Decreased Gs alpha mRNA levels accompany the fall in Gs and adenylyl cyclase activities in compensated left ventricular hypertrophy. In heart failure, only the impairment in adenylyl cyclase activation progresses.

We have previously reported that there is a global reduction in adenylyl cyclase associated with a decrement in Gs functional activity in cardiac sarcolemma from animals with pressure overload-induced hypertrophy and heart failure. This study was performed to determine whether hypertrophy alone in the absence of heart failure is sufficient to promote these changes and whether the superimposition of heart failure intensified these changes. Basal and stimulated adenylyl cyclase and Gs activity, as determined in the S49 cyc- reconstitution assay, were measured in sarcolemma from normal (NL), left ventricular hypertrophy (LVH) and heart failure (HF) animals. Simultaneously, we measured the mRNA level encoding for the Gs alpha subunit. These studies indicate that Gs activity and Gs alpha mRNA are decreased by approximately 30% both in the failing heart and even in the heart with compensated hypertrophy before heart failure develops (Gs activity, pmol cyclic AMP/10 min per microgram, NL 4.2 +/- 0.4, LVH 3.0 +/- 0.2, HF 3.2 +/- 0.3; Gs alpha mRNA, pg/10 micrograms RNA, NL 131 +/- 9.0, LVH 104 +/- 7.4, HF 97.4 +/- 9.1; P less than 0.05 as compared with NL for LVH and HF). Accompanying this decrement in Gs activity is a fall in adenylyl cyclase, both basal and stimulated. However, we also identified a further decrease in adenylyl cyclase without any additional change in Gs or in its alpha subunit mRNA level. This is seen only in the sarcolemma from animals with heart failure as compared with those with compensated LV hypertrophy (e.g., NaF-stimulated activity, pmol cyclic AMP/min per mg, NL 420.2 +/- 17.5, LVH 347.1 +/- 29.6, HF 244.2 +/- 27.3; P less than 0.05 compared with NL for LVH and HF, P less than 0.05 compared with LVH for HF). In summary, these studies indicate that both Gs and adenylyl cyclase activities fall in parallel with the development of LV hypertrophy followed by a further decrement in adenylyl cyclase, independent of Gs, in the setting of heart failure.     

Functional selectivity of dopamine receptor agonists. I. Selective activation of postsynaptic dopamine D2 receptors linked to adenylate cyclase

Dihydrexidine (DHX), the first high-affinity D(1) dopamine receptor full agonist, is only 10-fold selective for D(1) versus D(2) receptors, having D(2) affinity similar to the prototypical agonist quinpirole. The D(2) functional properties of DHX and its more D(2) selective analog N-n-propyl-dihydrexidine (PrDHX) were explored in rat brain and pituitary. DHX and PrDHX had binding characteristics to D(2) receptors in rat striatum typical of D(2) agonists, binding to both high- and low-affinity sites and being sensitive to guanine-nucleotides. Consistent with these binding data, both DHX and PrDHX inhibited forskolin-stimulated cAMP synthesis in striatum with a potency and intrinsic activity equivalent to that of quinpirole. Unexpectedly, however, DHX and PrDHX had little functional effect at D(2) receptors expressed on dopaminergic neurons that mediate inhibition of cell firing, dopamine release, or dopamine synthesis. Quantitative receptor competition autoradiography demonstrated that DHX bound to D(2) receptors in striatum (predominantly postsynaptic receptor sites) with equal affinity as D(2) sites in the substantia nigra (autoreceptor sites). The data from these experiments, coupled with what is known about the location of specific dopamine receptor isoforms, lead to the hypothesis that DHX, after binding to D(2L) and D(2S) receptors, causes agonist-typical functional changes only at some of these receptors. This phenomenon (herein termed "functional selectivity") suggests that drugs may be targeted not only at specific receptor isoforms but also at separate functions mediated by a single isoform, yielding novel approaches to drug discovery.     

Neural activation of alpha-2 adrenoceptors in cat cutaneous vasculature.

