Article

A study of α1-adrenoceptors in rat renal cortex: Comparison of [3H]-prazosin binding with the α1-adrenoceptor modulating gluconeogenesis under physiological conditions

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Abstract

1 A comparison has been made of the alpha 1-adrenoceptor controlling gluconeogenesis in tubules from rat renal cortex and [3H]-prazosin binding in membranes prepared from the same tissue under physiological conditions. 2 In renal tubules the alpha-adrenoceptor agonists, oxymetazoline, (--)-noradrenaline, (--)-alpha-methylnoradrenaline and (--)-phenylephrine, stimulated gluconeogenesis from pyruvate. Oxymetazoline was the most potent agonist (EC50 15.7 nM) but produced only 61% of the maximum response elicited by (--)-noradrenaline. 3 The alpha-adrenoceptor antagonists, BE2254, prazosin, indoramin and phentolamine inhibited (--)-noradrenaline-mediated increases in gluconeogenesis. The alpha 1-adrenoceptor selective compounds, BE2254 and prazosin, were the most effective antagonists with KB values of 0.74 and 1.47 nM respectively. 4 [3H]-prazosin binding to membranes prepared from rat renal cortex in physiological saline at 37 degrees C was best described by a two site model. High affinity, but not low affinity sites had characteristics consistent with alpha-adrenoceptors. 5 High affinity [3H]-prazosin binding could be completely displaced by the alpha-adrenoceptor agonists, oxymetazoline, (--)-noradrenaline, (--)-phenylephrine, and (--)-alpha-methylnoradrenaline. Slope factors for the displacement curves were all significantly less than unity. The concentrations of agonists required to displace [3H]-prazosin binding were markedly higher than those required to stimulate gluconeogenesis. 6 High-affinity [3H]-prazosin binding was also displaced by the alpha-adrenoceptor antagonists, prazosin, BE2254, phentolamine and indoramin. Slope factors for the displacement curves were close to unity. Ki values calculated from the binding experiments were very similar to KB values obtained in the gluconeogenesis studies. These results suggest that in rat renal cortex the alpha 1-adrenoceptor labelled by [3H]-prazosin is probably that which stimulates gluconeogenesis.

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... Activation of receptors is followed by the rapid hydrolysis of PIP2 by phospholipase C, and the production of at least two putative second messengers, DAG and IP3 which induce an increase in cellular activity (Berridge, 1984; Nishizuka, 1984). A characteristic feature of this mechanism is the multifunctional nature of the tion of these responses is seen in kidney, where activation of a,ondary effects adrenoceptors leads to increased sodium reabsorption for multiple (Osborn et al., 1983; Hesse & Johns, 1984), prostanoid production (Cooper & Malik, 1985), gluconeogenesis (Kessar & Saggerson, 1980; McPherson & Summers, 1982), renal vasoconstriction (Schmitz et al., 1981; Smyth et al., 1984; Cooper & Malik, 1985) and inhibition of renin release (Matsumura et al., 1985). In the light of the large quantity of physiological data, it is of great interest that the present study demonstrated that renal a,-adrenoceptors are linked to PI metabolism and that the products of the PI cycle can now be investigated as possible mediators of the diverse array of physiological processes occurring in response to a,adrenoceptor activation in the kidney. ...
... The indomethacin-sensitive component of the PI response may be only one component of the Ca2"-stimulated increased control levels of [3HJ- IP's. Gluconeogenesis is a calcium-dependent process stimulated by activation of a,-adrenoceptors in rat kidney (Kessar & Saggerson, 1980; McPherson & Summers, 1982). Glucose is able to cause a PI effect in liver (Fex & Lernmark, 1972) and so may also warrant consideration as a candidate for the role of an endogenous stimulant of PI turnover. ...
... The response to NA was much larger in the cortex than in medulla supporting the findings of the binding andautoradiographic studies. In competition studies, pA2 values for all drugs were in close agreement with those obtained for inhibiting gluconeogenesis in rat kidney (McPherson & Summers, 1982 ) and for inhibiting NAinduced increases in [3H]-inositol metabolism in rat cerebral cortex (Brown et al., 1984; Minneman & Johnson, 1984). The concentration-response curves for the agonist-stimulated PI hydrolysis lie at least one order of magnitude to the right compared to those obtained for gluconeogenesis (McPherson & Summers, 1982) (Table 2). ...
Article
The molecular events which follow activation of alpha 1-adrenoceptors in rat kidney were investigated by measuring inositol phospholipid hydrolysis. Slices were labelled with [3H]-inositol (0.25 microM) and the accumulation of [3H]-inositol phosphates ([3H]-IP's) was measured after stimulation with alpha-adrenoceptor agonists. Phospholipid labelling was both time- and Ca2+-dependent. In kidney, Ca2+ (1 mM) increased the incorporation of [3H]-inositol by 49% and in cerebral cortex reduced it by 46%. Following addition of noradrenaline (NA, 1 mM), accumulation of [3H]-IP's increased linearly for at least 60 min. In Ca2+-free buffers a 2.1 fold increase in [3H]-IP accumulation was observed and further increases in stimulated and control levels were produced in the presence of Ca2+ (2.5 mM). These responses were attenuated by the inclusion of indomethacin (10 microM) and abolished in the presence of EGTA (0.5 mM). Responses to (-)-NA were more than 4 fold higher in the renal cortex than in the medulla. Separation of the IP's which accumulate after alpha-adrenoceptor agonists showed that after 60 min stimulation the major products were glycerophosphoinositol and inositol-phosphate with smaller amounts of inositol-bisphosphate and inositol-trisphosphate. The most effective agonists tested for stimulation of accumulation of [3H]-IP's were (-)-NA greater than phenylephrine greater than methoxamine, (+)-NA. Clonidine and (-)-isoprenaline were ineffective at concentrations up to 100 microM. The order of effectiveness of alpha-adrenoceptor antagonists was prazosin greater than BE2254 greater than phentolamine greater than idazoxan greater than rauwolscine. The results indicate that alpha 1-adrenoceptors in rat kidney are linked to phosphoinositide hydrolysis and that this response is localized mainly to the renal cortex.
... The possibility of a direct effect of the sympathetic nervous system on tubular fluid transport was a controversial issue, until electron microscopic and histochemical studies identified adrenergic nerve terminals surrounding proximal and distal tubules (Muller and Barajas, 1972). More recently, al-and az-adrenergic receptors have been identified at the basolateral membrane of renal tubular cells and have been characterized by means of radioligand binding studies (McPherson and Summers, 1982;Schmitz et al., 1981;Snavely and Insel, 1982). On the basis of this information, studies were performed to correlate al-adrenergic stimulation with changes of fluid transport (Bello-Reuss et al., 1975DiBona, 1977;Gottschalk, 1979;Prosnitz and DiBona, 1978;Osswald and Greven, 1981;Osborne et al., 1983;Tomlinson and Wood, 1976;Wood and Tomlinson, 1974). ...
... In renal tubular cells, the mechanism of action is unknown. PE ultimately produces an increase in fluid transport and gluconeogenesis (Exton, 1985;Williamson et al., 1985;McPherson and Summers, 1982). ...
Article
This study was designed to examine the role of changes in cytoplasmic free calcium concentration ([Ca2+]i) during the response to α1-adrenergic agonists in cultured renal proximal tubular cells. Experiments were carried out on primary cultures of canine proximal tubular cells grown in defined culture medium on a solid support, on collagen-coated polycarbonate membranes, or on collagen-coated glass coverslips. Quin-2 and fura-2 were used to monitor [Ca2+]i. The basal level of [Ca2+]i was 101 nM, as measured with quin-2, and 122 nM, as determined using fura-2. Fluorescence flow cytometry revealed that about 85% of the population of proximal tubular cells responded to phenylephrine with an increase in [Ca2+]i. Phenylephrine (10−5 M) caused an immediate actual increase in [Ca2+]i by 18 and 24%, as determined with quin-2 and fura-2, respectively, with the peak increase in [Ca2+]i averaging 22% and 44% over the basal level (180–300 sec). This effect did not require extracellular calcium. The effect of phenylephrine was abolished by prazosin and verapamil. Fluorescence microscopy of quin-2 or fura-2 loaded cells revealed punctate areas of fluorescence within the cytoplasm suggesting vesicular uptake of the dyes. Pinocytotic entrapment of the dyes was demonstrated by the transfer of cell-impermeant fura-2 across tubular cell monolayers mounted in Ussing chambers. The transfer of the dye was similar to that of a marker of fluid-phase pinocytosis, Lucifer Yellow (LY). This pinocytotic entrapment of Ca2+-indicators would lead to underestimation of the actual calcium transients. Microfluorometric study of single proximal tubular cells “scrape-loaded” with fura-2 revealed a four-fold increase in [Ca2+]i concentration following stimulation with phenylephrine.
