Effects of clenbuterol on central beta-1 and beta-2 adrenergic receptors of the rat

Repeated administration of the centrally acting beta adrenoceptor agonist, clenbuterol, to rats reduced the ability of isoproterenol to increase the concentration of cyclic AMP (cAMP) in slices of cerebellum. This reduced responsiveness to isoproterenol was accompanied by a marked reduction in the density of beta adrenoceptors as measured by the binding of the beta adrenoceptor antagonist [125I]iodopindolol. In addition, the agonist-binding properties of remaining cerebellar beta adrenoceptors were altered after clenbuterol treatment. The clenbuterol-induced reduction in the density of beta adrenoceptors in the cerebellum is in marked contrast to its inability to do this in cerebral cortex. Comparison of the ability of clenbuterol to that of isoproterenol to increase levels of cAMP in slices of cerebral cortex or cerebellum showed that clenbuterol is a weakly potent agonist in both brain regions. The increase in cAMP induced by isoproterenol in the cortex was significantly reduced in the presence of the selective beta-1 adrenoceptor antagonist, ICI 89,406. In contrast, the clenbuterol-induced increase in cortical cAMP was unchanged by ICI 89,406 but was reduced significantly by the beta-2 adrenoceptor antagonist, ICI 118,551. In cerebellum, both isoproterenol- and clenbuterol-stimulated accumulation of cAMP were antagonized much more potently by ICI 118,551 than by ICI 89,406. Furthermore, clenbuterol antagonized the cAMP response induced by isoproterenol in the presence of ICI 118,551 in a concentration-dependent manner. In terms of measurement of cAMP in brain slices, clenbuterol is weakly potent as an agonist at beta-2 adrenoceptors and has antagonist properties at beta-1 adrenoceptors.     

Phorbol ester and staurosporine modulation of antagonist and agonist binding to alpha-2 adrenergic receptors: differential influence on Na+ versus guanylylnucleotide regulation.

The modulatory influence of the protein kinase C (PKC) activator phorbol dibutyrate (pDBu) and the PKC inhibitor staurosporine on the binding of the antagonist rauwolscine and the agonist (-)-epinephrine to alpha-2 adrenergic receptors was studied in plasma membranes from bovine aorta. In control membranes [3H]rauwolscine binding exhibited high (KDH = 110 pM) and low (KDL = 2.4 nM) affinity components. The addition of 0.1 mM 5'-guanylylimidodiphosphate [Gpp(NH)p] reduced binding to a single component (KD = 1.3 nM) and the addition of 140 mM NaCl increased the proportion of high affinity sites from 7 to 15%, whereas the combination of both Gpp(NH)p and NaCl did not differ from values for NaCl alone. PDBu pretreatment had little effect on [3H]rauwolscine binding with the exception of a small increase in KD in the presence of Gpp(NH)p. Staurosporine pretreatment, however, eliminated the high-affinity component in the absence of Gpp(NH)p or NaCl and rendered Gpp(NH)p ineffective. NaCl was able to restore two components of [3H]rauwolscine binding to the same extent as in untreated membranes. Epinephrine displaced [3H]rauwolscine in a biphasic manner (KDH = 93 nM, KDL = 3.5 microM; %RH = 42). In untreated membranes Gpp(NH)p reduced epinephrine affinity, but did not alter the %RH. NaCl alone increased KDL and caused a partial decrease in %RH, whereas the combination of Gpp(NH)p and NaCl was required to produce a single, low-affinity state (KD = 11.9 microM). PDBu pretreatment reduced epinephrine affinity and blocked the effectiveness of Gpp(NH)p, but the action of NaCl was more pronounced than in untreated membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of agonist exposure on the coupling of beta 1 and beta 3 adrenergic receptors to adenylyl cyclase in isolated adipocytes.