The present study was designed to assess the relative contribution of postjunctional alpha-1 and alpha-2 adrenoceptors in neurally evoked cutaneous vasoconstrictor responses in anesthetized cats. Preganglionic stimulation of the thoracic sympathetic nerve trunk produced an intensity-related decrease of digital skin blood flow as measured by laser-Doppler flowmetry. Sympathetic-cholinergic sudomotor (electrodermal) responses were recorded simultaneously as an additional index of neuronal activation. Vasoconstrictor responses were not altered by pretreatment with i.v. propranolol (1 mg/kg) or atropine (1 mg/kg) and were refractory to low doses (2 mg/kg) of hexamethonium. As expected, atropine abolished evoked skin potential responses which were also sensitive only to higher doses (20 mg/kg) of hexamethonium. Pretreatment with either phentolamine (1 mg/kg i.v.), yohimbine (0.5 mg/kg i.v.) or prazosin (0.3 mg/kg i.v.) produced significant reduction of sympathetic vasoconstriction with blockade by phentolamine and yohimbine being far greater than that seen with prazosin. In animals previously given prazosin (0.3 mg/kg), subsequent administration of yohimbine (0.5 mg/kg) almost totally blocked the remaining evoked cutaneous vasoconstriction. These results demonstrate that both alpha-1 and alpha-2 adrenoceptors are present in the cutaneous bed of the cat with the predominant effect mediated by innervated alpha-2 adrenoceptors.     

D-2 dopamine receptors in aging mouse striatum: determination of high- and low-affinity agonist binding sites

Striatal D-2 dopamine receptors in homogenates from aged male C57BL/6J mice were examined for high and low-affinity agonist binding. High-affinity dopamine binding requires the ternary complex of the D-2 receptor and a guanine nucleotide binding regulatory protein (N). Thus, changes in the interaction of D-2 and N could contribute to previously reported changes in agonist binding in aged rodents and humans. Qualitative experiments indicated no age-change in the ability of guanine nucleotides to reduce the apparent potency of dopamine at D-2 receptors. Also, no age differences were observed in the ability of guanine nucleotides to decrease N-[3H]propylnorapomorphine binding, suggesting that the ability of guanine nucleotides to dissociate D-2 and N was similar with age. Quantitative determination of the high- (RH) and low-affinity (RL) agonist binding components of striatal D-2 dopamine receptors in the absence of guanine nucleotides indicated differences in the density of RH, and the percentage of D-2 receptors measured as RH, between the ages of 3 and 12 months. No changes in RH or percentage of RH occurred after midlife. In contrast, the total D-2 receptor population, [3H]spiperone maximum binding, declined progressively from 3 to 24 months. Age-changes were restricted to D-2 receptor density; the dissociation constants for agonist and antagonist binding were similar across age. The data suggest that age-changes in striatal D-2 dopamine receptors can occur in the density of the D-2 receptor and in the mechanism that confers the property of high-affinity agonist binding upon the D-2 receptor.     

Strain differences in basal and cocaine-evoked dopamine dynamics in mouse striatum

In vivo microdialysis was used to characterize basal dopamine (DA) dynamics and cocaine-evoked DA levels in the striatum of 129/Sv-ter, C57BL/6J, DBA/2J, and Swiss-Webster mice. Basal dialysate levels of DA did not differ in the four strains tested. Similarly, the no net flux method of quantitative microdialysis revealed no difference in extracellular levels between strains. However, the in vivo extraction fraction of DA was significantly less in 129/Sv-ter (53%) mice compared with C57BL/6J (68%), DBA/2J (69%), and Swiss-Webster (67%) mice, indicating a lower rate of basal DA uptake in the 129/Sv-ter strain. Perfusion of K(+) (60 and 100 mM) through the microdialysis probe significantly increased dialysate DA levels and there was no difference between strains in the magnitude of this effect. The acute administration of cocaine (5-20 mg/kg i.p.) increased DA levels in the four strains tested. Cocaine-evoked DA levels (in nanomoles) were significantly greater in 129/Sv-ter compared with C57BL/6J, DBA/2J, or Swiss-Webster mice after administration of either 5, 10, or 20 mg/kg cocaine. However, the percentage increase in DA did not differ across strains. These data demonstrate that there are strain-related differences in basal DA dynamics in the striatum of the mouse. Basal DA uptake was reduced in striatum of 129/Sv-ter mice compared with C57BL/6J, DBA/2J, or Swiss-Webster mice. In addition, the response of DA levels to cocaine may be enhanced in 129/Sv-ter compared with C57BL/6J, DBA/2J, or Swiss-Webster mice.     

Inhibition of protein phosphorylation by opioid-inhibited adenylyl cyclase in rat brain membranes.

