Kisspeptin treatment induces gonadotropic responses and rescues ovulation in a subset of preclinical models and women with polycystic ovary syndrome.

STUDY QUESTION: Can kisspeptin treatment induce gonadotrophin responses and ovulation in preclinical models and anovulatory women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Kisspeptin administration in some anovulatory preclinical models and women with PCOS can stimulate reproductive hormone secretion and ovulation, albeit with incomplete efficacy. WHAT IS KNOWN ALREADY: PCOS is a prevalent, heterogeneous endocrine disorder, characterized by ovulatory dysfunction, hyperandrogenism and deregulated gonadotrophin secretion, in need of improved therapeutic options. Kisspeptins (encoded by Kiss1) are master regulators of the reproductive axis, acting mainly at GnRH neurons, with kisspeptins being an essential drive for gonadotrophin-driven ovarian follicular maturation and ovulation. Altered Kiss1 expression has been found in rodent models of PCOS, although the eventual pathophysiological role of kisspeptins in PCOS remains unknown. STUDY DESIGN, SIZE, DURATION: Gonadotrophin and ovarian/ovulatory responses to kisspeptin-54 (KP-54) were evaluated in three preclinical models of PCOS, generated by androgen exposures at different developmental windows, and a pilot exploratory cohort of anovulatory women with PCOS. PARTICIPANTS/MATERIALS, SETTING, METHODS: Three models of PCOS were generated by exposure of female rats to androgens at different periods of development: PNA (prenatal androgenization; N = 20), NeNA (neonatal androgenization; N = 20) and PWA (post-weaning androgenization; N = 20). At adulthood (postnatal day 100), rats were subjected to daily treatments with a bolus of KP-54 (100 µg/kg, s.c.) or vehicle for 11 days (N = 10 per model and treatment). On Days 1, 4, 7 and 11, LH and FSH responses were assessed at different time-points within 4 h after KP-54 injection, while ovarian responses, in terms of follicular maturation and ovulation, were measured at the end of the treatment. In addition, hormonal (gonadotrophin, estrogen and inhibin B) and ovulatory responses to repeated KP-54 administration, at doses of 6.4-12.8 nmol/kg, s.c. bd for 21 days, were evaluated in a pilot cohort of anovulatory women (N = 12) diagnosed with PCOS, according to the Rotterdam criteria. MAIN RESULTS AND THE ROLE OF CHANCE: Deregulated reproductive indices were detected in all PCOS models: PNA, NeNA and PWA. Yet, anovulation was observed only in NeNA and PWA rats. However, while anovulatory NeNA rats displayed significant LH and FSH responses to KP-54 (P < 0.05), which rescued ovulation, PWA rats showed blunted LH secretion after repeated KP-54 injection and failed to ovulate. In women with PCOS, KP-54 resulted in a small rise in LH (P < 0.05), with an equivalent elevation in serum estradiol levels (P < 0.05). Two women showed growth of a dominant follicle with subsequent ovulation, one woman displayed follicle growth but not ovulation and desensitization was observed in another patient. No follicular response was detected in the other women. LIMITATIONS, REASONS FOR CAUTION: While three different preclinical PCOS models were used in order to capture the heterogeneity of clinical presentations of the syndrome, it must be noted that rat models recapitulate many but not all the features of this condition. Additionally, our pilot study was intended as proof of principle, and the number of participants is low, but the convergent findings in preclinical and clinical studies reinforce the validity of our conclusions. WIDER IMPLICATIONS OF THE FINDINGS: Our first-in-rodent and -human studies demonstrate that KP-54 administration in anovulatory preclinical models and women with PCOS can stimulate reproductive hormone secretion and ovulation, albeit with incomplete efficacy. As our rat models likely reflect the diversity of PCOS phenotypes, our results argue for the need of personalized management of anovulatory dysfunction in women with PCOS, some of whom may benefit from kisspeptin-based treatments. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by research agreements between Ferring Research Institute and the Universities of Cordoba and Edinburgh. K.S. was supported by the Wellcome Trust Scottish Translational Medicine and Therapeutics Initiative (STMTI). Some of this work was undertaken in the MRC Centre for Reproductive Health which is funded by the MRC Centre grant MR/N022556/1. M.T.-S. is a member of CIBER Fisiopatología de la Obesidad y Nutrición, which is an initiative of Instituto de Salud Carlos III. Dr Mannaerts is an employee of Ferring International PharmaScience Center (Copenhagen, Denmark), and Drs Qi, van Duin and Kohout are employees of the Ferring Research Institute (San Diego, USA). Dr Anderson and Dr Tena-Sempere were recipients of a grant support from the Ferring Research Institute, and Dr Anderson has undertaken consultancy work and received speaker fees outside this study from Merck, IBSA, Roche Diagnostics, NeRRe Therapeutics and Sojournix Inc. Dr Skorupskaite was supported by the Wellcome Trust through the Scottish Translational Medicine and Therapeutics Initiative 102419/Z/13/A. The other authors have no competing interest.

Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin.

Although trafficking and degradation of several membrane proteins are regulated by ubiquitination catalyzed by E3 ubiquitin ligases, there has been little evidence connecting ubiquitination with regulation of mammalian G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) function. Agonist stimulation of endogenous or transfected beta2-adrenergic receptors (beta2ARs) led to rapid ubiquitination of both the receptors and the receptor regulatory protein, beta-arrestin. Moreover, proteasome inhibitors reduced receptor internalization and degradation, thus implicating a role for the ubiquitination machinery in the trafficking of the beta2AR. Receptor ubiquitination required beta-arrestin, which bound to the E3 ubiquitin ligase Mdm2. Abrogation of beta-arrestin ubiquitination, either by expression in Mdm2-null cells or by dominant-negative forms of Mdm2 lacking E3 ligase activity, inhibited receptor internalization with marginal effects on receptor degradation. However, a beta2AR mutant lacking lysine residues, which was not ubiquitinated, was internalized normally but was degraded ineffectively. These findings delineate an adapter role of beta-arrestin in mediating the ubiquitination of the beta2AR and indicate that ubiquitination of the receptor and of beta-arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR.

Molecular and cellular actions of platelet-activating factor in rat heart cells.

Platelet-activating factor (PAF) is a phospholipid with cardiovascular actions at low concentrations (1-100 nM) but with uncertain direct myocardial actions. We investigated the cellular and molecular effects of PAF on heart cells using isolated adult and neonatal rat myocytes. Addition of PAF, in the superfusion solution, decreased twitch amplitude and contractile velocity in both systems. Concentrations of PAF below 1 nM stimulated reproducible responses with maximal effects seen at 100 nM. These functional actions of PAF could be blocked by the known PAF antagonist, BN 50739, in a dose-dependent manner. Parallel biochemical studies showed that nanomolar PAF rapidly stimulated the phosphoinositide pathway in cultured myocytes, evidenced by the accumulation of [3H]inositol phosphates in prelabeled cultured myocytes. The potency and specificity of PAF, as well as the time course, for the response were nearly identical in the biochemical and functional assays. PAF produced no functional changes in protein kinase C-depleted myocytes, but it did stimulate inositol trisphosphate accumulation in such cells. We conclude that: (a) PAF exerts a direct negative inotropic effect on myocardial tissue; (b) the effects of PAF are mediated by a specific, high affinity cardiac receptor; (c) an underlying biochemical mechanism for the action of PAF includes the activation of the phospholipase C/phosphatidylinositol intracellular signaling pathway, which leads to activation of protein kinase C.

Ca(2+) channel modulation by recombinant auxiliary beta subunits expressed in young adult heart cells.

