Vincent Florio - Academia.edu (original) (raw)

Papers by Vincent Florio

cGMP-hydrolytic activity and its inhibition by sildenafil in normal and failing human and mouse m... more cGMP-hydrolytic activity and its inhibition by sildenafil in normal and failing human and mouse myocardium*

Journal of Biological Chemistry, 1989

Purified muscarinic receptors (0.5-10 nmol of L-[3H]quinuclidinyl benzilate-binding sites/mg of p... more Purified muscarinic receptors (0.5-10 nmol of L-[3H]quinuclidinyl benzilate-binding sites/mg of protein) from bovine brain and the GTP-dependent regulatory protein, Go, were reconstituted with a lipid mixture of phosphatidylcholine and cholesterol. Essentially all of the receptors could interact with Go as evinced by increases in affinity for agonist as large as 800-fold. Both the alpha and beta gamma subunits of Go were required for this effect. Similarly, both subunits were required for the stimulation of guanine nucleotide exchange by agonists. This latter action of the receptor on Go was catalytic and potentiated markedly by prior treatment with dithiothreitol. Initially, agonist stimulation of association of GTP and guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to Go was small and variable due to high basal rates. Prior addition of excess GDP inhibited the basal rate of exchange but allowed stimulation by agonists. Under these conditions, oxotremorine stimulated the rates of association of GTP gamma S up to 10-fold. This selective effect was not mimicked by GTP which inhibited both the basal and hormone-dependent rates. Direct examination of the association of GTP and GDP to Go demonstrated that agonist caused either stimulation or marked inhibition, respectively. These results indicate that receptors stimulate guanine nucleotide exchange on G proteins by both increasing the rates of dissociation of nucleotides and altering their relative affinities such that binding of GTP becomes highly favored over GDP. This would ensure the activation of G proteins by receptors in the presence of both nucleotides.

Journal of Biological Chemistry, 1979

Angiotensin II, catecholamines, and vasopressin are thought to stimulate hepatic glycogenolysis a... more Angiotensin II, catecholamines, and vasopressin are thought to stimulate hepatic glycogenolysis and gluconeogenesis via a cyclic AMP-independent mechanism that requires calcium ion. The present study explores the possibility that angiotensin II and vasopressin control the activity of regulatory enzymes in carbohydrate metabolism through Ca2+-dependent changes in their state of phosphorylation. Intact hepatocytes labeled with [32P]PO43- were stimulated with angiotensin II, glucagon, or vasopressin and 30 to 33 phosphorylated proteins resolved from the cytoplasmic fraction of the cell by electrophoresis in sodium dodecyl sulfate polyacrylamide slab gels. Treatment of the cells with angiotensin II or vasopressin increased the phosphorylation of 10 to 12 of these cytosolic proteins without causing measurable changes in cyclic AMP-dependent protein kinase activity. Glucagon stimulated the phosphorylation of the same set of 11 to 12 proteins through a marked increase in cyclic AMP-dependent protein kinase activity. The molecular weights of three of the protein bands whose phosphorylation was increased by these hormones correspond to the subunit molecular weights of phosphorylase (Mr = 93,000), glycogen synthase (Mr = 85,000), and pyruvate kinase (Mr = 61,000). Two of these phosphoprotein bands were positively identified as phosphorylase and pyruvate kinase by affinity chromatography and immunoprecipitation, respectively. Incubation of hepatocytes in a Ca2+-free medium completely abolished the effects of angiotensin II and vasopressin on protein phosphorylation but did not alter those of glucagon. Treatment of hepatocytes with angiotensin II, glucagon, or vasopressin stimulated phosphorylase activity by 250 to 260%, inhibited glycogen synthase activity by 50%, and inhibited pyruvate kinase activity by 30 to 35% (peptides) to 70% (glucagon). The effects of angiotensin II and vasopressin on the activity of all three enzymes were completely abolished if the cells were incubated in a Ca2+-free medium while those of glucagon were not altered. The results imply that angiotensin II, catecholamines, and vasopressin control hepatic carbohydrate metabolism through a Ca2+-requiring, cyclic AMP-independent pathway that leads to the phosphorylation of important regulatory enzymes.

