SLC-1 receptor mediates effect of melanin-concentrating hormone on feeding behavior in rat: a structure-activity study.

Several studies have shown that melanin-concentrating hormone (MCH) is an orexigenic peptide in rat. In the present study, a structure-activity relationship with MCH analogs was performed in rat, both in vitro and in vivo. On rat recombinant SLC-1 receptor, both cAMP inhibition and [(125)I]S36057 binding were measured. In vivo, these analogs were injected intracerebroventricularly in rats and their effects were evaluated upon food intake. First, data obtained with the rat recombinant receptor were highly correlated with those obtained from its human counterpart. Second, agonist potencies in the cAMP assay were also highly correlated with binding affinities. These peptides could be classified into several groups according to their potency at the SLC-1 receptor (from subnanomolar activity to complete inactivity). Indeed, there was a strong correlation between their effects upon food intake and the results obtained at the rat SLC-1 receptor. The present report describes for the first time the rat SLC-1 receptor pharmacology and clearly establishes the relevance of the SLC-1 receptor in feeding behavior.

Structure-activity relationship studies of melanin-concentrating hormone (MCH)-related peptide ligands at SLC-1, the human MCH receptor.

Melanin-concentrating hormone (MCH) is a cyclic nonadecapeptide involved in the regulation of feeding behavior, which acts through a G protein-coupled receptor (SLC-1) inhibiting adenylcyclase activity. In this study, 57 analogues of MCH were investigated on the recently cloned human MCH receptor stably expressed in HEK293 cells, on both the inhibition of forskolin-stimulated cAMP production and guanosine-5'-O-(3-[(35)S]thiotriphosphate ([(35)S]- GTPgammaS) binding. The dodecapeptide MCH-(6-17) (MCH ring between Cys(7) and Cys(16), with a single extra amino acid at the N terminus (Arg(6)) and at the C terminus (Trp(17))) was found to be the minimal sequence required for a full and potent agonistic response on cAMP formation and [(35)S]- GTPgammaS binding. We Ala-scanned this dodecapeptide and found that only 3 of 8 amino acids of the ring, namely Met(8), Arg(11), and Tyr(13), were essential to elicit full and potent responses in both tests. Deletions inside the ring led either to inactivity or to poor antagonists with potencies in the micromolar range. Cys(7) and Cys(16) were substituted by Asp and Lys or one of their analogues, in an attempt to replace the disulfide bridge by an amide bond. However, those modifications were deleterious for agonistic activity. In [(35)S]- GTPgammaS binding, these compounds behaved as weak antagonists (K(B) 1-4 microm). Finally, substitution in MCH-(6-17) of 6 out of 12 amino acids by non-natural residues and concomitant replacement of the disulfide bond by an amide bond led to three compounds with potent antagonistic properties (K(B) = 0.1-0.2 microm). Exploitation of these structure-activity relationships should open the way to the design of short and stable MCH peptide antagonists.

Removal of endogenous leptin from the circulation by the kidney.

OBJECTIVE: This study was performed to test the hypothesis that the kidneys play a primary role in the clearance of endogenous leptin from the circulation of obese rats. DESIGN: Zucker (fa/fa) obese rats were anaesthetized and subjected to various surgical manipulations of the kidneys. One hour after surgery arterial blood samples were taken at 1 h intervals for times upto 8 h. Plasma leptin concentrations were determined by radioimmunoassay. RESULTS: Bilateral nephrectomy induced a rapid increase in plasma leptin concentrations above control values. In contrast, continuous intravenous re-injection of voided urine did not increase circulating leptin concentrations, indicating that leptin is not present in the urine in large quantities. This conclusion was confirmed by the very low levels of detectable leptin in urine. Leptin is not metabolized across the renal circulation and is extracted intact by the kidney. Simultaneous measurement of renal plasma flow established renal leptin extraction at approximately 59 ng/ min for both kidneys. Following intravenous infusion of leptin, renal clearance and whole body clearance were equal. This finding indicates that the kidneys alone are responsible for the systemic elimination of leptin in Zucker rats. Seven hours after bilateral ureteral ligation, a procedure which lowers glomerular filtration, plasma leptin concentrations were elevated. The renal extraction of leptin did not change over a wide range of plasma leptin concentrations suggesting that renal leptin extraction is a high capacity, non-saturable process most probably glomerular filtration. CONCLUSION: Endogenous leptin is rapidly cleared from the circulation by the kidney by glomerular filtration followed by metabolic degradation in the renal tubules.