... Early studies reported that the oxymetazoline was able to displace the binding of prazosin ( 3 H) in membranes of renal tubules [76], liver, submaxillary gland [77] and brain [78]. In addition, it has been reported that alpha-1 adrenergic receptors antagonists such as prazosin were able to reverse the antinociceptive effects induced by alpha-1 adrenergic receptors agonists such as oxymetazoline [79]. ...
Article
Cocaine abuse and dependence are a global public health problem. To date, no effective therapy has been established to treat cocaine dependence but mirtazapine-as well as prazosin used in preclinical and clinical trials-has been shown to decrease cocaine behavioral effects. Therefore, our hypothesis was that the effectiveness of mirtazapine might improve when used in combination with prazosin. This study investigated the combined effect of mirtazapine and prazosin on cocaine-induced locomotor activity impairment in rats subjected to locomotor sensitization testing. We found that chronic treatment with the mirtazapine-prazosin combination significantly improved the effect of single mirtazapine dosing on cocaine-induced locomotor activity and on the induction and expression of cocaine sensitization. These results suggest that the combined use of mirtazapine and prazosin may be a potentially effective treatment to attenuate induction and expression of locomotor sensitization to cocaine.
... Renal al-receptors may be localized on both vascular and tubular elements, mediating vasoconstriction (Schmitz et al., 1981) or gluconeogenesis (McPherson and Summers, 1982), presumably via a Ca2+-dependent mechanism involving phosphatidyl inositol metabolism (Tyroler et al., 1986). The present study describes multiple affinity states of the al-receptor site, and the modulation of these apparent affinity states by ions and nucleotides. ...
Article
Full-text available
In order to characterize putative high- and low-affinity states of the renal alpha 1-adrenoceptor, binding sites for the selective antagonist radioligand [3H]prazosin were examined in washed membranes prepared from rat renal cortex and medulla. Norepinephrine competition curves at [3H]prazosin sites were biphasic and were best fit by a two-site model. Na+ and GTP selectively decreased the proportion of sites exhibiting a high affinity for norepinephrine. In contrast, Mg2+ facilitated high-affinity interactions of norepinephrine at the renal alpha 1-receptor. Guanine nucleotides and Na+ increased the affinity of some antagonists [( 3H]prazosin, WB-4101), but not others (phentolamine). Mg2+ again had opposite effects. The effects of ions and nucleotides on both agonist and antagonist interactions were concentration-dependent. The order of potencies for monovalent cations (Na+ greater than Li+ much greater than K+), divalent cations (Mn2+ greater than Mg2+) and nucleotides (Gpp (NH)p, GTP much greater than GMP, ATP) were similar to those reported for cyclase-coupled receptor systems. However, unlike other divalent cations Ca2+ decreased both agonist and antagonist binding, possibly due to a Ca2+-sensitive proteinase. Receptor binding properties were similar in renal cortex and medulla. Renal alpha 1-receptor sites appear to display high- and low-affinity states with respect to agonists, and the equilibrium between these states may be modulated by guanine nucleotides and mono- and divalent metal ions. Some antagonists appear to bind preferentially to sites with low agonist affinity, and this effect is probably independent of retained endogenous catecholamines.
... The pharmacological characterization of the adrenoceptor involved in the control of renal tubular sodium excretion has been the subject of increasing attention but has resulted in conflicting views. Radioligand binding studies applied to kidney membranes indicate the presence of a,-and x,-adrenoceptors in the rat renal cortex (U'Prichard & Snyder, 1979; McPherson & Summers, 1981; 1982a) and almost exclusively oc2-adrenoceptors in the guinea-pig kidney (McPherson & Summers, 1982b). Such an analysis gives no indication of the function of these aadrenoceptors and the question therefore arises as to which subtype(s) of a-adrenoceptor are involved in the increased reabsorption of sodium induced by the adrenergic nervous system. ...
Article
A study was undertaken in the anaesthetized rabbit to classify the alpha-adrenoceptor subtypes responsible for increasing renal tubular sodium reabsorption and renin secretion. Intrarenal administration of noradrenaline, at doses which did not change renal blood flow or glomerular filtration rate, significantly decreased urine flow, absolute and fractional sodium excretion by between 26% and 29%. These renal responses to noradrenaline were abolished by the selective alpha 1-adrenoceptor antagonist, prazosin, but not by the selective alpha 2-adrenoceptor antagonist, idazoxan. Noradrenaline, given intrarenally, increased renin secretion between two and three fold and this response was not modified by either prazosin or idazoxan. Intrarenal administration of the selective alpha-adrenoceptor agonists, phenylephrine and methoxamine, at dose rates which did not change renal haemodynamics, significantly reduced urine flow, absolute and fractional sodium excretion by 15% to 33%, but at doses which reduced blood flow and filtration rate, by between 11% and 26%, urine flow, absolute and fractional sodium excretion decreased between 42% and 49%. Infusion of guanabenz (selective alpha 2-adrenoceptor agonist), at doses with no renal haemodynamic action, increased urine flow, absolute and fractional sodium excretion by 11% to 15%, while at doses which decreased blood flow by 7%, these other variables did not change. Administration of UK 14304 (selective alpha 2-adrenoceptor agonist) reduced blood flow and filtration rate by 3% and 9% respectively but had no other measurable action. At higher doses, which decreased blood flow by 14% and filtration rate by 24%, urine flow, absolute and fractional sodium excretion fell by between 27% and 50%. Renin secretion was significantly increased by the high doses of phenylephrine and UK 14304 but not by the low doses of these drugs. These studies show that adrenergic stimulation of renal tubular sodium reabsorption involves the activation of alpha 1- but not alpha 2-adrenoceptors. Further, adrenergic activation of the juxtaglomerular cells to release renin does not appear to involve either alpha 1- or alpha 2-adrenoceptors.
... As far as the receptor density the low-affinity component prevailed, the number of receptor being about 40 times more than the high-affinity component. These results are in agreement with those of McPherson and coworkers [9] who found that 3H-prazosin binds to rat renal cortex with two binding sites, a low and a high-affinity one, the low-affinity having the higher density. Alpha-2 receptor appears to consist of a single class of high-affinity binding sites. ...
Article
Binding to several receptors was compared in kidneys from 3- and 24-month-old rats. In crude membrane preparations of aged rat kidneys, the number of beta 2-adrenergic receptors was significantly reduced but the number of total beta-adrenoceptors was unchanged. The high-affinity alpha 1-adrenoceptor component was significantly reduced in old rats, whereas the low-affinity component was unchanged. The number of alpha 2-adrenoceptors showed a non-significant decrease. 3H-spiperone binding sites were similar in young and old rats. For each receptor binding the KD values were the same in young and old animals. The D1 dopamine receptor was significantly reduced in old rats. In our experiments, age-related changes of specific binding sites in the kidney were selective for some receptors studied and did not seem to be due to general aging-induced membrane modifications. Moreover, the renal and central receptors were sensitive to aging differently in the same animal model.
... Qualitative assessment using histochemical localization of proximal convoluted tubules, revealed that the alpha, and alpha2 receptors were localized on the proximal convoluted tubules. The function of alpha1 adrenergic receptors on the proximal convoluted tubule is still a matter of some controversy, but evidence indicates that this receptor may be involved in gluconeogenesis [17], and sodium and calcium reabsorption [181 in the rat. Recently, it has been shown that alpha, receptor activation in kidney slices is associated with an increase in phosphatidylinositol turnover in rat kidney [19]. ...
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The adrenergic nervous system is active in kidney function, and the kidney has large numbers of adrenergic receptor subtypes. Because of the cellular complexity of the kidney, it is difficult to obtain direct assessments of adrenergic receptor binding characteristics over specific tissue compartments. Qualitative autoradiography allows the localization of adrenergic receptors over tissue types in the kidney, but quantitative autoradiography allows direct comparison of adrenergic receptor number over different cellular compartments. The purpose of this study was to obtain direct assessments of alpha 1, alpha 2, and beta adrenergic receptor numbers over different tissue compartments of the kidney using quantitative autoradiography. Sections of Sprague-Dawley rat kidney were incubated in several concentrations of 3H-dihydroalprenolol to label beta receptors, 3H-prazosin to label alpha 1 receptors and 3H-rauwolscine to label the alpha 2 receptors. Sections of rat heart incubated in 3H-dihydroalprenolol were included as standards. The sections were then prepared for receptor autoradiography. After processing, the grains were then quantified on an image analysis system, and binding curves constructed from the specific binding. In some animals, the proximal tubules were stained to localize the proximal convoluted tubules. Significant Scatchard analyses were obtained in the glomeruli with dihydroalprenolol (5.18 X 10(9) receptors/mm3) and with rauwolscine (2.48 X 10(9) receptors/mm3). Significant Scatchard analyses were obtained in the cortex with rauwolscine (9.47 X 10(9) receptors/mm3) and with prazosin (3.9 X 10(9)). In addition, specific binding was seen with rauwolscine and prazosin to the kidney arterioles.(ABSTRACT TRUNCATED AT 250 WORDS)
... Alpha and beta adrenoreceptors have been identified in the renal cortex [60][61][62][63]. Cyclic AMP is the second messenger for beta-I and beta-2 agonists; alpha-2 agonists inhibit adenylate cyclase and cAMP formation. ...