The coupling of beta 1 and beta 3 adrenergic receptors to adenylyl cyclase was examined in membranes of isolated white adipocytes. The activation of adenylyl cyclase by isoproterenol (ISO) was biphasic. The high-affinity activation of adenylyl cyclase, which occurred at submicromolar concentrations of ISO, was mediated by beta 1 receptors, whereas low-affinity activation was mediated by beta 3 receptors. The relative activation of adenylyl cyclase by beta 3 receptors was less when ISO was used to stimulate activity, compared to when the beta 3-selective agonist BRL 37344 was used. These data indicate that both receptor subtypes can stimulate the same adenylyl cyclase in membranes of control cells, and that the activation of adenylyl cyclase by beta 1 receptors by low concentrations of catecholamines can obscure the activation by beta 3 receptors by high concentrations of catecholamines. Exposure of adipocytes to ISO at concentrations that either selectively stimulate beta 1 receptors or nonselectively stimulate both beta receptor subtypes greatly decreased the ability of beta 1, but not beta 3, receptors to activate adenylyl cyclase. In contrast, the exposure of cells to the beta 3-selective agonist BRL only slightly desensitized beta 1 receptors and did not affect beta 3 receptor activation of adenylyl cyclase. These data indicate that acute agonist exposure desensitizes beta 1, but not beta 3, receptors.     

Phorbol ester-induced modulation of agonist binding to alpha-1 adrenergic receptors in bovine aortic membranes

Effects of the protein kinase C-activating phorbol ester, phorbol dibutyrate (PDBu) on the binding behavior of the alpha-1 adrenergic receptor were determined from radioligand binding assays at 25 and 2 degrees C. Membranes prepared from PDBu-treated bovine aorta exhibited a 16% reduction in [3H]prazosin binding capacity, whereas [3H]prazosin affinity was unchanged. This may reflect a role for protein kinase C-mediated receptor phosphorylation in determining receptor turnover and surface density. After PDBu treatment, the affinity of epinephrine for [3H]prazosin sites was altered in two respects. Control membranes exhibited both high and low affinity epinephrine binding (KDH, 20 nM; KDL, 1086 nM) whereas, PDBu-treated membranes exhibited only a single class of low affinity sites (KDL, 655 nM). The inclusion of 5'-guanylylimidodiphosphate caused the loss of high affinity sites in control membranes but had no effect on PDBu-treated membranes (KDL, 681 nM). Thus, protein kinase C blocks the ability of the agonist-receptor complex to couple to a GTP binding regulatory protein. In binding studies conducted at 2 degrees C epinephrine also bound to high (KDH, 34 nM) and low affinity (KDL, 1920 nM) sites although the percentage of high affinity sites was higher (percentage of RH, 80) than at 25 degrees C (percentage of RH, 19). PDBu-treated membranes also exhibited two agonist affinity states in 2 degrees C studies although affinity was slightly reduced (KDH, 74 nM; KDL, 2405 nM). 5'-Guanylylimidodiphosphate was without effect at 2 degrees C. These results indicate that a high affinity agonist binding state can still be achieved after PDBu treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-1 adrenergic receptors in renal medullary collecting duct cells.

The stimulation of alpha-1 adrenergic receptors in the mammalian nephron increases sodium reabsorption. In this study, alpha-1 adrenergic receptors in the inner medullary collecting duct (IMCD) cells were examined by radioligand binding technique. The IMCD cells were prepared from the rabbit kidney by incubating the inner medullary slices with collagenase and treating the isolated cells with hypotonic solution to lyse cells other than IMCD cells. The equilibrium binding of [3H]prazosin to IMCD cell homogenate was measured after incubation for 30 min at 25 degrees C in the absence (total binding) and the presence (nonspecific binding) of 100 microM phentolamine. The specific binding (the difference between total and nonspecific binding) of [3H]prazosin was saturable with a Bmax of 30 fmol/mg of protein and Kd of 0.9 nM. The displacement of [3H]prazosin binding to IMCD cells by adrenergic antagonists and agonists displayed the order of potency: beta-4-hydroxyphenyl-ethyl-amino-tetralone greater than phentolamine greater than naphazoline greater than epinephrine greater than yohimbine greater than norepinephrine greater than phenylephrine greater than propranolol. Because IMCD cells in the kidney have a hypertonic environment, the specific binding of [3H] prazosin to IMCD cells was also measured in a buffer that was made hypertonic (1200 mOsmol/kg of water) with NaCl and urea, the major solutes of the renal medulla. The hyperosmolality increased the Kd of [3H]prazosin to 5.2 mM without a change in its Bmax.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor interactions of imidazolines. IX. Cirazoline is an alpha-1 adrenergic agonist and an alpha-2 adrenergic antagonist

