Kjetil Hodne

Molecular Basis of Calpain Cleavage and Inactivation of the Sodium-Calcium Exchanger 1 in Heart Failure

Background: Sodium-calcium exchanger 1 (NCX1) and calpain are up-regulated in heart failure (HF). Molecular mechanisms and functional consequences of NCX1 cleavage by calpain are not known. Results: Calpain anchors to two NCX1 regions and cleaves at methionine-369, leading to inactivation. Conclusion: NCX1 inhibition by calpain might improve cardiac function. Significance: Calpain might play a pivotal role in NCX1 regulation during HF. Cardiac sodium (Na+)-calcium (Ca2+) exchanger 1 (NCX1) is central to the maintenance of normal Ca2+ homeostasis and contraction. Studies indicate that the Ca2+-activated protease calpain cleaves NCX1. We hypothesized that calpain is an important regulator of NCX1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in the heart. NCX1 full-length protein and a 75-kDa NCX1 fragment along with calpain were up-regulated in aortic stenosis patients and rats with heart failure. Patients with coronary artery disease and sham-operated rats were used as controls. Calpain co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes and left ventricle lysate. Immunoprecipitations, pull-down experiments, and extensive use of peptide arrays indicated that calpain domain III anchored to the first Ca2+ binding domain in NCX1, whereas the calpain catalytic region bound to the catenin-like domain in NCX1. The use of bioinformatics, mutational analyses, a substrate competitor peptide, and a specific NCX1-Met369 antibody identified a novel calpain cleavage site at Met369. Engineering NCX1-Met369 into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met369 inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met369 cleavage site was modeled into the narrow active cleft of human calpain. Inhibition of NCX1 activity, such as we have observed here following calpain-induced NCX1 cleavage, might be beneficial in pathophysiological conditions where increased NCX1 activity contributes to cardiac dysfunction.

Expression and Putative Function of Kisspeptins and Their Receptors During Early Development in Medaka

Kisspeptins (Kiss1 and Kiss2) and their receptors (putatively Gpr54-1 and Gpr54-2) have emerged as key players in vertebrate reproduction owing to their stimulatory effect on the brain-pituitary-gonadal axis. Virtually nothing is known, however, about their role during embryogenesis. Using medaka (Teleostei) as a model system, we report, for the first time in vertebrates, an early developmental expression and putative function of kisspeptins. Expression analyses and knockdown experiments suggest that early actions of kisspeptins are probably mediated by binding to maternally supplied Gpr54-1 and late action by both Gpr54-1 and Gpr54-2. Knockdown of maternally provided kiss1 and gpr54-1 arrested development during gastrulation, before establishment of any germ layers, whereas knockdown of zygotically provided kiss1 and gpr54-1 disrupted brain development. A similar phenotype was observed for gpr54-2 knockdown embryos, suggesting a critical role for kiss1, gpr54-1, and gpr54-2 in neurulation. These data demonstrate that kisspeptin signaling is active both maternally and zygotically and is involved in embryonic survival and morphogenesis.

Signal transduction involved in GnRH2-stimulation of identified LH-producing gonadotropes from lhb-GFP transgenic medaka (Oryzias latipes).

We have characterized the response to gonadotropin-releasing hormone 2 (GnRH2) in luteinizing hormone producing cells from gfp-transgenic medaka. Teleosts have separate cells producing the two types of gonadotropins, enabling us for the first time to study the intracellular signaling that controls secretion of each gonadotropin separately. Pituitary cell cultures were prepared, and lhb-producing cells were selected by their GFP expression. Cytosolic Ca(2+) imaging revealed three response patterns to GnRH2, one monophasic and two types of biphasic patterns. The Ca(2+) sources were examined by depleting intracellular Ca(2+) stores and preventing influx of extracellular Ca(2+). Both treatments reduced response amplitude, and affected latency and time to peak. Blocking L-type Ca(2+) channels reduced amplitude and time to peak, but did not remove extracellular Ca(2+) contribution. Patch-clamp recordings showed spontaneous action potentials in several cells, and GnRH2 increased the firing frequency. Presence of Ca(2+)-activated K(+) channels was revealed, BK channels being the most prominent.