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... However, it would be expected that these sites would saturate at low concentrations of prazosin which has very high affinity for a,-adrenoceptors in kidney (McPherson & Summers, 1981; Snavely & Insel, 1982). A more feasible explanation may involve numerous low affinity sites (KD 11 nM) labelled by [3H]-prazosin in rat kidney (McPherson & Summers, 1982) which are increased in density in the presence of EDTA (McPherson, 1982) conditions used in the experiments described here. This large population of low affinity sites identified by prazosin in rat kidney may provide attachment points from which prazosin can compete with [3H]-rauwolscine binding to a2-adrenoceptors. ...
Article
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... The functions of renal a~-receptors are poorly understood, and these receptors have been thought to be located primarily on the renal vasculature (20,41). However, recent studies indicate that renal a~-receptors mediate gluconeogenesis (27), which probably occurs in tubule cells (17,26). Other work suggests that renal tubule areceptors may regulate ion and water reabsorption by the nephron (6,20,32,35). ...
Article
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[3H]prazosin not only specifically and homogeneously labels alpha 1-adrenoceptors, but also binds to glass surfaces and non-linearly to the glass-fibre filters, commonly used in radioligand binding experiments. Binding to filters can be modulated by unlabeled alpha-adrenergic compounds and cations. If no correction is applied for displaceable filter binding, analysis of [3H]prazosin binding experiments leads to erroneous results. Analysis of [3H]prazosin saturation experiments on guinea-pig cerebral cortex membranes with correction for filter binding before the non-linear fit procedure indicated that [3H]prazosin labels a homogeneous population of alpha 1-adrenoceptors (Rtot: 8.33 fmol.mg-1 wet tissue) with a dissociation constant of 1.28 x 10(-10) M. However, analysis of the same data after correction for non-specific binding, (determined in parallel experiments by adding 10 microM phentolamine to the incubation medium) resulted in a best fit to a model in which [3H]prazosin labels two alpha 1-adrenoceptor subpopulations (R1: 15.0 fmol.mg-1 and R2: 14.6 fmol.mg-1 wet tissue) with dissociation constants of respectively 1.78 x 10(-10) and 5.63 x 10(-9) M. The discrepancy between the two methods of analysis is due to displacement of the radioligand from the filters by phentolamine. Prazosin and oxymetazoline are also able to displace filter-bound [3H]prazosin. The extent to which displaceable filter binding distorts the proper results depends on the actual magnitude of the error and also on the method of analysis.
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To evaluate the effect of hormones on renal phospholipid metabolism and turnover, we studied the changes in 32P-labeling of phospholipids in rat cortical tubule suspension. Angiotensin II, phenylephrine and parathyroid hormone (PTH) stimulate 32P incorporation into PC by 25, 29 and 26% and into PI by 189, 328 and 33% above control rates, respectively, whereas phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate labeling was not affected. However, when phospholipids were prelabeled with [32P]Pi, addition of angiotensin II led to a significant decrease in phosphatidylinositol 4,5-bisphosphate labeling in the first 2 min with no effect on the other phospholipid fractions. The phenylephrine effect on phospholipid labeling was blocked by prazosin but not by yohimbine, indicating an alpha 1-mediated action. In contrast, the effect of angiotensin II was not inhibited by either antagonist. The stimulating effect of substrates on 32P incorporation reported in the preceding paper was additive to that of hormones. Our results confirm previous studies on renal gluconeogenesis that catecholamines act by an alpha 1-type receptor on proximal tubules, and indicate that phenylephrine and angiotensin II act by different receptor sites exerting the same metabolic effect. The additivity of hormone effects with that of renal substrates indicates that the former are not secondary to release of precursors for phospholipid biosynthesis. The rapid decrease in phosphatidylinositol 4,5-bisphosphate labeling after angiotensin II suggests that the polyphosphoinositide is degraded after hormone binding to the receptor and that PI labeling is a secondary event.
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The binding of [3H]prazosin and [3H]clonidine to rat jejunal epithelial cell membranes has been studied. The membrane preparation was enriched in baso-lateral components as determined by Na+, K+ ATPase and alkaline phosphatase activities. The membranes possessed two saturable specific binding sites for [3H]prazosin, a high affinity (Kd 0.17 nM) low capacity (Bmax 27.3 fmole bound per mg protein) and a low affinity (Kd 5.0 nM) high capacity (Bmax 276 fmole bound per mg protein) site. The specificity of both sites was similar and was related to alpha 1-adrenoceptors. [3H]Clonidine bound to the membranes in a saturable fashion (Kd 7.3 nM). The specificity of this site was related to alpha 2-adrenoceptors. The [3H]clonidine binding site was present in the membranes in much lower density (Bmax 22.8 fmole bound per mg protein) suggesting that alpha 1-adrenoceptors predominate in this tissue.
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The affinities of several ergot derivatives for rat cerebral cortex alpha 1- and alpha 2-adrenoceptors were assessed using radioligand binding techniques. In most cases both [3H]prazosin (labelling alpha 1-adrenoceptors) and [3H]rauwolscine (labelling alpha 2-adrenoceptors) binding were displaced by the ergot derivatives tested, but with markedly different potency. Generally compounds displayed selectivity toward the alpha 2-adrenoceptor, particularly CQ 32-084, which had a Ki value against [3H]rauwolscine binding of 35 nM, but had little effect on [3H]prazosin binding at concentrations in excess of 5 microM. Lisuride was the most potent displacer of [3H]rauwolscine binding with a Ki value of 0.54 nM. Only bromocriptine was relatively alpha 1-selective with a Ki against [3H]prazosin binding of 18 nM and against [3H]rauwolscine binding of 120 nM. The results indicate that the ergot derivatives tested display a marked affinity for alpha-adrenoceptors and that their actions at this receptor class should be considered when interpreting their pharmacological activity in vivo.
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Binding of the alpha 2-adrenoceptor radioligands [3H]clonidine and [3H]rauwolscine but not the alpha 1-adrenoceptor radioligand [3H]prazosin was enhanced in membranes prepared from rat isolated renal glomeruli. [3H]Rauwolscine binding to glomeruli was stereoselective with respect to the (-)-isomer of noradrenaline and the order of potency of a series of antagonists for displacement of binding indicated that the alpha 2-adrenoceptors in this preparation differ somewhat from those in some other species and tissues. Chemical sympathectomy produced no significant change in the number of sites labelled by [3H]rauwolscine indicating that few of the alpha 2-adrenoceptors in glomerular membranes are associated with sympathetic nerve terminals.
Article
The Madin-Darby canine kidney (MDCK) cell line, derived from distal tubule/collecting duct, expresses differentiated properties of renal tubule epithelium in culture. We studied the expression of adrenergic receptors in MDCK to examine the role of catecholamines in the regulation of renal function. Radioligand-binding studies demonstrated, on the basis of receptor affinities of subtype-selective adrenergic agonists and antagonists, that MDCK cells have both alpha 1- and beta 2-adrenergic receptors. To determine whether these receptor types were expressed by the same cell, we developed a number of clonal MDCK cell lines. The clonal lines had stable but unique morphologies reflecting heterogeneity in the parent cell line. Some clones expressed only beta 2-adrenergic receptors and were nonmotile, whereas others expressed both alpha 1- and beta 2-receptors and demonstrated motility on the culture substrate at low cell densities. In one clone, alpha- and beta-receptor expression was stable for more than 50 passages. Catecholamine agonists increased phosphatidylinositol turnover by activating alpha-adrenergic receptors and cellular cyclic adenosine monophosphate accumulation by activating beta-adrenergic receptors. Guanine nucleotide decreased the affinity of isoproterenol for the beta 2-receptor but did not alter the affinity of epinephrine for the alpha 1-receptor. These results show that alpha 1- and beta 2-receptors can be expressed by a single renal tubular cell and that the two receptors behave as distinct entities in terms of cellular response and receptor regulation. Heterogeneity of adrenergic receptor expression in MDCK clones may reflect properties of different types of renal tubule cells.
Article
The beta adrenoceptor antagonist radioligand [3H] dihydroalprenolol (DHA) has been used to characterise beta adrenoceptors in membranes prepared from rat renal glomeruli. Association of the ligand was rapid and had reached equilibrium within 10 mins at 37 degrees C. Dissociation occurred in two distinct phases, a rapidly dissociating phase (low affinity site) and a slowly dissociating phase (high affinity site). The KD value for the high affinity site calculated from the kinetic experiments was 0.8 nM. Saturation analysis of binding gave comparable values for KD (1.77 nM) and demonstrated that membranes from glomeruli had four times the density of binding sites measured in renal cortex. In all saturation studies Hill coefficients were not significantly different from unity. Binding was stereoselective with respect to the (-) isomers of isoprenaline and propranolol and the potency of the selective displacing agents betaxolol (beta 1 adrenoceptors) and ICI 118,551 (beta 2 adrenoceptors) indicated that the receptors are of the beta 1 subtype.
