Annual Review of Physiology - Volume 63, 2001

The study of intermediary metabolism in biomolecules has been given new directions by recent experiments in human muscle and brain by ¹³C NMR. Labeled substrates, generally glucose, have enabled the fluxes to be determined in vivo, whereas the naturally abundant ¹³C has enabled concentrations to be measured. In muscle the glycogen synthesis pathway has been measured and the flux control determined by metabolic control analysis of data, which shows that this pathway is mainly responsible for insulin-stimulated glucose disposal and that a deficiency in the glucose transporter in the pathway is responsible for hyperglycemia in non-insulin-dependent diabetics. From a physiological point of view the most surprising result was that the heavily regulated allosteric enzyme, glycogen synthase, does not control flux but is needed to maintain homeostasis during flux changes. This novel role for a phosphorylated allosteric enzyme is proposed to be a general phenomenon in metabolic and signaling pathways, which physiologically link different cellular activities.

In human and rat brains ¹³C NMR measurements of the flow of labeled glucose into glutamate and glutamine simultaneously determine the rate of glucose oxidation and glutamate neurotransmitter cycling and reveal a 1:1 stoichiometry between the two fluxes. Implications for the interpretation of functional imaging studies and for psychology are discussed.

These results demonstrate how intermediary metabolism serves to connect biochemistry with systemic physiology when measured and analyzed by in vivo NMR methods.

Gastrin, produced by G cells in the gastric antrum, has been identified as the circulating hormone responsible for stimulation of acid secretion from the parietal cell. Gastrin also acts as a potent cell-growth factor that has been implicated in a variety of normal and abnormal biological processes including maintenance of the gastric mucosa, proliferation of enterochromaffin-like cells, and neoplastic transformation. Here, we review the models used to study the effects of gastrin on cell proliferation in vivo and in vitro with respect to mechanisms by which this hormone might influence normal and cancerous cell growth. Specifically, human and animal models of hypergastrinemia and hypogastrinemia have been described in vivo, and several cells that express cholecystokinin (CCK)B/gastrin receptors have been used for analysis of intracellular signaling pathways initiated by biologically active amidated gastrins. The binding of gastrin or CCK to their common cognate receptor triggers the activation of multiple signal transduction pathways that relay the mitogenic signal to the nucleus and promote cell proliferation. A rapid increase in the synthesis of lipid-derived second messengers with subsequent activation of protein phosphorylation cascades, including mitogen-activated protein kinase, is an important early response to these signaling peptides. Gastrin and CCK also induce rapid Rho-dependent actin remodeling and coordinate tyrosine phosphorylation of cellular proteins including the non-receptor tyrosine kinases p125^fak and Src and the adaptor proteins p130^cas and paxillin. This article reviews recent advances in defining the role of gastrin and CCK in the control of cell proliferation in normal and cancer cells and in dissecting the signal transduction pathways that mediate the proliferative responses induced by these hormonal GI peptides in a variety of normal and cancer cell model systems.

The intracellular signaling mechanisms by which cholecystokinin (CCK) and other secretagogues regulate pancreatic acinar function are more complex than originally realized. CCK couples through heterotrimeric G proteins of the Gq family to lead to an increase in intracellular free Ca²⁺, which shows spatial and temporal patterns of signaling. The actions of Ca²⁺ are mediated in part by activation of a number of Ca²⁺-activated protein kinases and the protein phosphatase calcineurin. By the process of exocytosis the intracellular messengers Ca²⁺, diacylglycerol, and cAMP activate the release of the zymogen granule content in a manner that is poorly understood. This fusion event most likely involves SNARE and Rab proteins present on zymogen granules and cellular membrane domains. More likely related to nonsecretory aspects of cell function, CCK also activates three MAPK cascades leading to activation of ERKs, JNKs, and p38 MAPK. Although the function of these pathways is not well understood, ERKs are probably related to cell growth, and through phosphorylation of hsp27, p38 can affect the actin cytoskeleton. The PI3K (phosphatidylinositol 3-kinase)-mTOR (mammalian target of rapamycin) pathway is important for regulation of acinar cell protein synthesis because it leads to both activation of p70^S6K and regulation of the availability of eIF4E in response to CCK. CCK also activates a number of tyrosyl phosphorylation events including that of p125^FAK and other proteins associated with focal adhesions.

