Rosa M. Solano

Expression, pharmacological, and functional evidence for PACAP/VIP receptors in human lung

Pituitary adenylate cyclase-activating peptide (PACAP) type 1 (PAC1) and common PACAP/vasoactive intestinal peptide (VIP) type 1 and 2 (VPAC1 and VPAC2, respectively) receptors were detected in the human lung by RT-PCR. The proteins were identified by immunoblotting at 72, 67, and 68 kDa, respectively. One class of PACAP receptors was defined from125I-labeled PACAP-27 binding experiments (dissociation constant = 5.2 nM; maximum binding capacity = 5.2 pmol/mg protein) with a specificity: PACAP-27 ≈ VIP > helodermin ≈ peptide histidine-methionine (PHM) ≫ secretin. Two classes of VIP receptors were established with 125I-VIP (dissociation constants of 5.4 and 197 nM) with a specificity: VIP ≈ helodermin ≈ PACAP-27 ≫ PHM ≫ secretin. PACAP-27 and VIP were equipotent on adenylyl cyclase stimulation (EC50 = 1.6 nM), whereas other peptides showed lower potency (helodermin > PHM ≫ secretin). PACAP/VIP antagonists supported that PACAP-27 acts in the human lung through either specific receptors or common PACAP/VIP receptors. The present results are the first demonstration of the presence of PAC1 receptors and extend our knowledge of common PACAP/VIP receptors in the human lung.Pituitary adenylate cyclase-activating peptide (PACAP) type 1 (PAC(1)) and common PACAP/vasoactive intestinal peptide (VIP) type 1 and 2 (VPAC(1) and VPAC(2), respectively) receptors were detected in the human lung by RT-PCR. The proteins were identified by immunoblotting at 72, 67, and 68 kDa, respectively. One class of PACAP receptors was defined from (125)I-labeled PACAP-27 binding experiments (dissociation constant = 5.2 nM; maximum binding capacity = 5.2 pmol/mg protein) with a specificity: PACAP-27 approximately VIP > helodermin approximately peptide histidine-methionine (PHM) >> secretin. Two classes of VIP receptors were established with (125)I-VIP (dissociation constants of 5.4 and 197 nM) with a specificity: VIP approximately helodermin approximately PACAP-27 >> PHM >> secretin. PACAP-27 and VIP were equipotent on adenylyl cyclase stimulation (EC(50) = 1.6 nM), whereas other peptides showed lower potency (helodermin > PHM >> secretin). PACAP/VIP antagonists supported that PACAP-27 acts in the human lung through either specific receptors or common PACAP/VIP receptors. The present results are the first demonstration of the presence of PAC(1) receptors and extend our knowledge of common PACAP/VIP receptors in the human lung.

Expression of functional PACAP/VIP receptors in human prostate cancer and healthy tissue.

Vasoactive intestinal peptide (VIP) is involved in prostate cell proliferation and function. VIP and pituitary adenylate cyclase-activating peptide (PACAP) are similarly recognized by VPAC(1)/VPAC(2) receptors whereas PACAP binds with higher affinity than VIP to PAC(1) receptor. Here we systematically studied the presence and distribution of functional PAC(1), VPAC(1) and VPAC(2) receptors in human normal and malignant prostate tissue. Functional PACAP/VIP receptors were detected in normal and malignant prostate by adenylyl cyclase stimulation with PACAP-27/38 and VIP. RT-PCR experiments showed PAC(1) (various isoforms due to alternative splicing), VPAC(1) and VPAC(2) receptor expression at the mRNA level, whereas Western blots found the three receptor protein classes in normal and pathological conditions. No conclusive differences could be established when comparing control and cancer tissue samples. Immunohistochemistry showed a weaker immunostaining in tumoral than in normal epithelial cells for the three receptor subtypes. In conclusion, we demonstrate the expression of functional PAC(1), VPAC(1) and VPAC(2) receptors in human prostate as well as its maintenance after malignant transformation.

Identification and functional properties of the pituitary adenylate cyclase activating peptide (PAC1) receptor in human benign hyperplastic prostate.