Opioid inhibition of adenylyl cyclase is a major second messenger system associated with opioid receptors in brain. To identify membrane phosphoproteins whose phosphorylation state is modulated by opioid inhibition of adenylyl cyclase, rat striatal membranes were preincubated with opioid agonists in the presence of 500 microM 5'-adenylyl-imidodiphosphate (which acted as a substrate for adenylyl cyclase, but not for protein kinase) before addition of [gamma-32P]ATP. Under these conditions, adenylyl cyclase in the membranes formed cyclic AMP, which stimulated cyclic AMP-dependent protein kinase. This process was confirmed by observing forskolin-stimulated phosphorylation of two bands of MW 85 and 63 kDa, which were also stimulated directly by cyclic AMP. Forskolin-stimulated phosphorylation of these two bands was inhibited by 15 to 30% by opioid agonists such as D-Ala2-Met5-enkephalinamide. This inhibition of phosphorylation was mediated by opioid receptors, because it required both sodium and GTP, and was blocked by naloxone. These results suggest that these two proteins may be primary targets of opioid-inhibited adenylyl cyclase in striatal membranes.     

Repeated amphetamine administration alters the interaction between D1-stimulated adenylyl cyclase activity and calmodulin in rat striatum

The effect of repeated intermittent treatment with amphetamine on the responses of rat striatal adenylyl cyclase to the D1 dopamine receptor agonist, SKF38393 [(+-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride], and potentiative interactions with the endogenous Ca(++)-binding protein, calmodulin, were investigated. Female Sprague-Dawley rats were treated with saline or an escalating dose regimen of amphetamine for 4 weeks and withdrawn from treatment for either 2 or 4 weeks. Thirty minutes before sacrifice, rats in both groups were given a challenge dose of either saline or 1.0 mg/kg of amphetamine. In striatal membranes from rats chronically treated with saline and withdrawn 4 weeks, calmodulin increased SKF38393-stimulated adenylyl cyclase activity by 195% over that seen in the presence of GTP alone. In contrast, this pronounced potentiative interaction was absent in rats chronically treated with amphetamine. The lack of potentiation by calmodulin was independent of length of withdrawal from the drug or amphetamine challenge. Although the repeated amphetamine treatment abolished the potentiative response to calmodulin, this treatment significantly increased the calmodulin content in the striatal cytosol by 40%. Heightened responsiveness of several dopamine-related neurochemical activities was evident after an amphetamine challenge to rats that had been treated repeatedly with amphetamine. A challenge dose of 1 mg/kg of amphetamine decreased SKF38393-stimulated adenylyl cyclase activity in amphetamine-treated but not saline-treated rats. The degree of desensitization induced by amphetamine challenge was heightened with increased length of withdrawal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of monoamine oxidase-B by (-)-deprenyl potentiates neuronal responses to dopamine agonists but does not inhibit dopamine catabolism in the rat striatum.

This report describes experiments designed to determine whether (-)-deprenyl potentiates dopaminergic transmission and whether its mechanism involves the inhibition of dopamine catabolism. Intraperitoneal administration of (-)-deprenyl (0.5-8 mg kg-1) produced a dose-dependent inhibition of striatal monoamine oxidase type B activity whereas monamine oxidase type A activity in the striatum was inhibited only by 8 mg kg-1 of (-)-deprenyl. Intraperitoneal administration of (-)-deprenyl (0.5-4 mg kg-1) did not alter the striatal concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) or homovanillic acid. DOPAC concentrations were decreased by 8 mg kg-1 of (-)-deprenyl. In contrast, administration of clorgyline (2 mg kg-1), a monoamine oxidase type A inhibitor, increased the striatal concentrations of DA and decreased the striatal concentrations of DOPAC and homovanillic acid. The striatal concentrations of 2-phenylethylamine (PE), a putative modulator of striatal DA transmission, were increased by (-)-deprenyl (1-8 mg kg-1) but were unaffected by clorgyline (2 mg kg-1). In electrophysiological studies, single caudate neuron responses to iontophoretically applied (-)-apomorphine and (+/-)-2-(N-phenethyl-N-propyl) amino-5-hydroxytetralin were potentiated by intracarotid injections of PE (30 micrograms kg-1) and i.p. injections of (-)-deprenyl (2 mg kg-1). Both PE and (-)-deprenyl reduced the IT50 of responses to apomorphine and (+/-)-2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptors for 5-hydroxytryptamine in rabbit blood vessels: activation of alpha adrenoceptors in rabbit thoracic aorta