In order to control cell functions, hormones and neurotransmitters generate an amazing diversity of Ca²⁺ signals such as local and global Ca²⁺ elevations and also Ca²⁺ oscillations. In pancreatic acinar cells, cholecystokinin (CCK) stimulates secretion of digestive enzyme and promotes cell growth, whereas acetylcholine (ACh) essentially triggers enzyme secretion. Pancreatic acinar cells are a classic model for the study of CCK- and ACh-evoked specific Ca²⁺ signals. In addition to inositol 1,4,5 trisphosphate (IP3), recent studies have shown that cyclic ADPribose (cADPr) and nicotinic acid adenine dinucleotide phosphate (NAADP) release Ca²⁺ in pancreatic acinar cells. Moreover, it has also been shown that both ACh and CCK trigger Ca²⁺ spikes by co-activation of IP3 and ryanodine receptors but by different means. ACh uses IP3 and Ca²⁺, whereas CCK uses cADPr and NAADP. In addition, CCK activates phospholipase A2 and D. The concept emerging from these studies is that agonist-specific Ca²⁺ signals in a single target cell are generated by combination of different intracellular messengers.

Gastric epithelial organization and function are controlled and maintained by a variety of endocrine and paracrine mediators. Peptides encoded by the gastrin gene are an important part of this system because targeted deletion of the gene, or of the gastrin-CCKB receptor gene, leads to decreased numbers of parietal cells and decreased gastric acid secretion. Recent studies indicate that the gastrin precursor, preprogastrin, gives rise to a variety of products, each with a distinctive spectrum of biological activity. The conversion of progastrin to smaller peptides is regulated by multiple mechanisms including prohormone phosphorylation and secretory vesicle pH. Progastrin itself stimulates colonic epithelial proliferation; biosynthetic intermediates (Gly-gastrins) stimulate colonic epithelial proliferation and gastric epithelial differentiation; and C-terminally amidated gastrins stimulate colonic proliferation, gastric epithelial proliferation and differentiation, and acid secretion. The effects of progastrin-derived peptides on gastric epithelial function are mediated in part by release of paracrine factors that include histamine, epidermal growth factor (EGF)–receptor ligands, and Reg. The importance of the appropriate regulation of this system is shown by the observation that prolonged moderate hypergastrinemia in transgenic mice leads to remodelling of the gastric epithelium, and in the presence of Helicobacter, to gastric cancer.

The traditionally accepted theory has been that most of the biological effects of growth hormone (GH) are mediated by circulating (endocrine) insulin-like growth factor-I (IGF-I). This dogma was modified when it was discovered that most tissues express IGF-I that can act via an autocrine/paracrine fashion. In addition, both GH and IGF-I had independent effects on various target tissues. Using tissue-specific gene deletion of IGF-I in the liver, it has been shown that circulating IGF-I is predominantly liver-derived but is not essential for normal postnatal growth. Therefore, it is proposed that non-hepatic tissue-derived IGF-I may be sufficient for growth and development. Thus the original somatomedin hypothesis has undergone further modifications.

There was a time when the classification of sex hormones was simple. Androgens were male and estrogens female. What remains true today is that in young adults androgen levels are higher in males and estrogen levels higher in females. More recently we have learned that estrogens are necessary in males for regulation of male sexual behavior, maintenance of the skeleton and the cardiovascular system, and for normal function of the testis and prostate. The importance of androgen in females was never in doubt, it is after all the precursor of estrogen as the substrate for aromatase, the enzyme that produces estrogen. In addition, the tissue distribution of androgen receptors suggests that androgens themselves are important in the ovary, uterus, breast, and brain.

New information promises to clarify some of the complex issues of the physiological roles of estrogen and the contribution of estrogen to the development of neoplastic diseases in humans. The discovery of the second estrogen receptor, the creation of mutant mice defective in both estrogen receptors and in the aromatase gene, the solution of the structures of the ligand-binding domains of estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), the finding of novel routes through which estrogen receptors can modulate transcription, and the identification of a man with a bi-allelic disruptive mutation of the ERα gene are but some of the milestones. This review focuses on the mechanistic aspects of signal transduction mediated by ERs and on the physiological consequences of deficiency of estrogen or estrogen receptor in the available mouse models.

Steroid hormone biosynthesis is acutely regulated by pituitary trophic hormones and other steroidogenic stimuli. This regulation requires the synthesis of a protein whose function is to translocate cholesterol from the outer to the inner mitochondrial membrane in steroidogenic cells, the rate-limiting step in steroid hormone formation. The steroidogenic acute regulatory (StAR) protein is an indispensable component in this process and is the best candidate to fill the role of the putative regulator. StAR is expressed in steroidogenic tissues in response to agents that stimulate steroid production, and mutations in the StAR gene result in the disease congenital lipoid adrenal hyperplasia, in which steroid hormone biosynthesis is severely compromised. The StAR null mouse has a phenotype that is essentially identical to the human disease. The positive and negative expression of StAR is sensitive to agents that increase and inhibit steroid biosynthesis respectively. The mechanism by which StAR mediates cholesterol transfer in the mitochondria has not been fully characterized. However, the tertiary structure of the START domain of a StAR homolog has been solved, and identification of a cholesterol-binding hydrophobic tunnel within this domain raises the possibility that StAR acts as a cholesterol-shuttling protein.

