Detection of PHLPP1α/β in Human and Mouse Brain by Different Anti-PHLPP1 Antibodies

Pleckstrin homology domain and leucine rich repeat protein phosphatase 1 (PHLPP1) is a member of the serine/threonine family of phosphatases. It has been studied in organs including brain, heart, pancreas, adipose, breast, and prostate. Human PHLPP1 encodes two splice variants - PHLPP1α (~140-150 kDa) and PHLPP1β (~180-190 kDa). Commercial antibodies are widely used to characterize PHLPP1 proteins in cells/tissues. Here we validate five different antibodies to detect PHLPP1α/β by Western blot using PHLPP1 WT/KO mice. All antibodies recognize PHLPP1β in brain. Only a single antibody (Cosmo Bio Co) detects PHLPP1α (~145-150 kDa). The other four antibodies detect a non-specific signal at ~150 kDa as evidenced by its abundance in PHLPP1 KO tissues. Results suggest Cosmo antibody is a better reagent to detect PHLPP1α by Western blot. In contrast, we found it unsuitable for immunofluorescence applications in brain. Our findings caution interpretation of the ~150 kDa band detected by some PHLPP1 antibodies in rodent and human tissues. Results also recapitulate the importance of including molecular weight standards in Western blot data to simplify retrospective analysis

Evidence for an endogenous cAMP-adenosine pathway in the rat kidney. J Pharmacol Exp Ther

ABSTRACT In the rat kidney, exogenous adenosine-3Ј-5Ј-monophosphate (cAMP) is converted to adenosine via the metabolism of cAMP to adenosine-5Ј-monophosphate by phosphodiesterase and adenosine-5Ј-monophosphate to adenosine by 5Ј-nucleotidase. Our purpose was to investigate whether in the rat kidney adenosine is synthesized from endogenous cAMP via the same pathway. Rat kidneys were perfused with Tyrode's solution, and stabilized for 3 hr to minimize basal renal purine secretion. In control experiments (n ϭ 6), the renal venous secretion rate of adenosine, inosine, hypoxanthine and ⌺purines (adenosine ϩ inosine ϩ hypoxanthine) did not change over the two 10-min experimental periods. In contrast, the beta adrenoceptor agonist (Ϯ)-isoproterenol (1 and 10 M added to the perfusate) caused a significant (1-factor analysis of variance with repeated measures; n ϭ 31) increase in the renal venous secretion of adenosine (P Ͻ .0001), inosine (P Ͻ .0007), hypoxanthine (P Ͻ .0007) and ⌺purines (P Ͻ .0001) as measured by high-performance liquid chromatography with ultraviolet detection. The ⌺purines was the most discriminating index of isoproterenol-induced changes in purine release, and the renal venous secretion of ⌺purines was significantly (2-factor analysis of variance with repeated measures) attenuated by inhibition of beta adrenoceptors with propranolol (.1 M, n ϭ 6; P Ͻ .05), phosphodiesterase with 3-isobutyl-1-methylxanthine (1 mM, n ϭ 5; P Ͻ .002) and 5Ј-nucleotidase with ␣,␤-methyleneadenosine-5Ј-diphosphate (0.1 mM, n ϭ 5; P Ͻ .03). Our data indicate that activation of beta adrenoceptors increases purine biosynthesis in the rat kidney via a mechanism that involves phosphodiesterase and 5Ј-nucleotidase. These results support the existence of an endogenous cAMP-adenosine pathway in the rat kidney