Mechanism of leptin removal from the circulation by the kidney.

This study was performed to test the hypothesis that the kidneys play a primary role in the clearance of endogenous leptin from the circulation. Lean male Sprague-Dawley rats were anesthetized and subjected to various surgical manipulations of the kidneys. Sixty minutes after surgery arterial blood samples were taken at 1-h intervals for up to 8 h. Plasma leptin levels were determined by radioimmunoassay. Bilateral nephrectomy induced a rapid increase in plasma leptin concentrations above control values, indicating that the kidneys are important for the elimination of leptin from the circulation. Leptin was not metabolized across the renal circulation and was extracted intact by the kidney. Simultaneous measurement of renal plasma flow established renal leptin extraction at approximately 6.5 ng/min for both kidneys. Compared with the quantities extracted from the plasma, leptin was only present in the urine in small quantities, indicating extensive metabolic degradation in the renal tubules. High plasma leptin levels were not maintained after binephrectomy indicating that pathways other than the kidneys are also responsible for leptin clearance. Seven hours after bilateral ureteral ligation, a procedure which lowers glomerular filtration, plasma leptin levels were slightly elevated. The renal extraction of leptin did not change over a wide range of plasma leptin concentrations suggesting that renal leptin extraction is a high capacity, non-saturable process most probably glomerular filtration. Endogenous leptin is rapidly cleared from the circulation by glomerular filtration followed by metabolic degradation in the renal tubules.

Angiotensin AT2 receptor mediated inhibition of particulate guanylate cyclase: a link with protein tyrosine phosphatase stimulation?

Ever since the identification of two distinct Ang II receptor subtypes, the function of the AT2 receptor has been a subject of debate. As opposed to the AT1 subtype, this receptor does not interact with G-proteins in most cell lines and tissues. We show here that, in intact PC12W cells which express only AT2 receptors, Ang II significantly decreases basal and atrial natriuretic peptide (ANP)-stimulated cGMP concentration. This effect is mimicked by the AT2 selective agonist CGP 42112, and is not prevented by the AT1 selective antagonist losartan, indicating that this is an AT2 receptor mediated response. The lack of effect of the phosphodiesterase (PDE) inhibitor IBMX shows that this mechanism does not involve PDE stimulation. This is confirmed by the finding that neither Ang II or CGP 42112 affect the Ca++/calmodulin dependent cGMP PDE activity. Furthermore Ang II and CGP 42112 have no effect on nitroprusside-stimulated cGMP levels in these cells, thus ruling out interactions between the AT2 receptor and soluble guanylate cyclase. These data indicate that the AT2 receptor mediated decrease of cGMP is due to the selective inhibition of particulate guanylate cyclase (pGC) activity. In an accompanying paper we report that interaction of Ang II with the AT2 receptor in the same cells results in the stimulation of phosphotyrosine phosphatase (PTPase) activity. Interestingly, the PTPase inhibitors sodium orthovanadate and phenylarsine oxyde, but not the Ser/Thr phosphatase inhibitor okadiac acid, inhibitthe Ang II and CGP 42112 induced decreases in cellular cGMP concentration. These findings suggest that stimulation of PTPase activity may be involved in the regulation of pGC activity via AT2 receptors.

Agonistic and antagonistic properties of angiotensin analogs at the AT2 receptor in PC12W cells.

Despite some recent reports describing the effects of AT2 receptor selective ligands in vitro and in vivo, the physiological function of this receptor is still a matter of debate. This problem stems amongst others from the difficulty in interpreting results from in vivo experiments with drugs of which it is not known whether they act as agonists or antagonists. We reported earlier that angiotensin II inhibits basal and atrial natriuretic peptide stimulated particulate guanylate cyclase activity through AT2 receptors in PC12W cells. We have used this parameter in intact PC12W cells in order to determine the pharmacological properties of different widely used angiotensin receptor ligands. We found CGP 42112 to behave as a full agonist in this system, whereas PD 123319 and Sar Ile angiotensin II act as antagonists. As expected, the AT1 antagonist losartan did not affect this response.

Responses to renin inhibition in conscious primates.