L-type Ca(2+) channels contribute importantly to the normal excitation-contraction coupling of physiological hearts, and to the functional derangement seen in heart failure. Although Ca(2+) channel auxiliary beta(1-4) subunits are among the strongest modulators of channel properties, little is known about their role in regulating channel behavior in actual heart cells. Current understanding draws almost exclusively from heterologous expression of recombinant subunits in model systems, which may differ from cardiocytes. To study beta-subunit effects in the cardiac setting, we here used an adenoviral-component gene-delivery strategy to express recombinant beta subunits in young adult ventricular myocytes cultured from 4- to 6-week-old rats. The main results were the following. (1) A component system of replication-deficient adenovirus, poly-L-lysine, and expression plasmids encoding beta subunits could be optimized to transfect young adult myocytes with 1% to 10% efficiency. (2) A reporter gene strategy based on green fluorescent protein (GFP) could be used to identify successfully transfected cells. Because fusion of GFP to beta subunits altered intrinsic beta-subunit properties, we favored the use of a bicistronic expression plasmid encoding both GFP and a beta subunit. (3) Despite the heteromultimeric composition of L-type channels (composed of alpha(1C), beta, and alpha(2)delta), expression of recombinant beta subunits alone enhanced Ca(2+) channel current density up to 3- to 4-fold, which argues that beta subunits are "rate limiting" for expression of current in heart. (4) Overexpression of the putative "cardiac" beta(2a) subunit more than halved the rate of voltage-dependent inactivation at +10 mV. This result demonstrates that beta subunits can tune inactivation in the myocardium and suggests that other beta subunits may be functionally dominant in the heart. Overall, this study points to the possible therapeutic potential of beta subunits to ameliorate contractile dysfunction and excitability in heart failure.

Augmentation of cardiac contractility mediated by the human beta(3)-adrenergic receptor overexpressed in the hearts of transgenic mice.

BACKGROUND: Stimulation of beta(1)- and beta(2)-adrenergic receptors (ARs) in the heart results in positive inotropy. In contrast, it has been reported that the beta(3)AR is also expressed in the human heart and that its stimulation leads to negative inotropic effects. METHODS AND RESULTS: To better understand the role of beta(3)ARs in cardiac function, we generated transgenic mice with cardiac-specific overexpression of 330 fmol/mg protein of the human beta(3)AR (TGbeta(3) mice). Hemodynamic characterization was performed by cardiac catheterization in closed-chest anesthetized mice, by pressure-volume-loop analysis, and by echocardiography in conscious mice. After propranolol blockade of endogenous beta(1)- and beta(2)ARs, isoproterenol resulted in an increase in contractility in the TGbeta(3) mice (30%), with no effect in wild-type mice. Similarly, stimulation with the selective human beta(3)AR agonist L-755,507 significantly increased contractility in the TGbeta(3) mice (160%), with no effect in wild-type mice, as determined by hemodynamic measurements and by end-systolic pressure-volume relations. The underlying mechanism of the positive inotropy incurred with L-755,507 in the TGbeta(3) mice was investigated in terms of beta(3)AR-G-protein coupling and adenylyl cyclase activation. Stimulation of cardiac membranes from TGbeta(3) mice with L-755,507 resulted in a pertussis toxin-insensitive 1.33-fold increase in [(35)S]GTPgammaS loading and a 1.6-fold increase in adenylyl cyclase activity. CONCLUSIONS: Cardiac overexpression of human beta(3)ARs results in positive inotropy only on stimulation with a beta(3)AR agonist. Overexpressed beta(3)ARs couple to G(s) and activate adenylyl cyclase on agonist stimulation.

Use of a PCR-based method to characterize protein kinase C isoform expression in cardiac cells.