Journal of Urology, 2005

tolerance, versus the individual's baseline +Gz tolerance. This study was approved by the Brooks ... more tolerance, versus the individual's baseline +Gz tolerance. This study was approved by the Brooks City-Base, Wilford Hall Medical Center, and USAF/Surgeon General IRBs. METHODS: Three human centrifuge rides in the 30-degree F-16 seat were performed on each of six healthy male subjects in a case-controlled manner. The first centrifuge testing session was performed with subjects naive to study medications. For testing sessions 2 and 3, order of administration of tamsulosin and terazosin was randomized and double-blinded. A one-week drug holiday was ensured between medication administrations. Gradual onset +Gz, rapid onset +Gz (with and without Anti-G Straining Maneuver) and Tactical Air Combat Maneuvers (T ACM) were performed without the aid of anti-Gz garments. Variables measured with each ride included +Gz induced loss of consciousness (GLOC), blood pressure, pulse, +Gz tolerance, and duration of the TACM. RESULTS: Half of the patients (3/6) on tamsulosin experienced +Gz-induced loss of consciousness. No patients experienced GLOC during unmedicated or terazosin rides. No statistically significant changes in blood pressure, pulse, +Gz tolerance, or duration of T ACM were identified in patients during tamsulosin or terazosin rides compared with unmedicated rides. Loss of consciousness could not be predicted by changes in blood pressure, pulse or + Gz tolerance when medicated rides were compared to unmedicated rides. CONCLUSIONS: The aviation community is justifiably concerned about the physiological impact of alpha-antagonists in the high-performance environment. Loss of consciousness, the most severe manifestation of impaired +Gz tolerance, appears to be increased in patients taking tamsulosin. Currently, we can identify no factors which might predict safe usage oftamsulosin in the high-+Gz environment. These agents warrant further study for potential use by multiplace aircraft pilots, as there were no deleterious effects of tamsulosin or terazosin in the low-+Gz environment.

The Journal of Sexual Medicine, 2006

Introduction. Tolerance can cause a decrease in drug efficacy during chronic therapy, possibly le... more Introduction. Tolerance can cause a decrease in drug efficacy during chronic therapy, possibly leading to treatment failures. Aim. The aim of this article is to determine whether tolerance developed to the effects of tadalafil on erectile function (EF) over a 6-month treatment period. Methods and Main Outcome Measures. Post hoc analysis of data from a multicenter, double-blind, randomized, placebo-controlled, parallel group study was performed. Men (≥18 years of age) with erectile dysfunction (ED) were randomized to treatment with placebo (N = 47) or 20-mg tadalafil (N = 93) taken as needed for 6 months. This report focuses on efficacy assessed with the Sexual Encounter Profile (SEP) diary (diaries were collected after a 4week treatment-free run-in period [baseline], and monthly for 6 months), and with the International Index of Erectile Function (IIEF) (administered at baseline, and at 3 and 6 months). Results. The mean per-patient percentage "yes" response on SEP question 3 (SEP3, successful intercourse) was 33 ± 4% at baseline, 74 ± 4% after 1 month, and 78 ± 4% after 6 months of tadalafil treatment. The IIEF EF domain score was 16.2 ± 0.7 at baseline, 24.3 ± 0.8 after 3 months, and 24.3 ± 0.9 after 6 months of tadalafil treatment. In a subgroup of patients who took tadalafil ≥3 times per week (N = 24), the SEP3 score was 87 ± 4% after 1 month and 93 ± 3% after 6 months of treatment, and the IIEF EF domain score was 27.3 ± 0.9 after 3 months and 28.5 ± 0.4 after 6 months. Of 16 tadalafil-treated patients who discontinued, three cited a lack of efficacy. Conclusions. Tadalafil treatment significantly improved SEP3 and IIEF EF domain scores. The efficacy of tadalafil, taken as needed, was maintained over a 6-month treatment period in men with ED. McMahon CG, Carson CC, Fischer CJ, Wang WC, Florio VA, and Bradley JD. Tolerance to the therapeutic effect of tadalafil does not occur during 6 months of treatment: A randomized, double-blind, placebo-controlled study in men with erectile dysfunction.