The alpha-1 and alpha-2 adrenergic effects of cirazoline were evaluated in guinea-pig aorta and field-stimulated guinea-pig ileum, respectively. Cirazoline was found to be a full agonist at alpha-1 receptors having an ED50, dissociation constant (KA) and relative efficacy similar to that of (-)-norepinephrine. In contrast, cirazoline does not possess agonist activity at presynaptic alpha-2 receptors in the guinea-pig ileum. Thus, whereas norepinephrine and cirazoline both inhibited the twitch response of the field-stimulated ileum, only the response to norepinephrine was blocked by the selective alpha-2 antagonist, yohimbine. The nonadrenergic inhibition of the twitch response observed in the ileum with cirazoline resulted from weak anticholinergic activity (antimuscarinic) at the level of the postsynaptic effector organ and was observed only at high concentrations. At concentrations far below the level required to inhibit the twitch response, cirazoline was found to competitively antagonize the alpha-2-mediated inhibition of the twitch response elicited by norepinephrine. A Schild plot analysis indicated that cirazoline is a potent competitive alpha-2 receptor antagonist characterized by a pA2 value (i.e., -log KB) of 7.56. These results indicate that cirazoline is unique among imidazolines in that it is a potent alpha-1 adrenergic receptor agonist and an even more potent alpha-2 receptor antagonist. This unusual combination of activities could make cirazoline a particularly effective vasoconstricting agent.     

Selective regulation of beta-1 and beta-2 adrenergic receptors by atypical agonists

The interactions of the atypical agonists pindolol and celiprolol with beta adrenergic receptors were compared with those of the full agonist, isoproterenol. Studies were carried out using intact cells as well as membranes prepared from C6 glioma cells. Computer-assisted analysis of dose-response curves resulting from the inhibition of the binding of [125I]iodopindolol by the beta-1 and beta-2 selective compounds ICI 89,406 and ICI 118,551 revealed that approximately one-third of the beta adrenergic receptors on these cells were beta-1 receptors. Addition of GTP to the binding assay simplified the dose-response curve for inhibition of the binding of [125I]iodopindolol by isoproterenol and diminished the potency of the agonist. GTP had no effect on the binding of pindolol or celiprolol, suggesting that these drugs do not induce the formation of a ternary complex with the receptor and the guanine nucleotide-binding protein for stimulation of adenylate cyclase activity. When added to the growth medium of intact C6 cells, isoproterenol induced a 40-fold increase in cyclic AMP accumulation. Pindolol and celiprolol, however, caused no elevation of enzyme activity. Addition of isoproterenol to the growth medium of intact cells resulted in an 80% decrease in the density of both beta-1 and beta-2 adrenergic receptors within 8 hr. Growing cells in the presence of pindolol or celiprolol induced a 50% decrease in the density of beta-2 receptors, which was inhibited by beta adrenergic antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective regulation of beta-2 adrenergic receptors by the chronic administration of the lipophilic beta adrenergic receptor agonist clenbuterol: an autoradiographic study

Many antidepressant drugs, when administered chronically to rats, have been shown to produce decreases in the density of beta adrenergic receptors in the central nervous system. The centrally active beta adrenergic receptor agonist clenbuterol is currently being evaluated clinically as an antidepressant. The chronic administration of this drug to rats resulted in a large decrease in the density of beta adrenergic receptors in some areas of the rat brain but not in others. Thus, autoradiographic studies revealed that the total density of beta adrenergic receptors in the molecular layer of the cerebellum, but not in layers 1 to 3 or layer 4 of the cerebral cortex, was decreased. To examine whether this regional selectivity occurred because of differences in plasticity of cerebellum and cortex or because cerebellum contains mainly beta-2 adrenergic receptors and cortex contains mainly beta-1 adrenergic receptors, separate analyses of the subtypes of beta adrenergic receptors were performed in each area. These experiments indicated that the decrease in receptor density was entirely specific for beta-2 adrenergic receptors, whereas the density of beta-1 receptors was unchanged. Thus, even in layers 1 to 3 and layer 4 of the cerebral cortex, beta-2 receptor density was decreased, with no change in beta-1 receptor density. Using the autoradiographic assay for ligand binding, it was shown that clenbuterol has equal affinity for beta-1 and beta-2 adrenergic receptors, indicating that the selective effect of this drug was not due to a selective affinity for beta-2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of pindolol and propranolol on beta adrenergic receptors on human lymphocytes