During the 1980s the purification, cloning, and expression of various forms of guanylyl cyclase (GC) revealed that they served as receptors for extracellular signals. Seven membrane forms, which presumably exist as homodimers, and four subunits of apparent heterodimers (commonly referred to as the soluble forms) are known, but in animals such as nematodes, much larger numbers of GCs are expressed. The number of transmembrane segments (none, one, or multiple) divide the GC family into three groups. Those with no or one transmembrane segment bind nitric oxide/carbon monoxide (NO/CO) or peptides. There are no known ligands for the multiple transmembrane segment class of GCs. Mutational and structural analyses support a model where catalysis requires a shared substrate binding site between the subunits, whether homomeric or heteromeric in nature. Because some cyclases or cyclase ligand genes lack specific GC inhibitors, disruption of either has been used to define the functions of individual cyclases, as well as to define human genetic disease counterparts.

Ionic currents activated by hyperpolarization and regulated by cyclic nucleotides were first discovered more than 20 years ago. Recently the molecular identity of the underlying channels has been unveiled. The structural features of the protein sequences are discussed and related to the mechanisms of activation, selectivity for cyclic nucleotides, and ion permeation. Coverage includes a comparison of the biophysical properties of recombinant and native channels and their significance for the physiological functions of these channels.

O2 sensing is a fundamental biological process necessary for adaptation of living organisms to variable habitats and physiological situations. Cellular responses to hypoxia can be acute or chronic. Acute responses rely mainly on O2-regulated ion channels, which mediate adaptive changes in cell excitability, contractility, and secretory activity. Chronic responses depend on the modulation of hypoxia-inducible transcription factors, which determine the expression of numerous genes encoding enzymes, transporters and growth factors. O2-regulated ion channels and transcription factors are part of a widely operating signaling system that helps provide sufficient O2 to the tissues and protect the cells against damage due to O2 deficiency. Despite recent advances in the molecular characterization of O2-regulated ion channels and hypoxia-inducible factors, several unanswered questions remain regarding the nature of the O2 sensor molecules and the mechanisms of interaction between the sensors and the effectors. Current models of O2 sensing are based on either a heme protein capable of reversibly binding O2 or the production of oxygen reactive species by NAD(P)H oxidases and mitochondria. Complete molecular characterization of the hypoxia signaling pathways will help elucidate the differential sensitivity to hypoxia of the various cell types and the gradation of the cellular responses to variable levels of PO2. A deeper understanding of the cellular mechanisms of O2 sensing will facilitate the development of new pharmacological tools effective in the treatment of diseases such as stroke or myocardial ischemia caused by localized deficits of O2.

Phosphagens are phosphorylated guanidino compounds that are linked to energy state and ATP hydrolysis by corresponding phosphagen kinase reactions: phosphagen + MgADP + H⁺ ↔ guanidine acceptor + MgATP. Eight different phosphagens (and corresponding phosphagen kinases) are found in the animal kingdom distributed along distinct phylogenetic lines. By far, the creatine phosphate/creatine kinase (CP/CK) system, which is found in the vertebrates and is widely distributed throughout the lower chordates and invertebrates, is the most extensively studied phosphagen system. Phosphagen kinase reactions function in temporal ATP buffering, in regulating inorganic phosphate (Pi) levels, which impacts glycogenolysis and proton buffering, and in intracellular energy transport. Phosphagen kinase reactions show differences in thermodynamic poise, and the phosphagens themselves differ in terms of certain physical properties including intrinsic diffusivity. This review evaluates the distribution of phosphagen systems and tissue-specific expression of certain phosphagens in an evolutionary and functional context. The role of phosphagens in regulation of intracellular Pi levels likely evolved early. Thermodynamic poise of the phosphagen kinase reaction profoundly impacts this capacity. Furthermore, it is hypothesized that the capacity for intracellular targeting of CK evolved early as a means of facilitating energy transport in highly polarized cells and was subsequently exploited for temporal ATP buffering and dynamic roles in metabolic regulation in cells displaying high and variable rates of aerobic energy production.

Terrestrial arthropods survive subzero temperatures by becoming either freeze tolerant (survive body fluid freezing) or freeze avoiding (prevent body fluid freezing). Protein ice nucleators (PINs), which limit supercooling and induce freezing, and antifreeze proteins (AFPs), which function to prevent freezing, can have roles in both freeze tolerance and avoidance. Many freeze-tolerant insects produce hemolymph PINs, which induce freezing at high subzero temperatures thereby inhibiting lethal intracellular freezing. Some freeze-tolerant species have AFPs that function as cryoprotectants to prevent freeze damage. Although the mechanism of this cryoprotection is not known, it may involve recrystallization inhibition and perhaps stabilization of the cell membrane. Freeze-avoiding species must prevent inoculative freezing initiated by external ice across the cuticle and extend supercooling abilities. Some insects remove PINs in the winter to promote supercooling, whereas others have selected against surfaces with ice-nucleating abilities on an evolutionary time scale. However, many freeze-avoiding species do have proteins with ice-nucleating activity, and these proteins must be masked in winter. In the beetle Dendroides canadensis, AFPs in the hemolymph and gut inhibit ice nucleators. Also, hemolymph AFPs and those associated with the layer of epidermal cells under the cuticle inhibit inoculative freezing. Two different insect AFPs have been characterized. One type from the beetles D. canadensis and Tenebrio molitor consists of 12- and 13-mer repeating units with disulfide bridges occurring at least every six residues. The spruce budworm AFP lacks regular repeat units. Both have much higher activities than any known AFPs.