Journal of Pharmacology and Experimental Therapeutics, 2009

In mouse models of cardiac disease, the type 5 (PDE5)-selective cyclic nucleotide phosphodiestera... more In mouse models of cardiac disease, the type 5 (PDE5)-selective cyclic nucleotide phosphodiesterase inhibitor sildenafil has antihypertrophic and cardioprotective effects attributable to the inhibition of cGMP hydrolysis. To investigate the relevance of these findings to humans, we quantified cGMP-hydrolytic activity and its inhibition by sildenafil in cytosolic and microsomal preparations from the left ventricular myocardium of normal and failing human hearts. The vast majority of cGMP-hydrolytic activity was attributable to PDE1 and PDE3. Sildenafil had no measurable effect on cGMP hydrolysis at 10 nM, at which it is selective for PDE5, but it had a marked effect on cGMP and

Journal of Lipid Research, 2004

This study assessed the effects of selective inhibitors of 3 ,5-cyclic nucleotide phosphodiestera... more This study assessed the effects of selective inhibitors of 3 ,5-cyclic nucleotide phosphodiesterases (PDEs) on adipocyte lipolysis. IC224, a selective inhibitor of type 1 phosphodiesterase (PDE1), suppressed lipolysis in murine 3T3-L1 adipocytes (69.6 ؎ 5.4% of vehicle control) but had no effect in human adipocytes. IC933, a selective inhibitor of PDE2, had no effect on lipolysis in either cultured murine 3T3-L1 adipocytes or human adipocytes. Inhibition of PDE3 with cilostamide moderately stimulated lipolysis in murine 3T3-L1 and rat adipocytes (397 ؎ 25% and 235 ؎ 26% of control, respectively) and markedly stimulated lipolysis in human adipocytes (932 ؎ 7.6% of control). Inhibition of PDE4 with rolipram moderately stimulated lipolysis in murine 3T3-L1 adipocytes (291 ؎ 13% of control) and weakly stimulated lipolysis in rat adipocytes (149 ؎ 7.0% of control) but had no effect on lipolysis in human adipocytes. Cultured adipocytes also responded differently to a combination of PDE3 and PDE4 inhibitors. Simultaneous exposure to cilostamide and rolipram had a synergistic effect on lipolysis in murine 3T3-L1 and rat adipocytes but not in human adipocytes. Hence, the relative importance of PDE3 and PDE4 in regulating lipolysis differed in cultured murine, rat, and human adipocytes.-Snyder, P. B., J. M. Esselstyn, K. Loughney, S. L. Wolda, and V. A. Florio. The role of cyclic nucleotide phosphodiesterases in the regulation of adipocyte lipolysis.

Journal of Histochemistry & Cytochemistry, 1999

We developed selective monoclonal antibodies and used them for Western and immunocytochemical ana... more We developed selective monoclonal antibodies and used them for Western and immunocytochemical analyses to determine the tissue and cellular distribution of the human cyclic GMP-stimulated phosphodiesterase (PDE2). Western analysis revealed PDE2A expression in a variety of tissue types, including cerebellum, neocortex, heart, kidney, lung, pulmonary artery, and skeletal muscle. Immunocytochemical analysis revealed PDE2A expression in a subset of tissue endothelial cells. PDE2A immunostaining was detected in venous and capillary endothelial cells in cardiac and renal tissue but not in arterial endothelial cells. These results were confirmed by in situ hybridization. PDE2A immunostaining was also absent from luminal endothelial cells of large vessels, such as aorta, pulmonary, and renal arteries, but was present in the endothelial cells of the vasa vasorum. PDE2A immunostaining was detected in the endothelial cells of a variety of microvessels, including those in renal and cardiac inte...