Single-Cell Isolation and Gene Analysis: Pitfalls and Possibilities

During the last two decades single-cell analysis (SCA) has revealed extensive phenotypic differences within homogenous cell populations. These phenotypic differences are reflected in the stochastic nature of gene regulation, which is often masked by qualitatively and quantitatively averaging in whole tissue analyses. The ability to isolate transcripts and investigate how genes are regulated at the single cell level requires highly sensitive and refined methods. This paper reviews different strategies currently used for SCA, including harvesting, reverse transcription, and amplification of the RNA, followed by methods for transcript quantification. The review provides the historical background to SCA, discusses limitations, and current and future possibilities in this exciting field of research.

Electrophysiological properties of pituitary cells in primary culture from atlantic cod (Gadus morhua)

The aim of the present study was to explore the electrophysiological properties of pituitary cells from Atlantic cod (Gadus morhua), as a basis for future studies of the signaling pathways involved in the control of pituitary secretion in this species. Primary cultures of pituitary cells from maturing Atlantic cod were prepared by trypsin treatment and mechanical dispersion. Electrophysiological recordings were performed using the perforated patch clamp method. A subpopulation of large cells were selected for recordings. Spontaneous action potentials were observed in about 30% of the cells. The action potentials displayed a fast initial spike followed by a prolonged plateau. Correspondingly, the inward current elicited by depolarizing steps consisted of both a transient, tetrodotoxin-sensitive Na+ component and a nifedipine-sensitive Ca2+ component that was sustained when Ba2+ replaced Ca2+ as current carrier. The outward current was partially blocked both by 5 mM tetraethylammonium and 10 mM 4-aminopyridine. The voltage-activated ion channels present in these cells largely correspond to the ion channels of pituitary cells in other teleosts (goldfish, Carassius auratus, and tilapia, Oreochromis mossambicus) and mammals, although differences exist regarding the shape and duration of action potentials.

RFamide Peptides in Early Vertebrate Development.

RFamides (RFa) are neuropeptides involved in many different physiological processes in vertebrates, such as reproductive behavior, pubertal activation of the reproductive endocrine axis, control of feeding behavior, and pain modulation. As research has focused mostly on their role in adult vertebrates, the possible roles of these peptides during development are poorly understood. However, the few studies that exist show that RFa are expressed early in development in different vertebrate classes, perhaps mostly associated with the central nervous system. Interestingly, the related peptide family of FMRFa has been shown to be important for brain development in invertebrates. In a teleost, the Japanese medaka, knockdown of genes in the Kiss system indicates that Kiss ligands and receptors are vital for brain development, but few other functional studies exist. Here, we review the literature of RFa in early vertebrate development, including the possible functional roles these peptides may play.

Optimized conditions for primary culture of pituitary cells from the Atlantic cod (Gadus morhua). The importance of osmolality, pCO2, and pH

Protocols for primary cultures of teleost cells are commonly only moderately adjusted from similar protocols for mammalian cells, the main adjustment often being of temperature. Because aquatic habitats are in general colder than mammalian body temperatures and teleosts have gills in direct contact with water, pH and buffer capacity of blood and extracellular fluid are different in fish and mammals. Plasma osmolality is generally higher in marine teleosts than in mammals. Using Atlantic cod (Gadus morhua) as a model, we have optimized these physiological parameters to maintain primary pituitary cells in culture for an extended period without loosing key properties. L-15 medium with adjusted osmolality, adapted to low pCO(2) (3.8mm Hg) and temperature (12°C), and with pH 7.85, maintained the cells in a physiologically sounder state than traditional culture medium, significantly improving cell viability compared to the initial protocol. In the optimized culture medium, resting membrane potential and response to releasing hormone were stable for at least two weeks, and the proportion of cells firing action potentials during spawning season was about seven times higher than in the original culture medium. The cells were moderately more viable when the modified medium was supplemented with newborn calf serum or artificial serum substitute. Compared to serum-free L-15 medium, expression of key genes (lhb, fshb, and gnrhr2a) was better maintained in medium containing SSR, whereas NCS tended to decrease the expression level. Although serum-free medium is adequate for many applications, serum supplement may be preferable for experiments dependent on membrane integrity.