Metabolism of cAMP to Adenosine in the Renal Vasculature 1

ABSTRACT We recently demonstrated that cAMP added to the perfusate increased the renal venous recovery of adenosine in the isolated rat kidney, an effect blocked by inhibition of ecto-phosphodiesterase and ecto-5Ј-nucleotidase. Although our previous study established the cAMP-adenosine pathway, i.e., the conversion of cAMP to adenosine, as a viable metabolic pathway within the kidney, that study did not determine whether conversion of arterial cAMP to adenosine recoverable in the venous effluent occurred in the tubules versus nontubular sites. In the current study, we addressed this issue by determining the effects of blocking cAMP transport into the renal tubules with probenecid (0.1, 0.3 and 1 mM) on the increase in renal venous output of adenosine induced by adding cAMP (30 M) to the perfusate of isolated rat kidneys. Addition of cAMP to the perfusate caused a marked increase in renal venous secretion of adenosine, an effect that was augmented, rather than inhibited, by probenecid. To test the hypothesis that the renal vasculature supports a cAMP-adenosine pathway, cultured rat preglomerular vascular smooth muscle cells were incubated with cAMP (30 M) for 1 hr in the presence and absence of 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor). Incubation with cAMP increased extracellular adenosine levels 41-fold, and this effect was abolished by 3-isobutyl-1-methylxanthine. In a third experimental series, addition of cAMP (0.3, 1, 3, 10 and 30 M) to the perfusate of isolated rat kidneys and mesenteric vascular beds increased the renal venous, but not mesenteric venous, output of AMP, adenosine and inosine. We conclude that the renal vasculature supports a cAMP-adenosine pathway, that administering cAMP into the renal artery and measuring adenosine in the venous effluent of the perfused rat kidney most likely monitors primarily the renal vascular cAMPadenosine pathway and that the quantitative importance of the cAMP-adenosine pathway is not equivalent in all vascular compartments. Renal adenosine participates importantly in the regulation of renin release, renal hemodynamics, tubuloglomerular feedback, erythropoietin production and tubular transport The existence of a cAMP-adenosine pathway in the kidneys is supported by two lines of evidence. First, infusion of IBMX (a phosphodiesterase inhibitor) into the renal cortical interstitium via a microdialysis probe decreases renal cortical interstitial levels of adenosine and inosine (a metabolite of adenosine) The above-mentioned studies in the isolated perfused rat kidney strongly suggest that in the kidney cAMP is converted to adenosine extracellularly. However, those studies do not determine whether conversion of perfusate cAMP to adenosine recoverable in the venous effluent occurs mostly in the tubules versus nontubular sites such as the renal vasculature. Several studies demonstrate that cAMP is efficiently transported by the probenecid-inhibitable organic anion transport system in the proximal tubul

Identification and Quantification of 2′,3′-cAMP Release by the Kidney

We recently developed a sensitive assay for 3′,5′-cAMP using high-performance liquid chromatography-tandem mass spectrometry. Using this assay, we investigated the release of 3′,5′-cAMP from isolated, perfused rat kidneys. To our surprise, we observed a dominant chromatographic peak that was because of an endogenous substance that had the same parent ion as 3′,5′-cAMP and that fragmented to the same daughter ion (adenine) as 3′,5′-cAMP. However, the retention time of this unknown was approximately 2.9 min, compared with 6.3 min for authentic 3′,5′-cAMP. We hypothesized that the unknown substance was an isomer of 3′,5′-cAMP. The unknown substance had the same retention time and mass spectral properties as authentic 2′,3′-cAMP. Renal venous secretion of 2′,3′-cAMP was greater in kidneys from 20-week-old genetically hypertensive rats compared with age-matched normotensive rats (12.49 ± 2.14 versus 5.32 ± 1.97 ng/min/g kidney weight, respectively; n = 18). Isoproterenol (1 μM; β-adrenoceptor agonist) increased renal venous 3′,5′-cAMP secretion (approximately 690% of control) but had no effect on 2′,3′-cAMP production. In contrast, rapamycin (0.2 μM; activator of mRNA turnover) and iodoacetate + 2,4-dinitrophenol (50 μM; metabolic inhibitors) increased the renal venous secretion of 2′,3′-cAMP (approximately 1000 and 4100% of control, respectively) while simultaneously decreasing the renal venous secretion of 3′,5′-cAMP. In conclusion, 2′,3′-cAMP is a naturally occurring isomer of 3′,5′-cAMP that is: 1) not made by adenylyl cyclase; 2) released from kidneys into the extracellular compartment; 3) released more by kidneys from rats with long-standing hypertension; 4) derived from mRNA turnover; and 5) increased by energy depletion