Studies determined the effects of chronic changes in sodium diet on the expression, regulation, and function of different angiotensin II (ANG II) receptor subtypes in renal resistance vessels. Rats were fed low-or high-sodium diets for 3 wk before study. Receptor function was assessed in vivo by measuring transient renal blood flow responses to bolus injections of ANG II (2 ng) into the renal artery. ANG II produced less pronounced renal vasoconstriction in rats fed a low-compared with high-sodium diet (16% vs. 56% decrease in renal blood flow, P Ͻ 0.001). After acute blockade of ANG II formation by iv enalaprilat injection in sodiumrestricted animals, ANG II produced a 40% decrease in renal blood flow, a level between untreated dietary groups and less than high salt diet. Intrarenal administration of angiotensin II receptor type 1 (AT 1 ) receptor antagonists losartan or EXP-3174 simultaneously with ANG II caused dosedependent inhibition of ANG II responses. Based on maximum vasoconstriction normalized to 100% ANG II effect in each group, AT 1 receptor antagonists produced the same degree of blockade in all groups, with an apparent maximum of 80-90%. In contrast, similar doses of the angiotensin II receptor type 2 (AT 2 ) receptor ligand CGP-42112 had only a weak inhibitory effect. In vitro equilibrium-saturation 125 I-ANG II binding studies on freshly isolated afferent arterioles indicated that ANG II receptor density was lower in the low-vs. high-sodium animals (157 vs. 298 fmol/mg, P Ͻ 0.04); affinity was similar (0.65 nM). Losartan and EXP-3174 displaced up to 80-90% of the ANG II binding; fractional displacement was similar in both diet groups. In contrast, the AT 2 receptor analogues PD-123319 and CGP-42112 at concentrations Ͻ 10 Ϫ 6 M had no effect on ANG II binding. RT-PCR assays revealed the expression of both angiotensin II receptor type 1A (AT 1A ) and angiotensin II receptor type 1B (AT 1B ) subtypes in freshly isolated afferent arterioles, while there was very little AT 2 receptor expression. Total AT 1 receptor mRNA expression was suppressed by low sodium intake to 66% of control levels, whereas it was increased to 132% of control by high-sodium diet, as indicated by ribonuclease protection assay. Receptor regulation was associated with parallel changes in AT 1A and AT 1B expression; the AT 1A /AT 1B ratio was stable at 3.7. We conclude that AT 1 receptors are the predominant ANG II receptor type in renal resistance vessels of 7-wk-old rats. Chronic changes in sodium intake caused parallel regulation of expression and amount of receptor protein of the two AT 1 receptor genes that modulate receptor function and altered reactivity of renal vessels to ANG II. ( J. Clin. Invest.

Tirzepatide (LY3298176), a dual GIP and GLP-1 receptor agonist, delivered superior glycemic control and weight loss compared to GLP-1 receptor (GLP-1R) agonism in patients with type diabetes. However, the mechanism by which tirzepatide improves efficacy and how GIP receptor (GIPR) agonism contributes is not fully understood. Here, we show that tirzepatide is an effective insulin sensitizer, improving insulin sensitivity in obese mice to a greater extent than GLP-1R agonism. To determine if GIPR agonism contributes, we compared the effect of tirzepatide in obese wild-type and Glp-1r null mice.In the absence of GLP-1R-induced weight loss, tirzepatide improved insulin sensitivity by enhancing glucose disposal in white adipose tissue (WAT). In support, a long-acting GIPR agonist (LAGIPRA) was found to enhance insulin sensitivity by augmenting glucose disposal in WAT. Interestingly, the effect of tirzepatide and LAGIPRA on insulin sensitivity was associated with reduced branched-chain amino (BCAAs) and keto-acids in the circulation. Insulin sensitization was associated with upregulation of genes associated with the catabolism of glucose, lipid and BCAAs in brown adipose tissue. Together, our studies show that tirzepatide improved insulin sensitivity in a weight-dependent andindependent manner. These results highlight how GIPR agonism contributes to the therapeutic profile of dual receptor agonism, offering mechanistic insights into the clinical efficacy of tirzepatide.