Pituitary adenylate cyclase activating peptide (PACAP) is a novel neuropeptide with regulatory and trophic functions that is related to vasoactive intestinal peptide (VIP). Here we investigate the expression of specific PACAP receptors (PAC1) and common VIP/PACAP receptors (VPAC1 and VPAC2) in the human hyperplastic prostate by immunological methods. The PAC1 receptor corresponded to a 60-KDa protein whereas the already known VPAC1 and VPAC2 receptors possessed molecular masses of 58 and 68 KDa, respectively. The heterogeneity of VIP/PACAP receptors in this tissue was confirmed by radioligand binding studies using [125I]PACAP-27 by means of stoichiometric and pharmacological experiments. At least two classes of PACAP binding sites showing different affinities could be resolved, with Kd values of 0.81 and 51.4 nM, respectively. The order of potency in displacing [125I]PACAP-27 binding was PACAP-27 approximately equal to PACAP-38 > VIP. PACAP-27 and VIP stimulated similarly adenylate cyclase activity, presumably through common VIP/PACAP receptors. The PAC1 receptor was not coupled to activation of either adenylate cyclase, nitric oxide synthase, or phospholipase C. It appears to be a novel subtype of PAC1 receptor because PACAP-27 (but not PACAP-38 or VIP) led to increased phosphoinositide synthesis, an interesting feature because phosphoinositides are involved via receptor mechanisms in the regulation of cell proliferation.

G‐proteins and β‐adrenergic stimulation of adenylate cyclase activity in the diabetic rat prostate

The consequences of experimental diabetes on membrane lipids, β‐adrenergic stimulation of adenylate cyclase activity, and G‐protein levels in the prostate gland are not defined.

Expression and distribution of pituitary adenylate cyclase-activating peptide in human prostate and prostate cancer tissues

The presence, expression and distribution of pituitary adenylate cyclase-activating peptide (PACAP) in human prostate cancer and healthy tissue were investigated by means of biochemical and morphological procedures. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated the presence of its precursor encoding mRNA in both normal and pathological conditions (amplification products with 577 or 226 bp were identified). Immunochemistry using an appropriate antibody served to detect in both classes of tissues a 19.9-kDa product corresponding to the PACAP preproprotein and another protein of 14.6 kDa that may represent a product partially processed by convertases. However, a 5-kDa band characteristic of PACAP-38 peptide was not observed. Immunohistochemistry on tissue sections indicated the location of PACAP in the epithelial layer of prostate glands (and in some scarce leucocytes) but not in the stroma, either in normal or carcinomatous tissues. No clear differences could be established when comparing samples from patients with different tumor Gleason grades. These results are the first demonstration of the localization of PACAP or its precursors and its mRNA in the human prostate gland and their presence during the progression of prostate carcinoma.

5-Hydroxytryptamine1A Receptor-Mediated Effects on Adenylate Cyclase and Nitric Oxide Synthase Activities in Rat Ventral Prostate

The rat ventral prostate possesses specific 5-hydroxytryptamine (5-HT1A) receptors coupled to adenylate cyclase. In vivo treatment of rats or in vitro preincubation of minced prostatic tissue with the 5-HT1A receptor agonist 8-hydroxy-2-(di-N-propylamino)-tetralin (8-OH-DPAT) in different experimental conditions shows the possibility of desensitisation mechanisms with switching from inhibitory to stimulatory pattern on adenylate cyclase activity. As in the majority of systems, we observed the inhibition of forskolin-stimulated adenylate cyclase activity as a functional correlate of 5-HT1A receptor activation. A similar feature occurred when the direct stimulation of the enzyme by the diterpene was replaced by a receptor-mediated activation with the neuropeptide vasoactive intestinal peptide. Furthermore, 8-OH-DPAT stimulated nitric oxide synthase (NOS) activity in a dose-dependent manner. Thus, serotonin appears to be able to act in the rat prostate gland through specific 5-HT1A receptors coupled to a complex system of signal transduction involving an inhibitory response of adenylate cyclase that can become stimulatory, as well as an enhancement of NOS activity.