Journal of Biological Chemistry, 2007

Isoforms in the PDE1 family of cyclic nucleotide phosphodiesterases were recently found to compri... more Isoforms in the PDE1 family of cyclic nucleotide phosphodiesterases were recently found to comprise a significant portion of the cGMP-inhibited cAMP hydrolytic activity in human hearts. We examined the expression of PDE1 isoforms in human myocardium, characterized their catalytic activity, and quantified their contribution to cAMP hydrolytic and cGMP hydrolytic activity in subcellular fractions of this tissue. Western blotting with isoform-selective anti-PDE1 monoclonal antibodies showed PDE1C1 to be the principal isoform expressed in human myocardium. Immunohistochemical analysis showed that PDE1C1 is distributed along the Z-lines and M-lines of cardiac myocytes in a striated pattern that differs from that of the other major dual-specificity cyclic nucleotide phosphodiesterase in human myocardium, PDE3A. Most of the PDE1C1 activity was recovered in soluble fractions of human myocardium. It binds both cAMP and cGMP with K m values of ϳ1 M and hydrolyzes both substrates with similar catalytic rates. PDE1C1 activity in subcellular fractions was quantified using a new PDE1-selective inhibitor, IC295. At substrate concentrations of 0.1 M, PDE1C1 constitutes the great majority of cAMP hydrolytic and cGMP hydrolytic activity in soluble fractions and the majority of cGMP hydrolytic activity in microsomal fractions, whereas PDE3 constitutes the majority of cAMP hydrolytic activity in microsomal fractions. These results indicate that PDE1C1 is expressed at high levels in human cardiac myocytes with an intracellular distribution distinct from that of PDE3A and that it may have a role in the integration of cGMP-, cAMP-and Ca 2؉mediated signaling in these cells.

International Journal of Impotence Research, 2006

The aim of this study was to determine, in an animal model, the effects of tadalafil on myocardia... more The aim of this study was to determine, in an animal model, the effects of tadalafil on myocardial infarct size (IS), hemodynamics and regional myocardial blood flow after myocardial ischemia and reperfusion. Patients with erectile dysfunction (ED) often have risk factors for coronary artery disease. Tadalafil, a long-acting inhibitor of the enzyme phosphodiesterase-5 (PDE5), is used for the treatment of ED; there are no previous data regarding tadalafil in the setting of coronary artery occlusion (CAO). Sprague-Dawley male rats were treated with tadalafil or vehicle (10 mg/kg, by gastric gavage), 2 h before a 30 min CAO. Heart rate was comparable between tadalafil and control groups. Tadalafil reduced mean arterial pressure (P ¼ 0.009), systolic (P ¼ 0.035) and diastolic (P ¼ 0.009) blood pressures during ischemia/reperfusion. Tadalafil significantly reduced IS (4272%) versus controls (5473%) (P ¼ 0.006). For the first time, we showed that the PDE5 inhibitor, tadalafil, was well tolerated and cardioprotective in the setting of an experimental myocardial infarction, by substantially reducing ischemic cell death.

International Journal of Impotence Research, 2005

Gene, 1999

A gene encoding a novel human 3', 5'-cyclic nucleotide phosphodiesterase (PDE) wa... more A gene encoding a novel human 3', 5'-cyclic nucleotide phosphodiesterase (PDE) was identified and characterized. PDE10A1 encodes a protein that is 779 amino acids in length. An incomplete cDNA for a second 5'-splice variant, PDE10A2, was isolated. The proteins encoded by the two variants share 766 amino acids in common. This common region includes an amino-terminal domain with partial homology to the cGMP-binding domains of PDE2, PDE5 and PDE6 as well as a carboxy-terminal region with homology to the catalytic regions of mammalian PDEs. Northern analysis revealed that PDE10A is widely expressed. The PDE10A gene was mapped to three yeast artificial chromosomes (YACs) that contain human DNA from chromosome 6q26-27. A recombinant protein corresponding to the 766 amino acid region common to PDE10A1 and PDE10A2 was expressed in yeast. It hydrolyzed both cAMP and cGMP. Inhibitors that are selective for other PDE families are poor inhibitors of PDE10A; however, PDE10A is inhibited by the non-specific PDE inhibitor, IBMX.