Activation of TGR5 via bile acids or bile acid analogs leads to the release of glucagon-like peptide-1 (GLP-1) from intestine, increases energy expenditure in brown adipose tissue, and increases gallbladder filling with bile. Here, we present compound 18, a non-bile acid agonist of TGR5 that demonstrates robust GLP-1 secretion in a mouse enteroendocrine cell line yet weak GLP-1 secretion in a human enteroendocrine cell line. Acute administration of compound 18 to mice increased GLP-1 and peptide YY (PYY) secretion, leading to a lowering of the glucose excursion in an oral glucose tolerance test (OGTT), while chronic administration led to weight loss. In addition, compound 18 showed a dose-dependent increase in gallbladder filling. Lastly, compound 18 failed to show similar pharmacological effects on GLP-1, PYY, and gallbladder filling in Tgr5 knockout mice. Together, these results demonstrate that compound 18 is a mouse-selective TGR5 agonist that induces GLP-1 and PYY secretion, and lowers the glucose excursion in an OGTT, but only at doses that simultaneously induce gallbladder filling. Overall, these data highlight the benefits and potential risks of using TGR5 agonists to treat diabetes and metabolic diseases.

The Rats developing genetic hypertension, such as the OkamotoAoki strain of spontaneously hypertensive rat (SHR), provide opportunities for insight into mechanisms involved in the pathogenesis of essential hypertension in humans. Crosstransplantation studies indicate that the kidneys play a pivotal role in the development of hypertension in genetically hypertensive rats (1-3). Alterations in renal vascular resistance, glomerular filtration rate, renal blood flow, and sodium and water retention have been described in 6-to 8-week-old SHRs compared with age-matched control Wistar-Kyoto rats (WKYs) (4-6). The abnormalities in renal hemodynamics and function become less pronounced as the hypertension advances to an established phase in 12-week-old SHRs (7,8).Genetic cosegregation studies reveal a direct relationship between increased renal vascular resistance and arterial hypertension (9). The mechanism(s) responsible for increased vascular resistance and reactivity have been the subject of intense investigation. Increased renal vascular resistance in adult animals is proportional to the increase in arterial pressure and may represent an appropriate autoregulatory response (7). In contrast, the reduced renal blood flow and glomerular filtration rate in young SHRs with minimally elevated arterial pressure are consistent with the participation of vasoconstrictor factor(s).Although circulating and intrarenal levels of renin are considered normal in SHRs, several lines of evidence support the notion that the renin-angiotensin system exerts a stronger than normal influence on the renal circulation in young SHR(s). Acute and chronic inhibition of angiotensinconverting enzyme prevents the development of hypertension in young SHR(s) (10). In previous studies we observed that renal vascular responses to angiotensin II (Ang-IT) are exaggerated in young SHRs compared with those in WKYs (10-13). This strain difference was not due to differences in the affinity and/or density of the Ang-II receptors found in the renal vasculature but rather was due to identified interactions with other vasoactive substances (12, 13).The increased vasoconstriction could be caused by reduced offsetting activity of a vasodilator system. Several renal vasodilators, such as prostaglandins E2 and I2 and the dopamine (DA1)-agonist fenoldopam, could not buffer the Ang-TIinduced vasoconstriction in the kidneys of 6-to 8-week-old SHRs. The same vasodilator agents were, however, able to almost completely abolish the Ang-II effect in kidneys of age-matched WKYs (13-15). The abnormality seemed specific to activators of the cAMP messenger system. Receptor agonists leading to increased nitric oxide production and activation of the cGMP pathway were equally effective in normotensive and hypertensive strains (13,14).The present study examined the mechanisms responsible for the inability of vasodilator autacoids/paracrine substances to counteract the Ang-1I-induced vasoconstriction in the renal vasculature of SHR(s) that are young and in the developmental ...