Molecular cloning has identified at least nine unique isozymes of protein kinase C (PKC) designated alpha, beta I, beta II, gamma, delta, epsilon, zeta, and eta/L, with the recent addition of the theta-isoform. Previous attempts to characterize PKC isoform expression in heart have been limited by low levels of protein and perhaps by the presence of novel isoforms. Thus to critically examine the diversity of PKC expression in cardiac cells, we developed a reverse transcriptase-polymerase chain reaction (RT-PCR) approach that would amplify regions of the target cDNA of all the PKC isozymes in a single reaction. Degenerate oligonucleotide primers were designed to recognize sequences in the conserved regions of the PKC sequence motif: the cysteine-rich and the ATP-binding regions. Amplification of target PKC cDNA sequences resulted in PCR products with unique sizes and restriction digestion properties. The system was validated by identifying PCR products that correspond to all of the PKC isoform transcripts, except PKC-zeta, in a single reaction with cDNA derived from hippocampus. Cardiac cDNA was RT-PCR amplified, and the products were analyzed by a combination of restriction mapping and DNA sequencing that revealed the presence of only the alpha, delta, epsilon, and eta isoforms in adult rat cardiac myocytes and cultured neonatal ventricular myocytes. A unique nondegenerate primer pair was synthesized to recognize PKC-zeta cDNA. Results with these primers show that PKC-zeta is present in both cardiac myocyte preparations as well. The RT-PCR method developed here is an efficient approach that is broadly useful to examine PKC expression in many tissues, including the identification of potentially novel isoforms.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II activates the Na+/HCO3- symport through a phosphoinositide-independent mechanism in cardiac cells.

Angiotensin II (AngII) is a hormone that alters contractility as well as myocyte growth in heart. Since many hormones that regulate cardiac contractility have also been found to modulate intracellular pH (pHi) the goal of this study was to determine if AngII altered pHi in cultured neonatal rat ventricular myocytes. Changes in pHi were monitored in single cells using the fluorescent pH indicator carboxy-seminaphthorhodafluor-1. Application of 100 nM AngII resulted in a rapid, receptor-mediated alkalinization of 0.08 +/- 0.02 pH unit. The Na+/H+ exchanger was not involved since the response was HCO3(-)-dependent and amiloride-insensitive. Ammonia rebound experiments showed AngII increased the initial rate of recovery from an imposed acid load by 3.15-fold and showed that the hormone led to the selective activation of the Na+/HCO3- symport. In contrast, phorbol ester activation of protein kinase C led to the selective activation of Na+/H+ antiport in these cells. Pharmacological studies showed that the alkalinization was independent of the AngII receptor subtype 1 (AT1) phosphoinositide signaling path. In contrast, AngII activation of the symport was blocked by nanomolar AT2 receptor antagonist PD 123319. Superfusion of the myocytes with exogenous arachidonic acid (5 microM) mimicked the AngII-mediated alkalinization, further suggesting that the AT2 signaling pathway underlies the response. In summary, while most of the known actions of AngII in heart are mediated through AT1 receptors, activation of the Na+/HCO3- symport occurs through a distinct alternative path that is likely related to fatty acid production.

Characterization of functional neuropeptide Y receptors in a human neuroblastoma cell line.

We identified receptors for neuropeptide Y (NPY) on an established human neuroblastoma cell line, SK-N-MC, which are functionally coupled to adenylate cyclase through the inhibitory guanine nucleotide-binding protein of adenylate cyclase, Gi. Intact SK-N-MC cells bound radiolabeled NPY with a KD of 2 nM and contained approximately 83,000 receptors/cell. Unlabeled porcine and human NPY and structurally related porcine peptide YY (PYY) competed with labeled NPY for binding to the receptors. NPY inhibited cyclic AMP accumulation in SK-N-MC cells stimulated by isoproterenol, dopamine, vasoactive intestinal peptide, cholera toxin, and forskolin. NPY inhibited isoproterenol-stimulated cyclic AMP production in a dose-dependent manner, with half-maximal inhibition at 0.5 nM NPY. Porcine and human NPY and porcine PYY gave similar dose-response curves. NPY also inhibited basal and isoproterenol-stimulated adenylate cyclase activity in disrupted cells. Pertussis toxin treatment of the cells completely blocked the ability of NPY to inhibit cyclic AMP production and adenylate cyclase activity. The toxin catalyzed the ADP-ribosylation of a 41-kDa protein in SK-N-MC cells that corresponds to Gi. The receptors on SK-N-MC cells appeared to be specific for NPY, as other neurotransmitter drugs, such as alpha-adrenergic, dopaminergic, muscarinic, and serotonergic antagonists, did not compete for either NPY binding or NPY inhibition of adenylate cyclase. Thus, SK-N-MC cells may be a useful model for investigating NPY receptors and NPY-mediated signal transduction.