Experiments were carried out to identify the receptors mediating the contraction of the rabbit thoracic aorta to 5-hydroxytryptamine (5-HT). Isolated aortic rings were mounted in tissue baths for the measurement of isometric contraction and 5-HT dose-response curves were obtained in the presence and absence of receptor antagonists. Prazosin, 1 X 10(-7) M, or 30-min pretreatment with 1 X 10(-5) M benextramine had no effect on the contractile response of aorta to 5-HT up to 1 X 10(-5) M, whereas 2 brom-D-lysergic acid diethylamide, 1 X 10(-7) M, shifted the 5-HT dose-response curve far to the right. Alpha receptor blockade with either prazosin or benextramine in the presence of 2 brom-D-lysergic acid diethylamide produced a greater blockade than that caused by 2 brom-D-lysergic acid diethylamide alone. When 5-HT dose-response curves were extended to 1 X 10(-3) M, three phases were identified. The first, a dose-related contraction, was mediated exclusively by serotonergic receptors. The second, a relaxation to approximately 40% of maximum, occurred at 1 X 10(-5) M and appeared to result from the rapid development of tachyphylaxis. The third phase was a dose-related contraction to concentrations of 5-HT above 1 X 10(-5) M and was inhibited by either prazosin or pretreatment with benextramine. Similar results were obtained in aortic rings from reserpine-pretreated rabbits. It is concluded that the contractile response to concentrations of 5-HT below 1 X 10(-5) M is mediated exclusively by serotonergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of alpha adrenoceptors in opossum internal anal sphincter.

The purpose of the present investigation was to examine the role of alpha adrenoceptors in the internal anal sphincter (IAS). Studies wer performed on alpha-chloralose anesthetized opossums. Resting pressure in the IAS (IASP) was recorded using low compliant continuously perfused catheters. The effects of the alpha-1 adrenoceptor agonist phenylephrine and alpha-2 adrenoceptor agonist clonidine and their corresponding selective antagonists, prazosin and yohimbine, respectively, were examined on the resting IASP, and on rectal balloon distension (RBD)-mediated IAS relaxation. Phenylephrine caused a rise in the IASP that was blocked by prazosin and not by yohimbine. Phenylephrine had no effect on IAS relaxation caused by RBD. Clonidine on the other hand caused significant suppression of IAS relaxation in response to RBD, but caused minimal changes in the resting IASP. The suppression of IAS relaxation by clonidine was selectively antagonized by yohimbine but not by prazosin. From these studies we conclude that alpha-2 adrenoceptors exert important neuromodulatory influences on rectoanal inhibitory reflex, while alpha-1 adrenoceptors may exert modulatory effects on the resting IAS tone.     

In vivo dopamine clearance rate in rat striatum: regulation by extracellular dopamine concentration and dopamine transporter inhibitors

Dopamine transporter (DAT) inhibitors are expected to decrease dopamine (DA) clearance from the extracellular space of the brain. However, mazindol and cocaine have been reported to "anomalously" increase DA clearance rate. To better understand in vivo DAT activity both in the absence and presence of DAT inhibitors, clearance of exogenously applied DA was measured in dorsal striata of urethane-anesthetized rats using high-speed chronoamperometry. As higher amounts of DA were ejected, DA signal amplitudes, but not time courses, increased. Clearance rates increased until near maximal rates of 0.3 to 0.5 microM/s were attained. Provided baseline clearance rates were relatively low (< 0.1 microM/s), local application of either nomifensine or cocaine markedly increased exogenous DA signal amplitudes and time courses. Relative to the low baseline group, locally applied nomifensine decreased clearance rate when baseline clearance was high ( approximately 0.4 microM/s). However, even when baseline clearance rates were high, systemic injection of nomifensine, mazindol, GBR 12909, or benztropine increased DA signal amplitudes to a greater extent than time courses, consistent with the observed increases in clearance rates. In contrast, despite low baseline clearance rates, systemic injection of cocaine, WIN 35,428, or d-amphetamine preferentially increased DA signal time course, consistent with the observed decreases in clearance rates. Our results emphasize that as extracellular DA concentrations increase, DAT velocity increases to a maximum, partially explaining the ability of DAT inhibitors to increase DA clearance rates. However, by itself, kinetic activation is not sufficient to explain the ability of certain systemically administered DAT inhibitors to anomalously increase DA clearance.     