The cardiovascular supersensitivity observed after discontinuation of administration of beta adrenergic receptor antagonists may be explained by an increase in the density of beta adrenergic receptors. Thus, a change in the density of receptors has been observed in human lymphocytes after administration of propranolol (Aarons et al., 1980). The effects of pindolol, a beta adrenergic receptor antagonist with intrinsic sympathomimetic activity, were compared with those of propranolol or placebo. Pindolol (10 mg q.i.d.), propranolol (40 mg q.i.d.) or placebo were administered to 12 subjects for 8 days. The density of beta adrenergic receptors was determined by Scatchard analysis of the specific binding of [125I]iodopindolol on membranes prepared from human lymphocytes. Administration of pindolol resulted in a 30 to 50% decrease in the density of beta adrenergic receptors. This decrease was apparent within 1 day of beginning pindolol administration and it persisted for at least 8 days after discontinuation of drug administration. The reversibility of the decrease in receptors observed after pindolol administration was studied in 27 subjects given propranolol, pindolol or placebo for 4 days in a double-blind cross-over trial. Propranolol consistently induced a small increase in the density of beta adrenergic receptors. The density of receptors returned to predrug values within 2 days after discontinuation of propranolol administration. Pindolol induced a 30 to 50% decrease in the density of receptors which, as observed previously, persisted for at least 10 days after discontinuation of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential modulation of protein kinase C by bryostatin 1 and phorbol ester

Bryostatin 1, a macrocyclic lactone, activated protein kinase C purified from mouse brain in a dose-dependent fashion to the same degree as phorbol 12-myristate 13-acetate (PMA). There was no significant difference in calcium and phosphatidylserine requirements for activation of protein kinase C between bryostatin 1 and PMA. We also found no significant difference in the inhibitory effect between staurosporine and H-7 known to be potent inhibitors of protein kinase C. These data suggest that bryostatin 1 and PMA activate protein kinase C in a similar way. We found, however, that negative modulation of protein kinase C with bryostatin 1 was weaker than that with PMA. The reason of this difference was unclear. It may possibly suggest that there is some difference in configuration of protein kinase C after binding between these activators.     

5-Hydroxytryptamine2 receptors coupled to phospholipase C in rat aorta: modulation of phosphoinositide turnover by phorbol ester

In rat aorta, 5-hydroxytryptamine (5-HT) stimulated phosphoinositide (PI) turnover and contraction (EC50 = 10 +/- 3 microM); these two responses were highly correlated (r = 0.95; P less than .01). We have characterized the inhibitory potency of a variety of 5-HT-antagonists against the stimulation of PI turnover elicited by 5-HT. Classic 5-HT2 antagonists mianserin, ketanserin, metergoline and pizotifen were found to inhibit this response in the low nanomolar range; amitryptiline and haloperidol were 10- to 20-fold less potent. The alpha-1 receptor antagonist, prazosin, was inactive in micromolar concentrations. The potency of the 5-HT2 antagonists was correlated with their ability to displace [3H] ketanserin binding from rat frontal cortex membranes (r = 0.90; P less than .05). The tumor promoter phorbol dibutyrate was found to inhibit 5-HT-stimulated PI turnover at low nanomolar concentrations whereas the biologically inactive substance 4-alpha-phorbol was ineffective. Pretreatment of rat aorta with phorbol dibutyrate at concentrations that inhibited 5-HT-induced PI turnover also attenuated the aortic contraction induced by 5-HT in the presence of a calcium channel blocker nitrendipine. Our results suggest that phorbol esters may desensitize 5-HT2-receptor-mediated PI turnover and contraction of rat aorta, possibly via an activation of protein kinase C.     

