na+,K+ pump and that TEA will antagonize the former effect. Chronic exposure (morphine pellets) of guinea pigs to morphine resulted in a partial depolarized state of myenteric neurons, as previously reported. Ouabain (5 microM), either with or without TEA, depolarized neurons from chronically morphine-treated guinea pigs very little (5-6 mV) in comparison with naive neurons (15-16 mV). This supports the conclusion that the depolarized state of morphine-tolerant neurons is associated with a reduction in electrogenic Na+,K+ pumping.

Mechanisms of adaptive supersensitivity: correlation of guinea pig atrial supersensitivity with modifications in adenylyl cyclase activity.

The possibility that the cellular mechanism underlying adaptive supersensitivity in right and left atria of the guinea pig may involve either adenylyl cyclase or components of that transduction process was examined in left and right atria obtained from controls or guinea pigs chronically treated with reserpine. Adenylyl cyclase activity and the abundance of alpha-subunits of several G-proteins (i.e. Gs, Gi, and Go) were quantified using standard techniques. Functional concentrations of Gs and Gi were compared in tissues from control and treated animals using pertussis- or cholera toxin-induced protein ribosylation. Chronic treatment with reserpine did not alter basal levels of adenylyl cyclase activity in left or right atrium but did increase significantly the ability of isoproterenol, 5'-guanylylimido diphosphate, and forskolin to activate adenylyl cyclase in the left atrium compared with the control. In contrast, treatment with reserpine increased the ability of only isoproterenol to active adenylyl cyclase in the right atrium. The increases in enzyme activation were not correlated with any detectable change in the concentrations of G-proteins or beta-adrenoceptors. The correlation between the specificity of changes in responsiveness and increased activation of adenylyl cyclase suggests that the cellular mechanism that underlies the development of adaptive supersensitivity in the guinea pig myocardium may involve a modification of adenylyl cyclase. The data also support the idea that the development of enhanced responsiveness in cardiac muscle may not only involve more than one cellular mechanism but may even differ between right and left atrium and ventricles of the same species.

Chronic morphine treatment of guinea pigs induces nonspecific sensitivity changes in the nucleus tractus solitarius in vitro.

Chronic morphine treatment results in the development of an opioid tolerance in guinea pig myenteric S-neurons that is nonspecific among pharmacologically unrelated inhibitory agonists and the concurrent development of a nonspecific super-sensitivity to unrelated excitatory agonists. The purpose of these studies was to extend this model of opioid tolerance in the guinea pig to central neurons, specifically to the medial nucleus tractus solitarius (mnTS), the primary brainstem relay for visceroceptive information. Pharmacological responses of the guinea pig mnTS neurons were examined in an in vitro brainstem slice preparation and compared between chronically morphine-treated animals and untreated controls. The spontaneous activity of guinea pig mnTS neurons was inhibited by gamma-aminobutyric acid (GABA), the GABAA-selective agonist muscimol, 2-chloroadenosine and clonidine and was excited by glutamate and elevations in extracellular potassium. Applied alone, morphine or the GABAA-selective antagonist bicuculline inhibited and excited approximately equal proportions of nucleus tractus solitarius (nTS) neurons. However, when applied in the presence of bicuculline, morphine inhibited most neurons tested. Reduced inhibitory responses to four agonists, i.e., morphine, muscimol, 2-chloroadenosine and clonidine, were observed in mnTS neurons in slices obtained from chronically morphine-treated animals. Increased excitation of these neurons by elevated extracellular potassium was observed. It is concluded that 1) guinea pig nTS neurons respond similarly to nTS neurons from other species in vitro, 2) opioids disinhibit some proportion of guinea pig nTS neurons in vitro through a GABAergic mechanism and 3) the development of opioid tolerance in guinea pig nTS neurons is qualitatively similar to that of guinea pig myenteric S-neurons.

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.

Adaptive supersensitivity and the Na+/K+ pump in the guinea pig vas deferens: time course of the decline in the alpha 2 subunit.