Differential coupling of somatostatin1 receptors to adenylyl cyclase in the rat striatum vs. the pituitary and other regions of the rat brain.

Subtypes of somatostatin (SRIF) receptors are expressed in the rat brain and may mediate the diverse actions of SRIF. In the present study we show that subtypes of SRIF receptors in different regions of the rat brain are differentially sensitive to the cyclic hexapeptide SRIF analog, MK 678. SRIF1 receptors are sensitive to MK 678 and found in high density in the cortex, hippocampus and striatum, as well as in the anterior pituitary. The pituitary appears to express only the SRIF1 receptor. The cortex, hippocampus and striatum also express SRIF2, or MK 678-insensitive, receptors. The proportion of SRIF1 receptors varies in different brain regions. In the cortex and hippocampus, SRIF1 receptors comprise approximately 50% of the total SRIF receptor population, whereas in the striatum SRIF1 receptors comprise the majority (86%) of SRIF receptors. SRIF1 receptors in the pituitary, cortex and hippocampus mediate, at least in part, the ability of SRIF to inhibit forskolin-stimulated adenylyl cyclase activity as MK 678 produced significant inhibition of activity in these tissues. However, in the striatum, MK 678 had no significant effect on forskolin-stimulated adenylyl cyclase activity, despite a significant inhibition produced by SRIF. The specific labeling of these receptors in the striatum by [125I]MK 678 is abolished in the presence of high concentrations of the nonhydrolyzable GTP analog, GTP gamma S, suggesting that SRIF1 receptors in this brain region are coupled to G proteins. The SRIF1 receptors in the striatum may be coupled via G proteins to cellular transducing systems other than adenylyl cyclase.     

Regulation of hormone-sensitive calcium influx by the adenylyl cyclase system in renal epithelial cells.

To study signaling pathways regulated by alpha s and alpha i1 in renal epithelial cells, we expressed mutant, activated forms of alpha s and alpha i1 in a continuous proximal tubule cell line (MCT cells). alpha sQ227L increased cAMP production, and alpha ilQ204L reduced forskolin-sensitive cAMP production. alpha ilQ204L increased and alpha sQ227L decreased bradykinin-induced Ca influx across the cell membrane, but neither mutant affected bradykinin-stimulated intracellular Ca release or basal Ca influx. Bradykinin-stimulated Ca influx was reduced by dibutyryl cAMP, isoproterenol, and forskolin. Expression of a mutant regulatory type I subunit for cAMP-dependent protein kinase with reduced affinity for cAMP and treatment with KT-5720, a specific cAMP-dependent protein kinase inhibitor, enhanced Ca influx to a degree similar to that in cells expressing alpha ilQ204L. Bradykinin-stimulated c-fos mRNA expression is partially dependent on extracellular Ca. alpha sQ227L reduced and alpha ilQ204L enhanced bradykinin-stimulated c-fos expression. Consequently, in bradykinin-stimulated cells, the adenylyl cyclase system regulates Ca influx through cAMP-dependent protein kinase, but not intracellular Ca release. Furthermore, the Ca influx mechanism acts as an integrator of two signaling pathways such that Ca-dependent signals are damped by activators of adenylyl cyclase and enhanced by inhibitors of adenylyl cyclase.     

Amphotericin B decreases adenylyl cyclase activity and aquaporin-2 expression in rat kidney

The present study was intended to examine whether the amphotericin-induced urinary concentration defect can be related to an altered regulation of aquaporin (AQP) water channels in the kidney. Male Sprague-Dawley rats were injected with amphotericin B (6 mg/kg/d, IP ) for 21 days. The protein expression of AQP1-3, Gsalpha, and adenylyl cyclase was determined in the kidney. To further specify the primary point of dysregulation of AQP channels that are activated by the arginine vasopressin/cyclic adenosine monophosphate (AVP/cAMP) pathway, different components of adenylyl cyclase complex were separately examined for their cAMP-generating activities. Amphotericin treatment resulted in kidney failure associated with decreased tubular water reabsorption and increased urinary flow rate. The expression of AQP2 proteins was significantly decreased in the outer medulla and inner medulla but not in the cortex. The expression of AQP2 proteins in the membrane fraction changed in parallel with that in the cytoplasmic fraction, suggesting a preserved targeting. Neither the expression of AQP1 nor that of AQP3 was significantly affected in the cortex, outer medulla, or inner medulla. The cAMP generation in response to AVP or sodium fluoride was decreased, whereas that to forskolin was not significantly altered. The expression of Gsalpha proteins was decreased in the inner medulla, whereas that of adenylyl cyclase VI remained unaltered. These findings indicate that the amphotericin-induced urinary concentration defect may in part be causally related to a reduced abundance of AQP2 channels in the kidney. It is also suggested that the primary impairment in the pathway leading to the activation of AQP channels that are regulated by the AVP/cAMP pathway lies at the level of G proteins.     