Prostaglandin synthesis elicited by adrenergic stimuli in rabbit aorta is mediated via alpha-1 and alpha-2 adrenergic receptors

The purpose of this study was to characterize the type of adrenergic receptor(s) involved in both prostaglandin (PG) synthesis and the contractile response elicited by adrenergic receptor agonists in the rabbit aorta. The synthesis of prostacyclin as measured by the production of 6-keto-PGF1 alpha was assessed in vitro after exposing the aortic rings to different adrenergic agonists. Norepinephrine (NE), selective alpha 1 adrenergic receptor agonists methoxamine (MET), phenylephrine (PHE) and cirazoline (CIR) and the alpha 2 adrenergic receptor agonists UK 14304 (UK) and xylazine (XYL), but not the beta adrenergic receptor agonist isoproterenol (ISP), enhanced 6-keto-PGF1 alpha synthesis in a concentration-dependent manner with following order of potency: NE greater than UK 14304 greater than XYL greater than PHE greater than MET greater than CIR. The NE-induced increased in 6-keto-PGF1 alpha synthesis was attenuated by the alpha 1 adrenergic receptor antagonists prazosin (PZ) and corynanthine (COR) and by the alpha 2 adrenergic receptor antagonists rauwolscine (RW) and yohimbine (YOH). MET-induced 6-keto-PGF1 alpha synthesis was reduced by PZ and COR but not by RW. UK-induced 6-keto-PGF1 alpha synthesis was reduced by RW, YOH, and PZ, which also acts as alpha-2B receptor antagonist, but not by COR. In rabbit aortic rings, adrenergic agonists produced contraction with the following order of potency: NE greater than PHE greater than MET greater than CIR greater than UK greater than XYL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic properties of agonist interactions with the beta adrenergic receptor-coupled adenylate cyclase system. I. High- and low-affinity states of agonist binding to membrane-bound beta adrenergic receptors

The properties associated with ligand interactions with membrane-bound beta adrenergic receptors prepared from L6 myoblasts were examined at five temperatures between 10 degrees and 30 degrees C. The interactions of antagonists with membrane-bound receptors were insensitive to temperature, whereas the interactions of agonists were temperature-dependent. The affinity constants for the low-affinity binding states of agonists (KL) decreased slightly with decreasing assay temperature. The small temperature-dependent changes in KL values were similar to the changes in Kd values observed in studies of the binding of agonists in the presence of GTP. The high-affinity dissociation constants (KH) for binding of full agonists to membrane-bound receptors in the absence of GTP were approximately 50-fold lower at 10 degrees than at 30 degrees C. The KH values for partial agonists also decreased with decreasing temperature, but the changes were smaller in magnitude. Thermodynamically, the binding of antagonists was primarily entropy-driven, whereas the binding of agonists was enthalpy-driven. The energetics of the low-affinity component of agonist binding to membrane-bound receptors were similar to the energetics for binding of agonists to membrane-bound receptors in the presence of GTP. Under these conditions, standard enthalpy change (delta H degree) and standard entropy change (delta S degree) values for binding of agonists were more negative than the corresponding values for binding of antagonists, possibly reflecting a conformational change in the receptor or an increased ordering of the lipids surrounding the receptor. The interaction of the receptor with the guanine nucleotide-binding protein to form the high-affinity component of agonist binding was thermodynamically described by larger negative changes in enthalpy and entropy than the values for formation of the low-affinity component of agonist binding. There was a correlation between the efficacies of ligands in activating adenylate cyclase and the delta H degree and delta S degree values for high-affinity binding of agonists. Thus, the extent or nature of the interaction between the guanine nucleotide-binding protein and the receptor may determine the efficacies of ligands.     

Prostaglandin synthesis elicited by adrenergic stimuli is mediated via alpha-2C and alpha-1A adrenergic receptors in cultured smooth muscle cells of rabbit aorta.