Article
The everted-sac technique was used to study the mechanism of action of noradrenaline on fluid absorption by rat jejunum. Noradrenaline (10(-3) M) significantly stimulated fluid absorption and this effect was dependent on the presence of calcium ions in the serosal fluid. Strontium, but not magnesium could substitute for calcium. Verapamil, manganese and neodymium, all inhibitors of calcium transport, blocked noradrenaline-stimulated fluid absorption when present in the serosal compartment without any effect on basal or glucose-stimulated absorption. Inhibitors of the translation stage of protein synthesis inhibited the response whereas blocking the transcription stage of protein synthesis was without effect. The noradrenaline response was not attenuated by tetrodotoxin suggesting that the response is not indirect due to noradrenaline altering endogenous intestinal nervous activity. It is concluded that noradrenaline acts by opening calcium channels in the basolateral membranes of epithelial cells, resulting in an influx of calcium which stimulates ribosomal protein synthesis to produce proteins involved in fluid transport.
Article
A study was undertaken in pentobarbitone anaesthetized rabbits, undergoing a saline diuresis, to determine the subtype of alpha-adrenoceptor mediating renal tubular sodium reabsorption. Stimulation of the renal nerves at low rates, to cause an 11% fall in renal blood flow, did not change glomerular filtration rate but significantly reduced urine flow rate, and absolute and fractional sodium excretions by approximately 40%. These responses were reproducible in different groups of animals and with time. Renal nerve stimulation during an intra-renal arterial infusion of prazosin, to block alpha 1-adrenoceptors, had no effect on the renal haemodynamic response but completely abolished the reductions in urine flow rate, and absolute and fractional sodium excretion. During intra-renal arterial infusion of yohimbine, to block renal alpha 2-adrenoceptors, stimulation of the renal nerves to cause similar renal haemodynamic changes resulted in significantly larger reductions in urine flow rate, and absolute and fractional sodium excretion of about 52-58%. These results indicate that in the rabbit alpha 1-adrenoceptors are present on the renal tubules, which mediate the increase in sodium reabsorption caused by renal nerve stimulation. They further suggest the presence of presynaptic alpha 2-adrenoceptors on those nerves innervating the renal tubules.
Article
The selective alpha1-adrenoreceptor antagonist radioligand [3H] prazosin has been used to localize alpha1-adrenoreceptors in slide mounted sections of rat, dog and human kidney. The biochemical characteristics of [3H] prazosin binding to rat kidney sections were examined and found to be saturable, reversible, stereoselective, with a KD of 0.12 nM and Bmax of 6.72 +/- 0.2 fmoles/section. 3H Ultrofilm images of [3H] prazosin binding to rat, dog, and human kidney revealed binding to the vasculature but in the rat additional receptors were confined to the renal cortex. In the rat kidney autoradiography using emulsion coated coverslips showed that binding in the renal cortex was largely to proximal tubules. In all three species the autoradiographic studies support a role for alpha1-adrenoreceptors in control of renal blood flow. In the rat the location of alpha1-adrenoreceptors suggests that they can also have an important influence on fluid and electrolyte balance, gluconeogenesis and production of prostanoids.
Article
[3H]prazosin (PRAZ) and [3H]rauwolscine (RAUW) were used to examine the ontogeny of renal alpha 1- and alpha 2-adrenoceptors in the inbred Dahl hypertension-sensitive (S/JR) and -resistant (R/JR) rat. PRAZ and RAUW each bound to a single population of non-interacting sites. The binding of each ligand was saturable and reversible. The greatest proliferation of each receptor subtype occurred between 5 and 25 days of age. During this period, a 4 to 5-fold increase in the density of each was observed. Adult levels of each were reached by 50 days of age. The alpha 2-adrenoceptor was the predominant subtype present in renal tissue. However, its ratio to the alpha 1 subtype was influenced by strain and age: the ratio was greatest in the S/JR strain and decreased with age in both strains. The profile of alpha 1-adrenoceptor development was similar in S/JR and R/JR rats. In contrast, the density of alpha 2-adrenoceptors was similar in S/JR and R/JR rats at 5 and 15 days of age but significantly greater in the S/JR rat between 25 and 150 days of age. The elevated density of alpha 2-adrenoceptors could not be explained by strain-related differences in blood pressure or alterations in the affinity of the receptor. The results suggest that a relationship may exist between elevated renal alpha 2-adrenoceptor density and the genetic predisposition to hypertension in the S/JR rat. However, because this relationship is not apparent during the neonatal period of development, the possibility that the elevated density of sites may be secondary to some other event should also be considered.
Article
[3H]Prazosin is a selective ligand for α1-adrenoceptors. Using an in vitro autoradiographic technique specific binding of [3H]prazosin has been demonstrated, mainly to the renal cortex of the rat. These results provide further evidence that, in the rat, there is a high density of α1-adrenoceptors within the renal cortex.
Article
The characteristics of [3H]-prazosin binding in renal cortical membranes of the rat have been assessed under a variety of buffer conditions. At 37 degrees, in Krebs' phosphate and Tris buffer, [3H]-prazosin bound to two sites, a small population of high affinity sites with properties of alpha1-adrenoceptors and a much larger population of low affinity sites with different characteristics. High affinity [3H]-prazosin binding was insensitive to Na+, K+, Ca2+ and Mg2+ ions, but low affinity [3H]-prazosin binding was markedly increased in Krebs' phosphate or sodium phosphate buffer and further enhanced in membranes pretreated with EGTA. Binding was decreased in the presence of Ca2+, the decrease in binding mainly being due to a decrease in the number of low affinity sites labelled by the ligand. Low affinity [3H]-prazosin binding was increased at 37 degrees and relatively insensitive to alpha-adrenoceptor antagonists which were weak competitors while catecholamines failed to compete for low affinity binding. Scatchard plots of [3H]-prazosin binding performed using (-)-noradrenaline (1 mM) to define non-specific binding defined binding only to alpha 1-adrenoceptors. This provides a means of differentiating high and low affinity [3H]-prazosin binding.
Chapter
A curious aspect of receptor function is that the relationship between the occupancy of a receptor pool by an agonist drug and production of a tissue response very often is not linear. In many tissues a maximal tissue response can be evoked when only a very small fraction of the receptor pool is occupied by agonist (Stephenson, 1956; Nickerson, 1956). The remaining receptors are referred to as “spare” receptors or receptor “reserve.” Since the extent of the receptor reserve can vary dramatically from tissue to tissue for the same receptor type, there is no predictable relationship between the concentration of agonist necessary to occupy a particular fraction of the receptor pool and the concentration necessary to cause the same fractional tissue response.
Article
The mechanisms underlying increase in blood pressure and the role of renal α1-adrenoceptors in the pathogenesis / maintenance of hypertension have not been established. We used a tail-cuff method to measure systolic blood pressure in conscious rats and radioligand binding to determine the number and affinity of mixed α1A-, α1B- and α1D-adrenoceptors in kidney cortex from Grollman type renal hypertensive rats. [3H]Prazosin saturation binding with and without inactivation of α1-adrenoceptors by chloroethylclonidine (CEC), and by BMY 7378 plus CEC showed a ratio of ≈70% α1A- and 30% α1B-adrenoceptors in normotensive (116 ± 3 mm Hg) rats, with no change in affinity. Competition binding with 5-methylurapidil showed a biphasic curve (pKiH 8.63 ± 0.10 and pKiL 6.56 ± 0.14), supporting the α1A-/α1B- adrenoceptors presence, while BMY 7378 yielded a monophasic curve (pKiL 6.57 ± 0.05), indicating the lack of α1D-adrenoceptors in these kidneys. In contrast, saturation binding in the presence of BMY 7378 plus CEC in membranes from renal-hypertensive (186 ± 5 mm Hg) rats showed a small, yet significant decrease in receptor number, with no change in affinity; competition binding showed a high (pKiH 8.80 ± 0.20) and a low (pKiL 6.37 ± 0.07) affinity sites for BMY 7378 indicating the presence of α1D-adrenoceptors. These results indicate that α1D-adrenoceptors emergence in renal hypertensive rats is associated with increased blood pressure and suggest that α1D-adrenoceptor appearance is at the expense of α1B-adrenoceptors.