Altered adenylyl cyclase activities and G-protein abnormalities in portal hypertensive rabbits.

Portal hypertension (PHT) is characterized by splanchnic hyperemia due to a reduction in mesenteric vascular resistance. We hypothesized that alterations in the activity of a guanine-nucleotide regulatory protein (G-protein) might be partially responsible for the marked circulatory disturbances observed in PHT. We, therefore, determined alterations in adenylyl cyclase/cAMP system in prehepatic portal hypertensive rabbits and correlated these changes to the activity of a G-protein. Basal and G-protein-stimulated adenylyl cyclase activities were lower in the PHT superior mesenteric artery (22-26%) and thoracic aorta (31-46%) membranes, but higher (178-321%) in portal vein. The functional activity of Gi alpha proteins (pertussis toxin-catalyzed ADP-dependent ribosylation) increased in the PHT superior mesenteric artery and thoracic aorta, but decreased in portal vein. Immunodetection revealed an increase in the Gi alpha protein subunits (Gi alpha 1/Gi alpha 2 and Gi alpha 3/Go alpha) in PHT thoracic aorta, without any change in Gs alpha proteins; and a decrease in the amount of Gi alpha proteins in PHT portal vein. There was no change in the amount of Gs alpha/Gi alpha in the PHT superior mesenteric artery. We conclude the hemodynamic alterations of PHT are associated with intrinsic alterations in G-protein-enzyme effector systems. These alterations are vessels specific and suggest a possible unique global derangement underlying the vasculopathy of PHT.     

Methylphenidate-induced increases in vesicular dopamine sequestration and dopamine release in the striatum: the role of muscarinic and dopamine D2 receptors

Methylphenidate (MPD) administration alters the subcellular distribution of vesicular monoamine transporter-2 (VMAT-2)-containing vesicles in rat striatum. This report reveals previously undescribed pharmacological features of MPD by elucidating its receptor-mediated effects on VMAT-2-containing vesicles that cofractionate with synaptosomal membranes after osmotic lysis (referred to herein as membrane-associated vesicles) and on striatal dopamine (DA) release. MPD administration increased DA transport into, and decreased the VMAT-2 immunoreactivity of, the membrane-associated vesicle subcellular fraction. These effects were mimicked by the D2 receptor agonist quinpirole and blocked by the D2 receptor antagonist eticlopride. Both MPD and quinpirole increased vesicular DA content. However, MPD increased, whereas quinpirole decreased, K(+)-stimulated DA release from striatal suspensions. Like MPD, the muscarinic receptor agonist, oxotremorine, increased K(+)-stimulated DA release. Both eticlopride and the muscarinic receptor antagonist scopolamine blocked MPD-induced increases in K(+)-stimulated DA release, whereas the N-methyl-d-aspartate receptor antagonist (-)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) was without effect. This suggests that D2 receptors mediate both the MPD-induced redistribution of vesicles away from synaptosomal membranes and the MPD-induced up-regulation of vesicles remaining at the membrane. This results in a redistribution of DA within the striatum from the cytoplasm into vesicles, leading to increased DA release. However, D2 receptor activation alone is not sufficient to mediate the MPD-induced increases in striatal DA release because muscarinic receptor activation is also required. These novel findings provide insight into the mechanism of action of MPD, regulation of DA sequestration/release, and treatment of disorders affecting DA disposition, including attention-deficit hyperactivity disorder, substance abuse, and Parkinson's disease.     

Vanadate stimulation of adenylyl cyclase: an index of tyrosine kinase vascular effects.