This study was performed to characterize the subtype of adrenergic receptor(s) (AR) involved in prostacyclin synthesis [measured as 6-keto-prostaglandin (PG)F1 alpha] elicited by AR agonists in cultured vascular smooth muscle cells of rabbit aorta. Both alpha-1 and alpha-2 AR agonists enhanced 6-keto-PGF1 alpha synthesis in a dose-dependent manner with the following order of potency: norepinephrine greater than BHT 933 greater than UK 14304 greater than xylazine greater than phenylephrine greater than or equal to methoxamine greater than cirazoline. Isoproterenol and oxymetazoline did not alter 6-keto-PGF1 alpha synthesis. Methoxamine-induced 6-keto-PGF1 alpha synthesis was not reduced by the alpha-2 AR antagonist rauwolscine. The affinities of AR antagonists (PA2 value) in inhibiting methoxamine-induced 6-keto-PGF1 alpha synthesis were of the following order: prazosin greater than WB 4101 greater than corynanthine greater than yohimbine. Administration of WB 4101 and the irreversible alpha-1B AR antagonist chloroethylclonidine reduced norepinephrine (in the presence of rauwolscine, 10(-8) M)- or methoxamine-induced 6-keto-PGF1 alpha synthesis; WB 4101 was more potent than chloroethylclonidine. UK 14304-induced 6-keto-PGF1 alpha synthesis was not reduced by chloroethylclonidine or BRL 44408, a selective alpha-2A AR antagonist, but it was inhibited by other alpha AR antagonists. The affinities of AR antagonists (PA2 values) in inhibiting UK 14304-induced 6-keto-PGF1 alpha synthesis were of the following order: rauwolscine greater than yohimbine greater than BAM 1303 greater than BRL 41992 greater than WB 4101 greater than ARC 239 greater than or equal to prazosin greater than SKF 104078 greater than or equal to corynanthine. The order of affinity of alpha-2 AR antagonists in inhibiting UK 14304-induced 6-keto-PGF1 alpha synthesis in vascular smooth muscle cells was similar to that derived from radioligand binding studies in opossum kidney cell line receptors classified as alpha-2C receptors. These data suggest that 6-keto-PGF1 alpha synthesis elicited by adrenergic stimuli in cultured vascular smooth muscle cells of rabbit aorta is mediated primarily via alpha-2C and to a lesser extent alpha-1A receptors.     

Defining the human targets of phorbol ester and diacylglycerol

Phorbol esters (PEs) and their derivatives are potent tumor-promoting agents. The best known receptors for these substances are the novel and classical isotypes of protein kinase C (PKC), which bind PE and the physiological second messenger diacylglycerol (DAG) by cysteine-rich domains, the C1 domains. However, PKC is not the sole receptor of PE, a concept that has been largely ignored in the past. PE (in addition to DAG) also targets C1-containing receptors unrelated to PKC. In order to get a better insight into DAG/PE-mediated signaling and the pathways involved, it is necessary to first determine all ligand-interacting proteins. Employing various sources of data, 66 different C1-containing human proteins are presented and predictions of their DAG/PE-binding potential are attempted. Defining the entire set of key mediators for the physiological DAG responses and for PE-induced tumorigenesis may aid our understanding of signal integration and can also help to design new strategies for therapeutic cancer intervention.     

Desensitization of adenylate cyclase and down regulation of beta adrenergic receptors after in vivo administration of beta agonist