Chapter
Although hormones, neurotransmitters, and growth factors can activate renal transport and metabolic functions, a detailed understanding of the role of these agents in the regulation of renal homeostasis has been difficult to ascertain. In large part, this is due to the complexity of the kidney. With its large variety of different cell types, the kidney presents a veritable myriad of potential target cells. One solution to this problem has been to attempt to isolate and study glomeruli, tubular segments, vascular tissue, or other anatomically distinct portions of the kidney. However, studies using these structures have largely ignored the heterogeneity of cell types within anatomically distinct regions. Thus, major questions remain unanswered regarding the sites and mechanisms of hormone responses within the kidney. These questions are readily amenable to examina tion by using cultured renal epithelial cells in which one can study hormone action in homogeneous cell populations (Handler et al., 1980). Although such cells lack the anatomical relationships and cell—cell interplay that exist in vivo, the advantages of being able to isolate variables, to control the cells’ ionic and hormonal milieu, and to draw “clean” conclusions regarding properties of a particular cell type have been powerful “drawing cards” for investigators interested in how hormones and neurotransmitters regulate the kidney.
Chapter
This chapter discusses that efferent renal nerve stimulation affect both proximal tubular transport of salt and water and the resistance of the renal vessels. It highlights the possibility of a neural mechanism, a renorenal reflex, being responsible for the coordination of the excretory activity of the two kidneys that occurs when the function of one of the kidneys is altered. The condition under which the mechanism becomes operative requires further identification, but it is not restricted to the anesthetized state. Renal chemoreceptors and the mechanoreceptors provide an afferent mechanism for ipsilateral and contralateral renorenal reflexes integrated at the spinal cord level. This appears to be the operative mechanism in at least one instance of coordination of renal excretory activity. The full extent of the neural mechanism, involving different neurotransmitters with opposing effects, almost certainly exceeds in complexity of the simple system.
Chapter
The sections in this article are:
Article
Norepinephrine stimulates renal tubular sodium reabsorption, probably through an alpha 1-adrenoceptor-mediated mechanism. Although the distribution of alpha 1-adrenoceptors in the kidney has been studied with autoradiography, the precise location of these receptors in isolated nephron segments is unclear. Using a microassay we determined the specific binding of [125I]iodoarylazidoprazosin ([125I]prazosin), a high specific radioactivity analog of the selective alpha 1-antagonist prazosin, to microdissected glomeruli and tubule segments. Specific binding of [125I]prazosin (3 nM) in the proximal convoluted tubule was time- and concentration-dependent, saturable, and reversible. In this segment the apparent KD by association and dissociation rate constants of [125I]prazosin binding was 0.47 nM, and the maximum receptor density was approximately 0.19 fmol/mm, or 720 fmol/mg protein. Binding specificity was verified in competition studies with excess (3 microM) unlabeled prazosin and probes for alpha 2- (yohimbine), beta- (propranolol), dopamine1- (SCH23390), and dopamine2- (S-sulpiride) receptors. [125I]Prazosin binding was inhibited significantly only by unlabeled prazosin. Mapping of prazosin binding along the nephron revealed that the highest density was in the proximal convoluted tubule, followed by the proximal straight tubule. Lesser binding was found in the thick ascending limb and in the distal convoluted tubule, whereas in the cortical and outer medullary collecting duct and in glomeruli, binding was not significantly different from zero.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
Rationale Levodopa (L-DOPA), the gold standard treatment for Parkinson’s disease (PD), eventually causes L-DOPA-induced dyskinesia (LID) in up to 80% of patients. In the 6-hydroxydopamine (6-OHDA) rat model of PD, L-DOPA induces a similar phenomenon, which has been termed abnormal involuntary movement (AIM). We previously demonstrated that BMY-14802 suppresses AIM expression in this model. Objectives Although BMY-14802 is widely used as a sigma-1 antagonist, it is also an agonist at serotonin (5-HT) 1A and adrenergic α-1 receptors. The current study was conducted to determine which of these mechanisms underlies BMY-14802’s AIM-suppressing effect. This characterization included testing the 5-HT1A agonist buspirone and multiple sigma agents. When these studies implicated a 5-HT1A mechanism, we subsequently undertook a pharmacological reversal study, evaluating whether the 5-HT1A antagonist WAY-100635 counteracted BMY-14802’s AIM-suppressing effects. Results Buspirone dose-dependently suppressed AIM, supporting past findings. However, no AIM-suppressing effects were produced by drugs with effects at sigma receptors, including BD-1047, finasteride, SM-21, DTG, trans-dehydroandrosterone (DHEA), carbetapentane, and opipramol. Finally, we show for the first time that the AIM-suppressing effect of BMY-14802 was dose-dependently prevented by WAY-100635 but not by the α-1 antagonist prazosin. Conclusions BMY-14802 exerts its AIM-suppressing effects via a 5-HT1A agonist mechanism, similar to buspirone. Other 5-HT1A agonists have failed clinical trials, possibly due to submicromolar affinity at other receptors, including D2, which may exacerbate PD symptoms. BMY-14802 is a promising candidate for clinical trials due to its extremely low affinity for the D2 receptor and lack of extrapyramidal effects during prior clinical trials for schizophrenia.
Article
A suboptimal fetal environment has been linked to increased risk of cardiovascular disease in adulthood. We investigated whether intrauterine stress (IUS) alters the development of adrenergic reactivity in different types of rat arteries. Intrauterine stress was induced by ligation of the uterine arteries at day 13 of pregnancy in Wistar rats. First-order mesenteric, renal, femoral and saphenous arteries of the 21-day-old male offspring were studied in a myograph. IUS in the rat changes arterial adrenergic reactivity in a regionally selective manner. Adrenoceptor-mediated responses are altered in the renal artery. Maximal contractile responses to phenylephrine were increased, while sensitivity to the alpha(1)-adrenoceptor agonist was decreased. Intrauterine stress significantly reduced contractile responses to norepinephrine and enhanced relaxing responses to isoproterenol in the renal artery. Adrenergic responses were not modified in mesenteric, femoral and saphenous arteries. In the kidneys the densities of [(3)H]prazosin binding sites, periarterial adrenergic nerves and of the glomeruli were not altered after intrauterine stress at day 13 of gestation. The observed regionally selective alterations in arterial reactivity might link a suboptimal fetal environment to the development of cardiovascular disease in the adult.
Article
Full-text available
1. Direct radioligand—binding techniques have been used to characterize and quantify adrenoreceptors in human peripheral lung tissue removed at thoracotomy from ten patients, nine of whom had evidence of obstructive airways disease. 2. [3H]Dihydroalprenolol was used to characterize β–adrenoreceptor sites and [3H]prazosin to identify α–adrenoreceptor sites. Binding of both ligands showed saturability, high affinity, rapid kinetics, reversibility and stereospecificity. The rank order of agonists and antagonists inhibiting specific binding correlated well with known physiological potencies. Specificity of [3H]dihydroal-prenolol binding suggested that the population of lung β–adrenoreceptors is predominantly of the β2 subtype.
Article
Full-text available
The renal medulla develops very differently among species, being more prominent in those with a high urinary concentrating capacity. Attempts to correlate structure and function must consider loops of Henle, collecting ducts, vessels, interstitium, and pelvis. Two types of loops of Henle, long and short, are distinguished. Numerical relationships between both differ among species. Based on the epithelial lining a short loop consists of a thick descending limb (pars recta of proximal tubule), a thin descending limb, and a thick ascending limb. Long loops, in addition, have a thin ascending limb; their descending thin limbs are different from those of short loops and are site of considerable interspecies differences. Collecting ducts form in the cortex by joining several nephrons. Patterns with and without arcade formation are distinguished. On entering inner medulla, collecting ducts fuse successively. Collecting duct epithelium consists of principal and intercalated cells whose individual functions are subject to debate. Blood vessels are arranged in a very strict pattern reflecting that, in addition to nourishment, unique requirements in maintaining the corticomedullary osmotic gradient are to be met. Ultrastructural organization of medullary vessels is less specific compared to cortical vessels. Two types of renal medulla are distinguished. The simple type has vascular bundles consisting only of de- and ascending vasa recta; in the complex type, descending thin limbs of short loops are also integrated into vascular bundles. Functional implications of this difference are considerable. Striking interspecies differences also occur in the renal pelvis.
Chapter
A wide variety of tissues undergo a change of functional state on exposure to noradrenaline or adrenaline. Those molecular constituents of the effector cells of a tissue with which molecules of these catecholamines must first interact in order to produce a change of state — or response — of the tissue, are the so-called adrenoceptors (also commonly called adrenergic receptors). For convenience, we refer to noradrenaline, adrenaline and other agents which produce responses in tissues by interacting with adrenoceptors, as adrenergic agonists. An agent which specifically inhibits a response produced by an adrenergic agonist is referred to as adrenergic blocking agent or adrenergic antagonist.
Article
1. Noradrenaline stimulates gluconeogenesis through an alpha-adrenoceptor in renal cortical tubule fragments from fed rats incubated with 5 mM-lactate. 2. The selective alpha 1-adrenoreceptor agonist methoxamine stimulated gluconeogenesis, but the selective alpha 2-adrenoceptor agonist clonidine was ineffective. 3. The selective alpha 1-adrenoceptor antagonist thymoxamine blocked the stimulatory effects on gluconeogenesis of noradrenaline and of oxymetazoline (a synthetic alpha-agonist). The selective alpha 2-adrenoceptor antagonist yohimbine was ineffective in this respect. 4. It is concluded that noradrenaline and oxymetazoline stimulate gluconeogenesis in rat kidney via an alpha 1-rather than an alpha 2-type of adrenoceptor.