beta-Arrestin 1 and 2 differentially regulate heptahelical receptor signaling and trafficking.

The two widely coexpressed isoforms of beta-arrestin (termed beta arrestin 1 and 2) are highly similar in amino acid sequence. The beta-arrestins bind phosphorylated heptahelical receptors to desensitize and target them to clathrin-coated pits for endocytosis. To better define differences in the roles of beta-arrestin 1 and 2, we prepared mouse embryonic fibroblasts from knockout mice that lack one of the beta-arrestins (beta arr1-KO and beta arr2-KO) or both (beta arr1/2-KO), as well as their wild-type (WT) littermate controls. These cells were analyzed for their ability to support desensitization and sequestration of the beta(2)-adrenergic receptor (beta(2)-AR) and the angiotensin II type 1A receptor (AT(1A)-R). Both beta arr1-KO and beta arr2-KO cells showed similar impairment in agonist-stimulated beta(2)-AR and AT(1A)-R desensitization, when compared with their WT control cells, and the beta arr1/2-KO cells were even further impaired. Sequestration of the beta(2)-AR in the beta arr2-KO cells was compromised significantly (87% reduction), whereas in the beta arr1-KO cells it was not. Agonist-stimulated internalization of the AT(1A)-R was only slightly reduced in the beta arr1-KO but was unaffected in the beta arr2-KO cells. In the beta arr1/2-KO cells, the sequestration of both receptors was dramatically reduced. Comparison of the ability of the two beta-arrestins to sequester the beta(2)-AR revealed beta-arrestin 2 to be 100-fold more potent than beta-arrestin 1. Down-regulation of the beta(2)-AR was also prevented in the beta arr1/2-KO cells, whereas no change was observed in the single knockout cells. These findings suggest that sequestration of various heptahelical receptors is regulated differently by the two beta-arrestins, whereas both isoforms are capable of supporting receptor desensitization and down-regulation.

Novel adenovirus component system that transfects cultured cardiac cells with high efficiency.

Although it is clear that gene transfection is a potentially valuable approach in the study of cardiac cell function and differentiation, classic transfection methods are limited by their poor efficiencies in cardiac cells. Recent studies show that recombinant replication-defective human adenovirus can transfect primary cardiac cultures with near 100% efficiency. Since such recombinants are time consuming to prepare, the goal of this study was to develop a plasmid/viral transfection system that would capitalize on the advantages of adenovirus. We have found that a "component system" formed by preincubation of Ad5dl312 adenovirus, poly-L-lysine, and an expression plasmid (lacZ reporter gene under control of the human cytomegalovirus (HCMV) major immediate early promoter) can transfect cultured cardiac cells. Optimal conditions were determined by quantifying beta-galactosidase expression. Histochemical analysis of cultures revealed that the component system transfected 70% of the cells under these conditions. LacZ-positive myocytes could be identified in intact myocytes with the fluorescent substrate C12-fluorescein di-beta-galactopyranoside. Functional studies with such cells indicated that contractile behavior was maintained in transfected cardiocytes. Furthermore, the component system was used to transfect a DNA vector expressing a physiologically relevant protein, protein kinase C delta. In summary, this powerful and simple approach can promote the expression of heterologous genes that can be studied at the biochemical and cellular level in cardiac cells.