In vitro incubation of cells with catecholamines leads to both down regulation of beta adrenergic receptor number and desensitization of agonist-stimulated adenylate cyclase activity. These same parameters, down regulation of beta adrenergic receptor number and desensitization of adenylate cyclase activity were assessed in rat lung membranes after in vivo administration of metaproterenol, a beta-2 selective agonist. In vivo treatment with metaproterenol leads to: 1) reduced beta adrenergic receptor number; 2) reduced isoproterenol-stimulated adenylate cyclase activity; 3) unaffected NaF or 5'-guanylylimidodiphosphate-stimulated adenylate cyclase activity; and 4) reduced affinity of the receptor for isoproterenol similar to the affinity observed in the presence of 5'-guanylylimidodiphosphate. The date suggest that in vivo metaproterenol administration results in an uncoupled receptor-adenylate cyclase complex. The effects of in vivo administration of the glucocorticoid, methylprednisolone, to metaproterenol-pretreated animals were also assessed. Glucocorticoid treatment was associated with 1) increased beta adrenergic receptor number in rats in which the receptors have been down regulated, 2) increased isoproterenol responsiveness in agonist-desensitized rats and 3) no effect on agonist affinity in desensitized animals. These data suggest that the restoration of agonist responsiveness by glucocorticoids in the catecholamine refractive state is not simply a reversal of receptor down regulation or adenylate cyclase desensitization.     

Fenoldopam is a partial agonist at dopamine-1 (DA1) receptors in LLC-PK1 cells.

Fenoldopam [6-chloro-7,8-dihydroxy-1-(4'-hydroxyphenyl)-2, 3,4,5-tetrahydro-(1H)-3-benzazepine] is a selective dopamine-1 (DA1) agonist with natriuretic/diuretic properties. A component of the natriuretic response to fenoldopam may involve direct DA1 receptor-mediated effects on proximal tubule sodium reabsorption, possibly through stimulation of adenylyl cyclase. Here, we compared the effects of fenoldopam and DA in stimulating cyclic AMP (cAMP) synthesis in LLC-PK1 cells, a renal epithelial cell line that has proximal tubule-like properties and expresses a DA1 receptor linked to stimulation of adenylyl cyclase. Fenoldopam stimulated cAMP accumulation in LLC-PK1 cells in a dose-dependent manner, an effect which could be blocked by the DA1-selective antagonist Sch 23390. Although fenoldopam was more potent than DA (EC50 55.5 +/- 7.75 nM vs. 1.65 +/- 0.64 microM) in stimulating cAMP accumulation in LLC-PK1 cells, the maximum stimulation obtained by fenoldopam was only 37% of the maximum stimulation obtained by DA(Emax 13.0 +/- 2.95 pmol/mg of protein vs. 35.6 +/- 10.19 pmol/mg of protein). Simultaneous incubation of DA and fenoldopam resulted in lower cAMP levels than with DA alone. Incubation of DA with increasing concentrations of fenoldopam produced parallel rightward shifts in the DA dose-response curves. Schild analysis further indicated that fenoldopam acted as a competitive antagonist in the presence of DA, with a pA2 value of 7.38 and a slope of unity. These results indicate that fenoldopam is a partial agonist with low efficacy at DA1 receptors linked to cAMP generation in the LLC-PK1 cells.     

(R)-nipecotic acid ethyl ester: a direct-acting cholinergic agonist that displays greater efficacy at M2 than at M1 muscarinic receptors

Previous reports have suggested that the ethyl ester of (R)-nipecotic acid ethyl ester [(R)-NAEE] displays cholinomimetic properties in vivo. The present study was undertaken to characterize more fully this action by examining the effects of (R)-NAEE in a number of pharmacological and biochemical tests of cholinergic action. (R)-NAEE was found to produce negative inotropic and chronotropic effects on the guinea pig atria (pD2 = 5.91 and 5.62, respectively), and was capable of stimulating contractions in the guinea pig ileum (pD2 = 5.95) and rat jejunum (pD2 = 5.40) at concentrations similar to bethanechol. Both the cardiac and intestinal effects of (R)-NAEE were reversed by atropine. Moreover, (R)-NAEE competed with N-[3H]methylscopolamine and [3H]pirenzepine for muscarinic binding sites in a variety of tissues. Like carbachol, (R)-NAEE inhibited GTP-stimulated adenylate cyclase in rat striatal membranes (EC50 = 52 microM), whereas, unlike carbachol, (R)-NAEE was unable to stimulate inositol phosphate accumulation in rat cerebral cortical slices, behaving as an antagonist in this latter system (pA2 = 5.0). The results indicate that (R)-NAEE interacts directly with cholinergic muscarinic receptors, being an agonist for the M2 subtype and an antagonist at M1 sites.