A method for the measurement of renal clearances was adapted in a novel manner to the conscious marmoset. Twenty-four hours before an experiment, animals underwent surgery for placement of both femoral arterial and venous catheters. A catheter was also implanted into the urinary bladder through the abdominal wall. The urinary catheter consisted of two tubes. One tube was connected to a pump, which removed the urine, while the other tube remained open to prevent the formation of a vacuum inside the bladder. Using this technique, we kept renal clearances stable for at least 3 h. All catheters were removed the day after the clearance experiment, and the bladder was reconstructed using microsurgical techniques. The bladder operation did not appear to produce lasting anatomical or functional changes, since the animals were able to void spontaneously and, after 3 mo, the bladder had regained its normal size and shape. Reimplantation of both the vessel and bladder catheters at this time allowed for an additional study of renal clearances within the same animals. Administration of the renin inhibitor CGP 29287 (1 mg/kg) to furosemide-pretreated conscious marmosets lowered blood pressure and increased renal blood flow. Glomerular filtration rate remained unchanged by CGP 29287, leading to a fall in the effective filtration fraction. Urinary volume and urinary sodium excretion also were unchanged by the renin inhibitor. We describe a novel method for the study of renal clearances in a small conscious primate and suggest that the renin-angiotensin system plays an important role in the control of renal function in the sodium-depleted marmoset.

Renal actions of a new adenosine agonist, CGS 21680A selective for the A2 receptor.

This study compares the renal actions of the A2 selective adenosine agonist, CGS 21680A, with the A1 selective adenosine agonist, N6-cyclopentyladenosine (CPA), and the nonselective agonist, 5'-N-ethylcarboxamide adenosine (NECA), in the anesthetized dog. Initial receptor binding studies in dog brain demonstrated that CPA and CGS 21680A were selective for the A1 and A2 adenosine receptor, respectively, whereas NECA displayed slightly greater affinity for A1 than A2 adenosine receptors in the canine brain. Intravenous infusion of CGS 21680A (0.25 and 2.5 micrograms/kg/min) decreased blood pressure (BP) and increased heart rate (HR). CGS 21680A transiently increased renal blood flow (RBF) and either did not change or, at the highest dose infused, decreased glomerular filtration rate (GFR). Both urine volume (UV) and urinary sodium excretion (UNaV) also were decreased by CGS 21680A. At the lowest infusion rate (0.025 micrograms/kg/min) CGS 21680A produced a slowly developing increase in RBF, no change in GFR and a significant decrease in sodium excretion. Intravenous infusion of CPA (15 micrograms/kg/min) lowered BP and HR RBF and GFR. UNaV, UV and renin release also were inhibited by CPA. At a lower infusion rate (2.5 micrograms/kg/min), CPA markedly inhibited UNaV in the absence of a significant change in either BP or renal hemodynamic parameters. Infusion of NECA (0.01 and 0.1 micrograms/kg/min) lowered BP but did not change HR. Furthermore, RBF was increased by NECA, whereas UV and UNaV were inhibited in the absence of a change in GFR. These results may be explained by the relative selectivity of each analog for A1 or A2 adenosine receptors.

Intrarenal actions of the new adenosine agonist CGS 21680A, selective for the A2 receptor.

The purpose of this study was to define the direct intrarenal actions of the new adenosine agonist CGS 21680A (hydrochloride "A" salt of (2-[p-2-carboxyethyl)phenethylamino]5'-N-ethyl-carboxamido adenosine), which is selective for the A2 receptor. CGS 21680A was infused into the left renal artery, while simultaneously measuring blood pressure (BP) and parameters of renal function from both the left and right kidneys. At doses higher than 0.05 micrograms/kg/min, CGS 21680A appeared to leak from the circulation of the infused kidney in pharmacologically significant quantities, inasmuch as BP fell and renal blood flow was increased and renal vascular resistance decreased in the noninfused contralateral kidney. At doses of 0.025 to 0.05 micrograms/kg/min, CGS 21680A appeared localized to the renal circulation, because neither BP nor any measured parameter of contralateral renal function was altered. Intrarenal infusion of 0.025 to 0.05 micrograms/kg/min of CGS 21680A increased renal blood flow but not glomerular filtration rate leading to a fall in the filtration fraction. Urine volume and urinary sodium excretion were unaffected by selective intrarenal infusion of CGS 21680A. Plasma renin activity and renin secretion rate were also not altered significantly by intrarenal infusion of 0.05 micrograms/kg/min of CGS 21680A. In contrast plasma renin activity increased significantly in response to the intrarenal infusion of 0.075 micrograms/kg/min of CGS 21680A, a dose which leaked from the kidney and lowered BP. The results presented suggest that the new adenosine agonist CGS 21680A exerts a direct intrarenal effect on renal hemodynamics, but does not affect urine volume or sodium excretion or directly influence renin release.