Article
3H-Prazosin binding to membranes from rat brain is saturable, Bmax, 77 fmol/mg protein, of high affinity, KD, 0.28 nM and with a drug specificity indicating that it labels α-adrenergic receptors. The ranking of α-antagonists for these binding sites suggests that 3H-prazosin may be useful in identifying a subpopulation of α-receptors (α1) in the central nervous system.
Article
1A study has been made of α-adrenoreceptors in the smooth muscle of the guinea-pig splenic capsule.2In this preparation, noradrenaline (NA), adrenaline (Adr) and α-methyl-NA were full agonists, while phenylephrine, dopamine and the catechol-imidazolidine (3,4-dihydroxyphenylamino)-2-imidazolidine, (DPI) were partial agonists.3The imidazolidines clonidine and oxymetazoline showed no agonist activity, but both inhibited the agonist activity of NA. pA2 values calculated from Arunlakshana and Schild plots were 6.88 and 6.95 for clonidine and oxymetazoline respectively. Slopes of the Schild plots were close to unity, indicating competitive antagonism.4The α-adrenoreceptor antagonists phentolamine, prazosin and yohimbine also inhibited the agonist activity of NA with mean pA2 values of 8.32, 9.22 and 6.90 respectively. Slopes of Schild plots were significantly less than 1, suggesting that the inhibition was not truly competitive in nature.5The irreversible α-adrenoreceptor antagonist phenoxybenzamine at low concentrations (10–9M) shifted the log concentration-response curve to NA to the right and greatly reduced the maximum response.6It is suggested that the adrenoreceptors in the guinea-pig splenic capsule are probably of the α1-type, and that this tissue possesses relatively few spare receptors.
Article
: Recent research has introduced into [alpha]-adrenoceptor pharmacology some interesting new aspects. In this article the presynaptic [alpha]-autoreceptors of noradrenergic neurones, and the subclassification of [alpha]-adrenoceptors into [alpha]1- and [alpha]2-types, are discussed. Presynaptic a-receptors occur at all noradrenergic axons where they have been sought. They subserve an autoinhibition in which released noradrenaline inhibits its own further release, and they are probably located on the terminal noradrenergic axons. It soon became clear that presynaptic [alpha]-receptors differed from classical postsynaptic a-receptors in their sensitivity to drugs. This led to a general pharmacological subclassification. [alpha]1-Adrenoceptors are the classical postsynaptic [alpha]-receptors of smooth muscle cells, but occur also elsewhere, e.g., in brain and liver. The prototypes of the [alpha]2-adrenoceptors are the presynaptic [alpha]-receptors, but similar receptors have since been identified outside terminal noradrenergic axons. For instance, in cholinergic neurones, [alpha]2-receptors mediate inhibition of acetylcholine release. The most unforeseen [alpha]2-adrenoceptors have recently been detected in smooth muscle cells, where, like the [alpha]1-receptors, they mediate contraction. The [alpha]1/[alpha]2 subclassification may have clinical implications, e.g., in the treatment of hypertension with [alpha]2-selective agonists and [alpha]1-selective antagonists. (C) Lippincott-Raven Publishers.
Article
Isolated kidney cortex tubules from starved rats have been used to study the actions of catecholamines on renal adenosine 3′:5′-monophosphate (Ado-3′:5′-P) levels and gluconeogenesis. In accordance with previous workers, norepinephrine was found to increase glucose formation from lactate and pyruvate and to a smaller degree from malate, succinate, fumarate, glutamate and glutamine. The stimulatory effect of 0.5 μM norepinephrine was additive to that of 0.1 mM Ado-3′:5′-P, indicating an Ado-3′:5′-P-independent mechanism of catecholamine action. The effects of parathyroid hormone and oleate on gluconeogenesis were also additive to that of norepinephrine. A comparative study of the actions of different catecholamine derivatives revealed that gluconeogenesis was stimulated in parallel to the α-adrenergic potency of the hormones, whereas Ado-3′: 5′-P levels were increased according to the known β-stimulatory potency of the agents. Although iso-proterenol was by far the most effective in raising Ado-3′: 5′-P levels, it was without effect on glucose formation from pyruvate, when added at 0.1 μM. At the same concentration, phenylephrine, which had no effect on Ado-3′:5′-P levels, was the best stimulator of gluconeogenesis. The α-receptor blocking agent phentolamine inhibited the stimulatory effect of catecholamines on gluconeogenesis with a 50 times higher potency than propranolol, a,β-blocking agent. The fact that the stimulatory effect of Ado-3′:5′-P was also blocked by propranolol, indicated an unspecific mechanism of action of this substance. The results indicate that the stimulatory effect of catecholamines on renal gluconeogenesis are mediated by an a-receptor and that they are independent from the stimulation of renal adenyl cyclase by these agents.
Article
Prazosin is know to block postsynaptic α-adrenoceptors. In this study 3H-prazosin has been used to label biochemically central α-adrenoceptors. In rat brain membranes 3H-prazosin bound specifically in a rapid, reversible and saturable manner to a single class of high affinity sites. The relative order of potencies for inhibition of 3H-prazosin binding was WB4101 > ARC 239 > phentolamine ≫ piperoxane > yohimbine which is a characteristic of the α1 type of adrenoceptors. In contrast, the relative order of potencies for inhibition of 3H-clonidine binding was yohimbine > piperoxane > WB4101 > ARC239 > prazosin which is a characteristic of the peripheral ‘α1’-and ‘α1’-adrenoceptors. These results indicate that 3H-prazosin binds to central ‘α1’ and 3H-clonidine to ‘α’2-receptor and confirm the presence of two classes of α-adrenoceptors in rat brain membranes.
Article
We have developed a general strategy and a versatile computer program for analysis of data from ligand-binding experiments (e.g., radioreceptor assay systems for hormones, neurotransmitters, drugs). This method provides optimal (weighted least squares) estimates of “binding parameters” (affinity constants, binding capacities, nonspecific binding) for any number of ligands reacting simultaneously with any number of receptors. This approach provides two major advantages compared with other available methods: (i): It uses an exact mathematical model of the ligand-binding system, thereby avoiding the possible biases introduced by several commonly used approximations. (ii) It uses a statistically valid, appropriately weighted least-squares curve-fitting algorithm with objective measurement of goodness of fit, thereby avoiding the subjective graphical or simplified statistical methods which may introduce bias. Additional important features include the following. (i) The level of nonspecific binding is regarded as an unknown parameter, subject to uncertainty, which must be estimated simultaneously with other parameters of the system by appropriate statistical methods. This approach provides a more accurate and precise estimate of the parameters and their standard errors. (ii) Selected parameters can be forced to share a common value, or be fixed at any desired constant value. This feature facilitates hypothesis testing by appropriate statistical methods e.g., testing whether a particular experimental manipulation results in a change in affinity (K), binding capacity (R), or both parameters. (iii) One can combine results from multiple experiments by introduction of explicit scaling or “correction” factors which compensate for the commonly observed large degree of between-experiment variation of the overall binding capacity (Bmax) while other properties of the system (e.g., K values, relative binding capacities for high- and low-affinity sites) are highly reproducible. (iv) One can characterize complex cross-reacting systems involving any number of ligands reacting simultaneously with any number of binding sites. This enables one to pool results from several curves obtained using several different ligands.
Article
A theoretical analysis has been made of the relationship between the inhibition constant (KI) of a substance and the (I50) value which expresses the concentration of inhibitor required to produce 50 per cent inhibition of an enzymic reaction at a specific substrate concentration. A comparison has been made of the relationships between KI and I50 for monosubstrate reactions when noncompetitive or uncompetitive inhibition kinetics apply, as well as for bisubstrate reactions under conditions of competitive, noncompetitive and uncompetitive inhibition kinetics. Precautions have been indicated against the indiscriminate use of I50 values in agreement with the admonitions previously described in the literature. The analysis described shows KI does not equal I50 when competitive inhibition kinetics apply; however, KI is equal to I50 under conditions of either noncompetitive or uncompetitive kinetics.
Article
1.1. Tubule fragments isolated from kidney cortex of fed rats by collagenase treatment were incubated with 5 mM lactate.2.2. By use of selective α-adrenergic agonists and blocking agents it was shown that renal gluconeogenesis is stimulated through an α1-type of adrenoceptor.3.3. Catecholamine stimulation of the process is not abolished by omission of extracellular Ca2+ and is not accompanied by any change in influx of 45Ca2+. On the other hand there is an α-adrenergic stimulation of 45Ca2+ efflux from tubule fragments preloaded with this isotope, suggesting mobilisation of some intracellular calcium store.4.4. Ouabain, and removal of extracellular K+, abolish noradrenaline stimulation of gluconeogenesis suggesting that Na+ and/or K+ are involved in transmission of the α-adrenergic stimulus.