This study aimed to characterize the interaction between nitric oxide (NO)- and cAMP-related pathways in the control of renal blood flow. Using the isolated perfused rat kidney model, we determined the effects of inhibition of NO formation by Nomega-nitro-L-arginine methyl ester (L-NAME; 1 mmol/L) and of NO administration by sodium nitroprusside (SNP, 10 micromol/L) on renal vascular resistance under conditions of elevated vascular cAMP levels. cAMP levels were increased either by adenylate cyclase activation via isoproterenol or by inhibition of cAMP phosphodiesterases (PDEs) 1, 3, and 4. We found that L-NAME markedly increased vascular resistance and that this effect was completely reversed by SNP. Both isoproterenol and inhibitors of the cAMP PDEs lowered basal vascular resistance. In the presence of isoproterenol (3 nmol/L) and inhibitors of PDE-1 [8-methoxymethyl-l-methyl-3-(2-methylpropyl)-xanthine; 8-MM-IBMX, 20 micromol/L] and PDE-4 (rolipram, 20 micromol/L), L-NAME again substantially increased vascular resistance, and this effect of L-NAME was completely reversed by SNP. In the presence of the PDE-3 inhibitors milrinone (20 micromol/L) and trequinsin (200 nmol/L), however, both L-NAME and SNP failed to exert any additional effects. Because PDE-3 is a cGMP-inhibited cAMP PDE and because the vasodilatory effect of SNP was abrogated by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) (20 micromol/L), our findings are compatible with the idea that an action of NO on PDE-3 could account for the vasodilatory properties of NO on the renal vasculature. Moreover, our findings suggest that PDE-3 activity is an important determinant of renal vascular resistance.

The kidney plays a central role in long-term regulation of arterial blood pressure and salt and water homeostasis. This is achieved in part by the local actions of paracrine and autacoid mediators such as the arachidonic acid-prostanoid system. The present study tested the role of specific PGE(2) E-prostanoid (EP) receptors in the regulation of renal hemodynamics and vascular reactivity to PGE(2). Specifically, we determined the extent to which the EP(2) and EP(3) receptor subtypes mediate the actions of PGE(2) on renal vascular tone. Renal blood flow (RBF) was measured by ultrasonic flowmetry, whereas vasoactive agents were injected directly into the renal artery of male mice. Studies were performed on two independent mouse lines lacking either EP(2) or EP(3) (-/-) receptors and the results were compared with wild-type controls (+/+). Our results do not support a unique role of the EP(2) receptor in regulating overall renal hemodynamics. Baseline renal hemodynamics in EP(2)-/- mice [RBF EP(2)-/-: 5.3 +/- 0.8 ml. min(-1). 100 g kidney wt(-1); renal vascular resistance (RVR) 19.7 +/- 3.6 mmHg. ml(-1). min. g kidney wt] did not differ statistically from control mice (RBF +/+: 4.0 +/- 0.5 ml. min(-1). 100 g kidney wt(-1); RVR +/+: 25.4 +/- 4.9 mmHg. ml(-1). min. 100 g kidney wt(-1)). This was also the case for the peak RBF increase after local PGE(2) (500 ng) injection into the renal artery (EP(2)-/-: 116 +/- 4 vs. +/+: 112 +/- 2% baseline RBF). In contrast, we found that the absence of EP(3) receptors in EP(3)-/- mice caused a significant increase (43%) in basal RBF (7.9 +/- 0.8 ml. min(-1). g kidney wt(-1), P < 0.05 vs. +/+) and a significant decrease (41%) in resting RVR (11.6 +/- 1.4 mmHg. ml(-1). min. g kidney wt(-1), P < 0.05 vs. +/+). Local administration of 500 ng of PGE(2) into the renal artery caused more pronounced renal vasodilation in EP(3)-/- mice (128 +/- 2% of basal RBF, P < 0.05 vs. +/+). We conclude that EP(3 )receptors mediate vasoconstriction in the kidney of male mice and its actions are tonically active in the basal state. Furthermore, EP(3) receptors are capable of buffering PGE(2)-mediated renal vasodilation.