Neuropeptide Y inhibits vasoactive intestinal peptide-stimulated adenylyl cyclase in rat ventral prostate

Neuropeptide Y (NPY), a peptide present in the prostate gland, was found to inhibit vasoactive intestinal peptide (VIP)-stimulated cyclic AMP accumulation in isolated rat prostatic epithelial cells as well as VIP-stimulated adenylyl cyclase activity in rat prostatic membranes. The inhibitory effect of NPY was selective for the VIP receptor/effector system since it was also observed when using pituitary adenylyl cyclase activating peptide (PACAP-27) which presumably recognizes VIP receptors in this gland, but not when using unrelated substances such as isoproterenol or forskolin. NPY did not modify either the general lipid membrane microviscosity or the VIP-receptor binding. The inhibitory effect of VIP was blocked by pretreatment of the prostatic membranes with pertussis toxin. These results suggest the presence of NPY receptors in rat ventral prostate coupled in an inhibitory manner to adenylyl cyclase through a guanine nucleotide regulatory Gi protein.

LOW EXPRESSION OF Gα PROTEIN SUBUNITS IN HUMAN PROSTATE CANCER

Purpose: Because of increasing interest in G protein regulation of cell growth, differentiation and oncogenesis, we studied the functionality and expression of different G protein subunits in human prostate adenocarcinoma.Materials and Methods: Surgical prostate specimens from control patients with bladder cancer and patients with prostate cancer were used. The functionality of αs and αi G protein subunits was evaluated by studying somatostatin or guanyl-5′-yl-imidotriphosphate regulation of forskolin stimulated adenylyl cyclase activity. The expression of αs, αi and β subunits was studied by reverse transcriptase-polymerase chain reaction and immunoblot analysis.Results: Adenylyl cyclase sensitivity to somatostatin inhibition decreased in prostate cancer. Low guanyl-5′-yl-imidotriphosphate doses inhibited forskolin stimulated adenylyl cyclase, whereas the opposite was true at high concentrations, evidencing the functionality of αi and αs, respectively, in normal and cancer tissue samples. Reverse transcr...

Effects of Pygeum africanum extract (Tadenan®) on vasoactive intestinal peptide receptors, G proteins, and adenylyl cyclase in rat ventral prostate

Tadenan® (a Pygeum africanum extract) is a drug used in the treatment of benign prostatic hyperplasia. Its effects on prostate fibroblast proliferation and bladder function after partial outlet obstruction have been demonstrated in various pharmacological studies. However, its effects at the molecular level are poorly documented.

Effect of flutamide-induced androgen-receptor blockade on adenylate cyclase activation through G-protein coupled receptors in rat prostate

The effect of the antiandrogen flutamide on the prostatic vasoactive intestinal peptide (VIP) receptor/effector system was studied in rats. Rats were s.c. injected with a daily dose of flutamide (15 mg/kg B.W.) or vehicle for 14 days. Drug treatment resulted in histological evidence of gland involution and increased plasma membrane fluidity as estimated by fluorescence spectroscopy. The number of VIP receptors and the stimulatory effect of VIP on adenylate cyclase activity in prostatic membranes decreased in flutamide-treated rats. However, the pattern of forskolin stimulation of the enzyme activity was not modified by this drug. Androgen-receptor blockade by flutamide also decreased the prostatic levels of alpha(s,) alpha(i1/2), and alpha(i3/0) G-protein subunits, as estimated by an immunological procedure. Whereas apoptotic DNA fragmentation was evidenced in prostate from 3-day castrated animals, a heterogeneous electrophoretic pattern was observed after flutamide treatment. Thus, androgen-receptor blockade by flutamide results in an important impairment of the components of the VIP receptor/effector system in rat prostate as well as in a modification of their coupling extent, which is presumably due to differences observed in plasma membrane fluidity. These results represent a crosstalk in the prostate between two mechanisms of signal transduction involved in cell proliferation.