Adaptive supersensitivity in the guinea pig vas deferens has been shown previously to be associated with decreases in transmembrane potential, Na+/K+-ATPase activity, [3H]ouabain binding sites, and density of the alpha 2 subunit of the pump. One of several procedures that induce adaptive supersensitivity in the guinea pig vas deferens is neurotransmitter depletion by chronic administration of reserpine. Guinea pigs were treated with reserpine (1.0 mg/kg/day, intraperitoneally) for 2, 5, or 8 days. Tissues were homogenized and the concentration of the alpha 2 subunit was quantified by use of the selective antibody McB2, slot blot analysis, enhanced chemiluminescence, and densitometric analysis. As reported previously, the concentration of the alpha 2 protein was reduced 41% after 5 days of pretreatment. The reduction was maintained at 8 days (37%). However, there was no change from control after 2 days of pretreatment with reserpine. Thus, the time course of the decline in the alpha 2 subunit is similar to that of the appearance of supersensitivity, depolarization, and the declines in Na+/K+-ATPase and [3H]ouabain binding established earlier. Based upon results in the literature for several different tissues and species, membrane depolarization and decreases in Na+/K+ pump sites may represent widely occurring adaptive mechanisms.

Sustained hypertension in Dahl rats. Negative correlation of agonist response to blood pressure.

The perfused mesenteric vasculature of Dahl salt-sensitive rats on a high salt diet for 5 days (prehypertensive or early hypertensive) is selectively supersensitive to norepinephrine. The present goal was to determine whether that supersensitivity was maintained as hypertension developed. Littermates of salt-sensitive and salt-resistant rats (Dahl Brookhaven strain) were followed on low or high salt for up to 6 weeks. Systolic blood pressure was elevated in the salt-sensitive, high salt rats after 3 or 6 weeks but not after 5 days of the diet. The perfused mesenteric vascular beds from salt-sensitive rats were supersensitive to norepinephrine and nerve stimulation but not to potassium chloride when the rats had been maintained for 5 days or 3 weeks on the high salt diet. However, responses to norepinephrine declined after 6 weeks of the high salt diet. To determine whether sustained high blood pressure has a negative effect on mesenteric vascular responses, we conducted additional experiments with perfused mesenteric vascular beds from salt-sensitive Brookhaven (high salt, 5 weeks) and Rapp (high salt, 6 weeks) animals. Both groups exhibited significant negative correlations between in vivo systolic pressure and maximal responses of mesenteric vessels to norepinephrine and potassium chloride. We suggest that sustained hypertension in Dahl rats has a negative effect on the contractility of the mesenteric arterial system that, by 5 to 6 weeks, masks the initial supersensitivity to norepinephrine. No effects of any diet on the dilating responses of the mesenteric vessels to acetylcholine were observed in any group.

Functional distribution and role of alpha-1 adrenoceptor subtypes in the mesenteric vasculature of the rat.

Because alpha-1 adrenoceptor subtypes are distributed differentially in different arteries, experiments were conducted to determine the functional contribution of these subtypes in conduit vs. resistance vessels. Concentration- or dose-response curves for norepinephrine were obtained from aortic rings, superior mesenteric artery rings or the isolated perfused mesenteric vasculature from male Sprague-Dawley rats. Frequency-response curves to transmural electrical stimulation were obtained from the latter two preparations. The effects of 5-methylurapidil (5-MU), an alpha-1a adrenoceptor antagonist, and chloroethyl-clonidine (CEC), an alpha-1b adrenoceptor antagonist, on contractile responses were determined. In artery rings, 5-MU (30 nM) had no effect on the EC50 of norepinephrine but reduced the maximum response of the mesenteric artery rings by nearly 25%. In the perfused mesenteric vasculature, however, 5-MU (30 nM) shifted the ED50 for norepinephrine about 40-fold while reducing the maximum response by 30%. 5-MU depressed the frequency-response curve in the perfused mesenteric vasculature by nearly 80%, but did not alter the response of artery rings. In aorta, pretreatment with CEC (10 microM) shifted the concentration-response curve of norepinephrine by 800-fold without effecting the maximum. In mesenteric artery rings and perfused mesenteric vasculature, CEC reduced the slope and maximum response of both frequency-response and norepinephrine dose-response curves. Responses to norepinephrine (10 microM) in the perfused mesentery were abolished by 5-MU and reduced only 25% by nifedipine. These data suggest that the density or role of alpha-1a adrenoceptors may be greater in resistance vessels than in conduit vessels.

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.