BACKGROUND: Beyond their mitogenic effects, hormones such as insulin, which activate receptor tyrosine kinases, regulate vascular tone. Further, we have demonstrated that receptor tyrosine kinase activation enhances adenylyl cyclase activation, a prominent mechanism that mediates vasodilation. However, whether tyrosine kinase-mediated human vascular responses parallel tyrosine kinase-mediated cellular effects on adenylyl cyclase activity is unknown. METHODS AND RESULTS: To assess tyrosine kinase-mediated vascular responses, vascular sensitivity to insulin was assessed with the dorsal hand vein linear variable differential transformer technique. Insulin infusion resulted in a dose-dependent relaxation in all subjects. Cellular responses were assessed by means of the insulinomimetic vanadate-mediated sensitization of vascular adenylyl cyclase activity. Vanadate stimulated a tyrosine kinase-dependent enhancement of adenylyl cyclase function in human and rat aortic vascular smooth muscle cells, human lymphocytes, and human aortic endothelial cells. Further, maximal insulin-mediated vasodilation was significantly positively correlated with maximal vanadate-mediated enhancement of human lymphocyte adenylyl cyclase activity. CONCLUSION: Insulin-mediated vasodilation is positively correlated with vanadate-mediated enhancement of adenylyl cyclase activity. Vanadate-mediated enhancement of adenylyl cyclase activity in lymphocytes may represent an index of tyrosine kinase-mediated vascular effects.     

Functional effects of activation of alpha-1 adrenoceptors by dexmedetomidine: in vivo and in vitro studies.

Dexmedetomidine, an alpha-2 adrenoceptor (AR) agonist, produces a biphasic hypnotic response in rats. Since central alpha-1 AR stimulation may reverse the hypnotic response produced by central alpha-2 AR stimulation, we have investigated, in both in vivo and in vitro models, the functional effects of dexmedetomidine on alpha-1 AR. For in vivo studies, stainless steel cannulas were inserted stereotaxically into the lateral ventricle of halothane-anesthetized rats to facilitate i.c.v. drug administration. Four to 7 days later, the alpha-1 AR antagonist prazosin (1 mg/kg-1) or saline was administered i.p. 15 min before i.c.v. injections of dexmedetomidine (10-333 micrograms) and the sleep-time (duration of loss of righting reflex) was assessed. The sleep-time increased, in a linear fashion, up to 33 micrograms; above this dose, there was a decrease in sleep-time. Pretreatment with prazosin prevented the decrease in sleep-time which was seen at higher doses. For in vitro studies, binding parameters of dexmedetomidine and its anesthetically inert L-isomer were determined from competition binding curves using [125I]2-[beta-(4-hydroxy-3-[125I]iodo- phenyl)ethylaminomethyl]-tetralone as the radiolabeled ligand and membranes prepared from HeLa cell lines stably expressing either alpha-1B or alpha-1C AR subtypes. Dexmedetomidine bound with equal affinity to both the alpha-1B (1178 +/- 63 nM) and the alpha-1C (1344 +/- 230 nM) isoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Supraspinal and spinal delta(2) opioid receptor-mediated antinociceptive synergy is mediated by spinal alpha(2) adrenoceptors.

Concurrent administration of low doses of [D-Ala(2), Glu(4)]deltorphin (DELT) in the spinal cord and rostral ventromedial medulla of the rat produces a synergistic antinociception in the tail-flick test. It was postulated that the synergistic antinociception results from an interaction of the intrathecally-administered DELT with norepinephrine released in the spinal cord as a result of the microinjection of DELT in the rostral ventromedial medulla. Three approaches were taken to test this hypothesis. The first experiment determined that microinjection of DELT in the rostral ventromedial medulla produced an increase in tail-flick latency that was partially attenuated by intrathecal administration of the alpha(2)-adrenoceptor antagonist yohimbine. These data indicated that microinjection of DELT in the medulla causes a release of norepinephrine in the spinal cord. The second experiment determined that intrathecal co-administration of DELT with the alpha(2)-adrenoceptor agonist dexmedetomidine in a 2:1 fixed dose ratio produced a synergistic antinociception in the tail-flick test. The final experiment determined that the antinociception produced by concurrent medullary and intrathecal administration of DELT was completely antagonized by intrathecal administration of yohimbine. Taken together, these findings support the hypothesis that the synergistic antinociception produced by concurrent activation of medullary and spinal delta(2) opioid receptors is mediated, in part, by endogenous norepinephrine release in the spinal cord. The norepinephrine, acting at alpha(2)-adrenoceptors, interacts in a synergistic manner with intrathecally administered DELT, acting at spinal delta(2) opioid receptors, to produce antinociception.