Article
The rabbit pulmonary artery contains postsynaptic α-adrenoceptors which mediate smooth muscle contraction; its noradrenergic nerves contain presynaptic α-adrenoceptors which mediate inhibition of the release of the transmitter evoked by nerve impulses. Dose-response curves for the pre- and postsynaptic effects of eight α-receptor agonists were determined on superfused strips of the artery in the presence of cocaine, corticosterone and propranolol.1. According to the concentrations which caused 20% of the maximal contraction (EC20 post), the postsynaptic rank order of potency was: adrenaline > noradrenaline > oxymetazoline > naphazoline > phenylephrine > tramazoline >α-methylnoradrenaline > methoxamine. The pA2 values of phentolamine against oxymetazoline, phenylephrine, α-methylnoradrenaline and methoxamine were 7.43, 7.48, 7.59 and 7.69, respectively. 2. For the investigation of presynaptic effects, the arteries were preincubated with 3H-noradrenaline. All agonists inhibited the overflow of tritium evoked by transmural sympathetic nerve stimulation. According to the concentrations which reduced the stimulation-induced overflow by 20% (EC20 pre), the rank order of potency was: adrenaline > oxymetazoline > tramazoline > α-methylnoradrenaline > noradrenaline > naphazoline > phenylephrine > methoxamine. 10−5 M phentolamine shifted the presynaptic dose-response curves for noradrenaline and oxymetazoline to the right. 3. The ratio EC20 pre/EC20 post was calculated for each agonist as an index of its relative post- and presynaptic potency. According to the ratios, the agonists were arbitrarily classified into three groups. Group 1 (ratio about 30; preferentially postsynaptic agonists) comprised methoxamine and phenylephrine; group 2 (ratio near 1; similar pre- and postsynaptic potencies) comprised noradrenaline, adrenaline and naphazoline; group 3 (ratio below 0.2; preferentially presynaptic agonists) comprised oxymetazoline, α-methylnoradrenaline and tramazoline (as well as clonidine). 4. Preferentially presynaptic and preferentially postsynaptic agonists had opposite effects on the vasoconstrictor response to nerve stimulation. Methoxamine and phenylephrine either did not change or enhanced, but never reduced, the response. In contrast, oxymetazoline, α-methylnoradrenaline and tramazoline at low concentrations selectively inhibited the response to stimulation at low frequency (0.25–2 Hz). 5. It is concluded that α-adrenoceptor agonists vary widely in their relative pre- and postsynaptic potencies, possibly because of structural differences between pre- and postsynaptic α-receptors. Pre- and postsynaptic components contribute to their overall postsynaptic effect in actively transmitting synapses. The preferential activation of presynaptic α-receptors results in α-adrenergic inhibition of synaptic transmission.
Article
1. Low frequency (0.1 Hz) electrical stimulation of the rat isolated vas deferens produced regular contractions that were inhibited by low concentrations of clonidine. 2. The inhibition of the vas deferens produced by clonidine was presynaptic in origin and involved alpha-adrenoceptors. 3. Presynaptic alpha-adrenoceptor antagonist activity was assessed by studying the effects of increasing concentrations of the antagonists on cumulative clonidine dose-response curves on the stimulated vas deferens. 4. Postsynaptic alpha-adrenoceptor antagonist activity was assessed by comparison of control cumulative noradrenaline dose-response curves with those in the presence of increasing concentrations of antagonists in the rat anococcygeus muscle. 5. The results indicate that yohimbine and phentolamine are more potent in blocking presynaptic than postsynaptic alpha-adrenoceptors. Phenoxybenzamine and prazosin block postsynaptic alpha-adrenoceptors preferentially. 6. The findings support the view that presynaptic and postsynaptic alpha-adrenoceptors differ in their sensitivity to alpha-adrenoceptor antagonists.
Article
The alpha adrenergic effects of a series of pharmacologically active imidazolines have been studied in relation to phenylephrine on isolated rat aortic strips. Dose-response relationships showed that all imidazolines but one [(3,4-dihydroxyphenylamino)-2-imidazoline, (DPI)] were partial agonists with respect to phenylephrine on the alpha adrenergic receptor. Most imidazolines, in addition to activating the receptor, were also potent competitive antagonists. Dissociation constants determined for each compound by several techniques closely agreed. The dissociation constants for agonist activity (KA) were identical to those calculated for antagonist activity (KB) (correlation coefficient = 0.98). As a class, the imidazolines possessed high affinity for the alpha adrenergic receptor and even the weakest imidazoline (DPI) was nearly as potent as phenylephrine. Many structural requirements of the receptor for the imidazolines were determined through consideration of the changes in receptor affinity that resulted from structural alterations. From the dissociation constants, efficacies (er) of the imidazolines were calculated relative to phenylephrine. Structure-action relationships based on er values were then evaluated. Most of the imidazolines possessed er values in the range of 0.004 to 0.040 relative to phenylephrine whose er was designated as 1.000. Substitution of a meta-hydroxyl group on xylometazoline, however, produced an increase in the er from 0.036 to 0.169. The formation of a catechol (DPI) from 2-phenylaminoimidazoline resulted in an even greater increase in er from 0.018 to 1.162 such that DPI was no longer a partial agonist but produced the same maximal response as phenylephrine. These results suggest that aromatic hydroxyl substitutions enhance the ability of an imidazoline to activate the alpha adrenergic receptor. The increase in efficacy that resulted from aromatic hydroxyl substitution was accompanied by a decrease in affinity indicating that, for the imidazolines, steric requirements governing affinity for the receptor are distinct from steric requirements for receptor activation.
Article
3H-Prazosin binding to membranes from rat brain is saturable, Bmax, 77 fmol/mg protein, of high affinity, KD, 0.28 nM and with a drug specificity indicating that it labels alpha-adrenergic receptors. The ranking of alpha-antagonists for these binding sites suggests that 3H-prazosin may be useful in identifying a subpopulation of alpha-receptors (alpha1) in the central nervous system.
Article
Tritiated prazosin was used to characterize high affinity binding sites with characteristics similar to α 1 adrenoceptors in rat brain membranes. These sites were compared with α 2 adrenoceptors labeled with tritiated clonidine. The prazosin sites had an association constant of 2 nM−1 and bound the ligand optimal around pH 7.0. The density of the sites was 300 fmoles per mg of protein; the half time of dissociation of prazosin was 7 min at 30° C. The order or potencies of agonists, determined from binding-inhibition experiments with labeled prazosin, was: naphazoline > clonidine > adrenaline > noradrenaline > phenylephrine > α-methylnoradrenaline > dophamine. The order of potencies of antagonists was: prazosin > phenoxybenzamine > phentolamine > clozapine > yohimbine. Sodium ions and divalent cations as well as guanyl nucleotides have little or no effect on the binding of the labeled antagonist. This is in contrast to the binding of the labeled agonist clonidine (Glossmann and Presek, 1979a, 1979b). Labeled prazosin may be a useful tool to characterize α 1 adrenoceptors.
Article
1. In incubated tubule fragments from renal cortex of fed rats gluconeogenesis from pyruvate was stimulated by adrenaline (1mum optimum) and by the selective alpha-adrenergic agonists oxymetazoline and amidephrine. The selective beta-agonists isoproterenol and salbutamol were ineffective at concentrations up to 10mum. 2. Stimulation of gluconeogenesis by 1mum-adrenaline was almost completely blocked by 10mum-phentolamine (alpha-antagonist), partially blocked by 10mum-phenoxybenzamine (alpha-antagonist) and unaffected by 10mum-propranolol (beta-antagonist). 3. Adrenaline stimulation of gluconeogenesis was rapid and was sustained for at least 1h. 4. Oxymetazoline (alpha-agonist) was extremely potent in stimulation of gluconeogenesis. This compound stimulated glucose production from pyruvate, lactate and glutamate, but not from succinate or glycerol. 5. In the absence of Ca(2+) oxymetazoline was ineffective, whereas some stimulatory effect of adrenaline on gluconeogenesis was still observed. 6. Glucagon had no effect on gluconeogenesis from pyruvate in the presence of 1.27mm-Ca(2+) and inhibited the process in the presence of 0.25mm-Ca(2+). Parathyrin (parathyroid hormone) stimulated gluconeogenesis at 1.27mm-Ca(2+). 7. In short incubations of tubule fragments glucagon, papaverine and adrenaline significantly increased 3':5'-cyclic AMP. Adrenaline also slightly decreased 3':5'-cyclic GMP. Oxymetazoline had no effect on the amount of either cyclic nucleotide. 8. At all concentrations tested, theophylline and papaverine decreased gluconeogenesis from pyruvate. 9. It is concluded that renal gluconeogenesis may be increased by alpha- but not beta-adrenergic stimuli and that this is probably independent of changes in 3':5'-cyclic AMP or 3':5'-cyclic GMP. An involvement of Ca(2+) in the action of oxymetazoline appears likely, but this is less certain with adrenaline.