Cellular Signalling, 1999

The PDE1A gene encodes a Ca 2ϩ /calmodulin-stimulated 3Ј,5Ј-cyclic nucleotide phosphodiesterase (... more The PDE1A gene encodes a Ca 2ϩ /calmodulin-stimulated 3Ј,5Ј-cyclic nucleotide phosphodiesterase (PDE). We have performed 5Ј and 3Ј RACE and identified two additional 5Ј-splice variants and one additional 3Ј-splice variant of the human PDE1A gene. The three known 5Ј-splice variants and the two known 3Ј-splice variants combine to generate six different PDE1A mRNAs. However, one of the 5Ј-splice variants exhibits alternate splicing in the 5Ј untranslated region. Thus the six mRNAs encode four different PDE1A proteins. Recombinant forms of the different human PDE1A isoforms were expressed in Sf9 cells. The kinetic properties and inhibitor sensitivities of the four PDE1A isoforms are very similar to one another.

Bioorganic & Medicinal Chemistry Letters, 2012

A series of 2-methoxyacylhydrazones were optimized to yield compounds with high affinity for PDE1... more A series of 2-methoxyacylhydrazones were optimized to yield compounds with high affinity for PDE10A. Several compounds demonstrated efficacy in animal models of schizophrenia, including conditioned avoidance response and a pro-psychotic phencyclidine hyperactivity model.

Biochemical and Biophysical Research Communications, 1992

... References 1. Gharib, SD, Wierman, ME, Shupnik, MA, and Chin, WW (1990) Endocr. Rev. 11: 1771... more ... References 1. Gharib, SD, Wierman, ME, Shupnik, MA, and Chin, WW (1990) Endocr. Rev. 11: 177199. 2. Clayton, RN (1989) J. Endocrinol. 120:1119. 3. Loumaye, E., and Catt, KJ (1982) Science 215: 983985. 4. Conn, PM, and Crowley, WF (1991) N. Engl. J. Med. 324: 93103. ...

The Australian and New Zealand Journal of Obstetrics and Gynaecology, 2006

The human endometrium is a dynamic, cyclically regenerating tissue. Because adult stem cells are ... more The human endometrium is a dynamic, cyclically regenerating tissue. Because adult stem cells are present in other regenerative tissues, a clonal analysis of purified endometrial epithelial and stromal cells derived from hysterectomy tissue was undertaken, as a first attempt to identify and characterise endometrial stem/progenitor cells. Rare populations of epithelial and stromal cells were clonogenic. Only those cells initiating large clones, 0.09% of epithelial cells and 0.02% of stromal cells, are likely to be endometrial stem/progenitor cells.

Journal of Biological Chemistry, 1992

The primary (alpha 1) subunit of purified skeletal muscle dihydropyridine-sensitive calcium chann... more The primary (alpha 1) subunit of purified skeletal muscle dihydropyridine-sensitive calcium channels is present in full-length (212 kDa) and truncated (190 kDa) forms which are both phosphorylated by cAMP-dependent protein kinase (cA-PK) in vitro. In the present study, phosphorylation of the purified calcium channel by cA-PK followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional phosphopeptide mapping revealed differential phosphorylation of the related 190- and 212-kDa forms. The 190-kDa form of the alpha 1 subunit was phosphorylated on three major and three minor tryptic phosphopeptides; the 212-kDa form was phosphorylated on all six of these phosphopeptides plus two that were unique. Time course experiments showed that a single site on the COOH-terminal portion of the full-length form of the alpha 1 subunit is most intensely and rapidly (within 10 s) phosphorylated. Phosphorylation occurs almost exclusively on this COOH-terminal site unless harsh conditions such as treatment with denaturing detergents are employed to expose phosphorylation sites within the 190-kDa segment of the molecule. Elution of phosphopeptides from the second dimension chromatograph followed by immunoprecipitation with an anti-peptide antibody (anti-CP1) directed against the COOH-terminal amino acid sequence enabled us to identify this major phosphorylation site as serine 1854. The nearby consensus sites for cA-PK phosphorylation at serines 1757 and 1772 were phosphorylated only after denaturation or proteolytic cleavage. Phosphorylation of serine 1854 may play a pivotal role in the regulation of calcium channel function by cA-PK.