Single-cell qPCR on dispersed primary pituitary cells -an optimized protocol

BackgroundThe incidence of false positives is a potential problem in single-cell PCR experiments. This paper describes an optimized protocol for single-cell qPCR measurements in primary pituitary cell cultures following patch-clamp recordings. Two different cell harvesting methods were assessed using both the GH4 prolactin producing cell line from rat, and primary cell culture from fish pituitaries.ResultsHarvesting whole cells followed by cell lysis and qPCR performed satisfactory on the GH4 cell line. However, harvesting of whole cells from primary pituitary cultures regularly produced false positives, probably due to RNA leakage from cells ruptured during the dispersion of the pituitary cells. To reduce RNA contamination affecting the results, we optimized the conditions by harvesting only the cytosol through a patch pipette, subsequent to electrophysiological experiments. Two important factors proved crucial for reliable harvesting. First, silanizing the patch pipette glass prevented foreign extracellular RNA from attaching to charged residues on the glass surface. Second, substituting the commonly used perforating antibiotic amphotericin B with β-escin allowed efficient cytosol harvest without loosing the giga seal. Importantly, the two harvesting protocols revealed no difference in RNA isolation efficiency.ConclusionDepending on the cell type and preparation, validation of the harvesting technique is extremely important as contaminations may give false positives. Here we present an optimized protocol allowing secure harvesting of RNA from single cells in primary pituitary cell culture following perforated whole cell patch clamp experiments.

Developmental tracing of luteinizing hormone β-subunit gene expression using green fluorescent protein transgenic medaka (Oryzias latipes) reveals a putative novel developmental function.

Background: Luteinizing hormone (LH) and follicle stimulating hormone (FSH), produced in gonadotrope cells in the adenohypophysis are key regulators of vertebrate reproduction. The differential regulation of these hormones, however, is poorly understood and little is known about gonadotrope embryonic development. We developed a stable transgenic line of medaka with the LH beta subunit gene (lhb) promotor driving green fluorescent protein (gfp) expression to characterize development of LH‐producing gonadotropes in whole larvae and histological sections. Additionally, developmental and tissue‐specific gene expression was examined. Results: The lhb gene is maternally expressed during early embryogenesis. Transcript levels increase by stage 21 (36 hours post fertilization [hpf]) and then decrease during continued larval development. Examination of the expression of pituitary marker genes show that LH‐producing cells are initially localized outside the primordial pituitary, and they were localized to the developing gut tube by 32 hpf. At hatching, lhb‐GFP is clearly detected in the gut epithelium and in the anterior digestive tract. lhb‐GFP expression later consolidate in the developing pituitary by 2 weeks postfertilization. Conclusions: During embryonic development, lhb is primarily expressed outside the central nervous system and pituitary. The novel expression of lhb in the embryonic gut suggests that LH has a hitherto unidentified developmental function. Developmental Dynamics 241:1665–1677, 2012.

Electrophysiological Differences Between fshb- and lhb-Expressing Gonadotropes in Primary Culture

Synthesis and release of FSH and LH are differentially regulated by GnRH, but the mechanisms by which this regulation is achieved are not well understood. Teleost fish are powerful models for studying this differential regulation because they have distinct pituitary cells producing either FSH or LH. By using pituitary cultures from Atlantic cod (Gadus morhua), we were able to investigate and compare the electrophysiological properties of fshb- and lhb-expressing cells, identified by single-cell quantitative PCR after recording. Both cell types fired action potentials spontaneously. The relative number of excitable cells was dependent on reproductive season but varied in opposing directions according to season in the 2 cell types. Excitable and quiescent gonadotropes displayed different ion channel repertoires. The dynamics of outward currents and GnRH-induced membrane responses differed between fshb- and lhb-expressing cells, whereas GnRH-induced cytosolic Ca²⁺ responses were similar. Expression of Ca²⁺-activated K⁺ channels also differed with cell type and showed seasonal variation when measured in whole pituitary. The differential presence of these channels corresponds to the differences observed in membrane response to GnRH. We speculate that differences in ion channel expression levels may be involved in seasonal regulation of hormone secretion as well as the differential response to GnRH in LH- and FSH-producing gonadotropes, through differences in excitability and Ca²⁺ influx.