In contrast to rat kidney cortex the glucogenic capacity of kidney cortex slices from normally treated guinea pigs was very low. Reduction of the pH of the incubation medium by either lowering the HCO3-concentration or by increasing the pCO2 resulted only in varying stimulatory effects on glucose production from endogeneous or exogeneous sources. Considerable rates of net synthesis of glucose from lactate, pyruvate, malate, 2-oxoglutarate, glutamate, and glycerol--but not from glutamine--were only observed in kidneys from animals with prolonged metabolic acidosis. Neither in experiments with normally treated animals nor in those with acidotic guinea pigs the glucose production decreased, when calcium was omitted from the incubation medium. Though glutamine was not converted into glucose, it served as a substrate for ammoniagenesis. On the basis of the presented results it is concluded that species differences exist in the regulation of renal gluconeogenesis.
Article
Phosphoenolpyruvate carboxykinase activity was determined in microdissected lyophilized structures of rat kidney. The determination of 32P incorporation from γ labeled 5' guanosine triphosphate into phosphoenolpyruvate allowed the determination of enzyme activity in tissue samples of 0.5 to 20 μg dry weight. A study of structures from kidneys of starved rats revealed that phosphoenolpyruvate carboxykinase activity was highest in the proximal contorted tubule, decreasing to less than half in the straight portion. Samples from the thick ascending limbs of Henle's loop contained nearly none and collecting ducts no activity. Because of the limited tissue available no determinations were done in the contorted part of the distal tubule. Since phosphoenolpyruvate carboxykinase in rat kidney is thought to be a gluconeogenic enzyme, these results suggest that this metabolic pathway is nearly exclusively localized in the proximal tubule of rat kidney.
Article
The alpha-adrenolytic activity of BE 2254 was investigated in in vitro as well as in vivo assays. On the isolated rat anococcygeus muscle, 2-[beta-(4-hydroxyphenyl)-ethyl-amino-methyl]tetralone(1) (BE 2254) shows a high affinity for postsynaptic alpha-adrenoceptors (pA2 = 8.9), in contrast to its much weaker potency (pA2 = 6.7) in inhibiting clonidine on the electrically driven rat vas deferens, thus suggesting a relative preference for postsynaptic alpha-adrenoceptors. BE 2254 effects on other catecholamine receptors are either negligible or not detectable. The hypotensive action of BE 2254 is shown to be solely due to alpha-blockade. All alpha-adrenolytic actions studied were of competitive nature.
Article
The results of this study indicate that both [3H]clonidine and [3H]prazosin bind with high affinity to sites in rat kidney. [3H]Clonidine binding is found in both renal cortex and medulla, whereas [3H]prazosin binding is localized to the cortex. These results are in contrast to those obtained in the guinea-pig, where [3H]clonidine binding is localized to the cortex and no significant [3H] prazosin binding is observed.
Article
1. A study has been made of alpha-adrenoreceptors in the smooth muscle of the guinea-pig splenic capsule. 2. In this preparation, noradrenaline (NA), adrenaline (Adr) and alpha-methyl-NA were full agonists, while phenylephrine, dopamine and the catechol-imidazolidine (3,4-dihydroxyphenylamino)-2-imidazolidine, (DPI) were partial agonists. 3. The imidazolidines clonidine and oxymetazoline showed no agonist activity, but both inhibited the agonist activity of NA. pA2 values calculated from Arunlakshana and Schild plots were 6.88 and 6.95 for clonidine and oxymetazoline respectively. Slopes of the Schild plots were close to unity, indicating competitive antagonism. 4. The alpha-adrenoreceptor antagonists phentolamine, prazosin and yohimbine also inhibited the agonist activity of NA with man pA2 values of 8.32, 9.22 and 6.90 respectively. Slopes of Schild plots were significantly less than 1, suggesting that the inhibition was not truly competitive in nature. 5. The irreversible alpha-adrenoreceptor antagonist phenoxybenzamine at low concentrations (10(-9)M) shifted the log concentration-response curve to NA to the right and greatly reduced the maximum response. 6. It is suggested that the adrenoreceptors in the guinea-pig splenic capsule are probably of the alpha 1-type, and that this tissue possesses relatively few spare receptors.
Article
The relative selectivity of a series of imidazoline derivatives and substituted guanidines structurally related to clonidine for α1 and α2 adrenoceptors has been examined in binding studies using membranes prepared from rat cerebral cortex and the radioligands [3H]prazosin and [3H]clonidine. There was a 150-fold difference between the most and least selective clonidine-like drugs in their relative affinity for α1 and α2 adrenoceptors.
Article
1. Noradrenaline stimulates gluconeogenesis through an alpha-adrenoceptor in renal cortical tubule fragments from fed rats incubated with 5 mM-lactate. 2. The selective alpha 1-adrenoreceptor agonist methoxamine stimulated gluconeogenesis, but the selective alpha 2-adrenoceptor agonist clonidine was ineffective. 3. The selective alpha 1-adrenoceptor antagonist thymoxamine blocked the stimulatory effects on gluconeogenesis of noradrenaline and of oxymetazoline (a synthetic alpha-agonist). The selective alpha 2-adrenoceptor antagonist yohimbine was ineffective in this respect. 4. It is concluded that noradrenaline and oxymetazoline stimulate gluconeogenesis in rat kidney via an alpha 1-rather than an alpha 2-type of adrenoceptor.
Article
[3H]Dihydroergocryptine, a nonselective alpha adrenergic antagonist, the alpha-1 selective antagonist, [3H]prazosin and the alpha-2 selective antagonist, [3H]yohimbine, were used to study binding sites in rat renal membranes. To establish a correlation between binding and a biological function, the ability of alpha adrenergic agents to stimulate or inhibit vasoconstriction was quantified in vitro using an isolated perfused kidney preparation. Binding with each radioligand was rapid, saturable and specific. Moreover, the order of potencies of a variety of adrenergic agents, determined by competitive inhibition studies, suggested that the binding of each radioligand was to sites with alpha adrenergic specificity. The total number of binding sites in these rat renal membranes. determined with [3H]dihydroergocryptine (Bmax, 212 fmol/mg of protein; KD, 12.8 nM) was approximately equal to the sum of binding site concentrations determined with the alpha-1 and alpha-2 selective radioligands (Bmax, 57 and 170 fmol/mg of protein; KD, 0.85 and 20 nM, respectively). However, the alpha receptor mediating renal arteriolar vasoconstriction appeared to be of the alpha-1 subtype as there was a close correlation between the in vitro results and the binding data determined with [3H]prazosin (r = 0.93). In addition, in both the functional and [3H]prazosin binding studies, unlabeled prazosin ws 5 to 40-fold more potent than the nonselective antagonist, phentolamine, and 400- to 1500-fold more potent than the alpha-2 antagonist, yohimbine. These studies suggest that rat renal plasma membranes contain binding sites with both alpha-1 and alpha-2 adrenergic receptor specificity, in a ratio of approximately 1:3. Despite the preponderance of alpha-2 receptors, the alpha receptor mediating renal vasoconstriction appears to be of the alpha-1 type.
Article
Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins (l), a number of modified analytical pro- cedures ut.ilizing this reagent have been reported for the determination of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and in insulin (10). Although the reagent would seem to be recommended by its great sen- sitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard t.o effects of variations in pH, time of reaction, and concentration of react- ants, permissible levels of reagents commonly used in handling proteins, and interfering subst.ances. Procedures are described for measuring pro- tein in solution or after precipitation wit,h acids or other agents, and for the determination of as little as 0.2 y of protein.
SUMMERS adrenoceptors in rat brain: Direct identification with prazosin
  • In Vascular
  • G A Mcpherson
In Vascular r184 G.A. McPHERSON & R.J. SUMMERS adrenoceptors in rat brain: Direct identification with prazosin. Naunyn-Schmiedebergs Arch. Pharmac., 30, 223-230
Zur Pharmakologie des α-Rezeptoren blockers BE2254 (HEAT)
  • HEINZ
HEINZ, V.N. & HOFFERBER, F. (1980). Zur Pharmakologie des a-Rezeptoren blockers BE2254 (HEAT).
Selectivity of blocking agents for pre-and post-synaptic aadrenoceptors
  • J C Smith
  • C F C Walker
DOXEY, J.C., SMITH, C.F.C. & WALKER, J.M. (1977). Selectivity of blocking agents for pre-and post-synaptic aadrenoceptors. Br. J. Pharmac., 60, 91-96.
[3H]-prazosin binds specifically to α1-adrenoceptors in rat brain. Naunyn-Schmiedebergs Arch
  • P J Dausse
  • J.-P Cardot
  • A Meyer
Hormonal control of gluconeogenesis in tubule fragments from renal cortex of fed rats
  • D W R Saggerson
Hormonal control of gluconeogenesis in tubule fragments from renal cortex of fed rats
  • MACDONALD