The purpose of this study was to determine the relative importance of calcium signaling pathways in angiotensin II (ANG II)-induced renal vasoconstriction in vivo. Nifedipine was used to antagonize dihydropyridine-sensitive voltage-dependent calcium channels; BAY K 8644 was employed to activate these calcium channels. Intracellular calcium mobilization was evaluated using TMB-8 or heparin to inhibit calcium release from sarcoplasmic reticulum. Renal blood flow was measured by electromagnetic flowmetry in anesthetized euvolemic Wistar-Kyoto rats. The animals were pretreated with indomethacin to avoid interactions with prostaglandins. ANG II (2 ng) or BAY K 8644 (1 microgram) was injected into the renal artery to produce a transient 30-50% decrease in renal blood flow without affecting arterial pressure. Coadministration of nifedipine with BAY K 8644 produced dose-dependent inhibition of the maximum renal vasoconstriction elicited by BAY K 8644. The calcium-channel antagonist had similar effects on ANG II-induced renal vasoconstriction. Nifedipine exerted maximum inhibition by blocking 50% of the peak ANG II response. To evaluate intracellular calcium mobilization, TMB-8 or heparin was coadministered with ANG II. Each agent produced dose-dependent inhibition of up to 50% of the maximum renal vasoconstriction produced by ANG II. The inhibitory effects of nifedipine and TMB-8 were additive; neither agent had an effect when ANG II AT1 receptors were antagonized with losartan. These observations indicate that one-half of the ANG II-induced constriction of renal resistance vessels is mediated by voltage-dependent L-type calcium channels responsive to the dihydropyridine nifedipine. The remaining 50% of the renal vasoconstriction elicited by ANG II is mediated by inositol 1,4,5-trisphosphate-mediated calcium mobilization from intracellular sources. The additive nature of the inhibitory effects indicates distinct mechanisms involving calcium mobilization and calcium entry signaling pathways that are of equal importance in ANG II activation of AT1 receptors to trigger constriction of renal resistance vessels under basal conditions.

The renal vasculature of young spontaneously hypertensive rats (SHR) responds to angiotensin II (ANG II) with exaggerated vasoconstriction, due in part to defective buffering by the adenosine 3',5'-cyclic monophosphate (cAMP) pathway. In vitro studies suggest greater activation of phospholipase C and protein kinase C (PKC) in cultured mesangial cells and vascular smooth muscle cells. The present studies evaluated the role of PKC activation in renal vascular responses to ANG II receptor activation and the relative contributions in SHR vs. Wistar-Kyoto control rats (WKY). Renal blood flow was measured in 8-wk-old anesthetized SHR and WKY pretreated with indomethacin. ANG II (2 ng) injection into the renal artery produced a transient 45-50% maximum reduction of renal blood flow in both rat strains. Intrarenal infusion of either staurosporine or chelerythrine into the renal artery effectively attenuated the vasoconstriction elicited by ANG II in a dose-dependent manner, with maximum inhibition of 60-70%. The PKC inhibitory effects were significant and independent of strain. Coadministration of the PKC inhibitors produced maximal inhibition similar to that observed with one agent, suggesting action via a common pathway. In other studies, the linkage of the PKC pathway to the AT1 receptor was evaluated using sub and maximal doses of losartan to antagonize 50-80% of ANG II-induced vasoconstriction. The same degree of inhibition was observed when a PKC inhibitor was coadministered with losartan. These findings support the views that the PKC system is a major intracellular signaling pathway coupled to the AT1 receptor in renal resistance vessels and that PKC activation is involved to similar degrees in the renal vasoconstriction elicited by ANG II in young WKY and SHR. Exaggerated vascular reactivity to vasoconstrictor agents in genetically hypertensive animals is probably due to a defect in cAMP generation in the presence of a normally operating PKC pathway.