Marine teleosts at high latitudes can encounter ice-laden seawater that is approximately 1°C colder than the colligative freezing point of their body fluids. They avoid freezing by producing small antifreeze proteins (AFPs) that adsorb to ice and halt its growth, thereby producing an additional non-colligative lowering of the freezing point. AFPs are typically secreted by the liver into the blood. Recently, however, it has become clear that AFP isoforms are produced in the epidermis (skin, scales, fin, and gills) and may serve as a first line of defense against ice propagation into the fish. The basis for the adsorption of AFPs to ice is something of a mystery and is complicated by the extreme structural diversity of the five antifreeze types. Despite the recent acquisition of several AFP three-dimensional structures and the definition of their ice-binding sites by mutagenesis, no common ice-binding motif or even theme is apparent except that surface-surface complementarity is important for binding. The remarkable diversity of antifreeze types and their seemingly haphazard phylogenetic distribution suggest that these proteins might have evolved recently in response to sea level glaciation occurring just 1–2 million years ago in the northern hemisphere and 10–30 million years ago around Antarctica. Not surprisingly, the expression of AFP genes from different origins can also be quite dissimilar. The most intensively studied system is that of the winter flounder, which has a built-in annual cycle of antifreeze expression controlled by growth hormone (GH) release from the pituitary in tune with seasonal cues. The signal transduction pathway, transcription factors, and promoter elements involved in this process are just beginning to be characterized.

This review discusses the rapidly progressing field of cardiomyocyte signal transduction and the regulation of the hypertrophic response. When stimulated by a wide array of neurohumoral factors or when faced with an increase in ventricular-wall tension, individual cardiomyocytes undergo hypertrophic growth as an adaptive response. However, sustained cardiac hypertrophy is a leading predictor of future heart failure. A growing number of intracellular signaling pathways have been characterized as important transducers of the hypertrophic response, including specific G protein isoforms, low-molecular-weight GTPases (Ras, RhoA, and Rac), mitogen-activated protein kinase cascades, protein kinase C, calcineurin, gp130-signal transducer and activator of transcription, insulin-like growth factor I receptor pathway, fibroblast growth factor and transforming growth factor β receptor pathways, and many others. Each of these signaling pathways has been implicated as a hypertrophic transducer, which collectively suggests an emerging paradigm whereby multiple pathways operate in concert to orchestrate a hypertrophic response

This review surveys a range of approaches using plasmid DNA encoding the 165-amino-acid isoform of vascular endothelial growth factor (phVEGF165) to therapeutically modulate micro- or macrovascular endothelial cells, focusing on strategies to augment postnatal collateral circulation in arterial insufficiency or to accelerate re-endothelialization after balloon angioplasty to prevent restenosis. We focus on intra-arterial and intramuscular/intramyocardial gene transfer of the VEGF165 gene, the options that have been most thoroughly studied to date in patients. We review developmental and postnatal significance of the endothelial-cell-specific mitogen VEGF that has stimulated these studies and present limitations of current knowledge as well as challenges for the future.

More children die from congenital heart defects (CHD) each year than are diagnosed with childhood cancer, yet the causes remain unknown. The remarkable conservation of genetic pathways regulating cardiac development in species ranging from flies to humans provides an opportunity to experimentally dissect the role of critical cardiogenic factors. Utilization of model biological systems has resulted in a molecular framework in which to consider the etiology of CHD. As whole genome sequencing and single nucleotide polymorphism data become available, identification of genetic mutations predisposing to CHD may allow preventive measures by modulation of secondary genetic or environmental factors. In this review, genetic pathways regulating cardiogenesis revealed by cross-species studies are reviewed and correlated with human CHD.

There is increasing evidence suggesting that formation of the tracheobronchial tree and alveoli results from heterogeneity of the epithelial-mesenchymal interactions along the developing respiratory tract. Recent genetic data support this idea and show that this heterogeneity is likely the result of activation of distinct networks of signaling molecules along the proximal-distal axis. Among these signals, fibroblast growth factors, retinoids, Sonic hedgehog, and transforming growth factors appear to play prominent roles. We discuss how these and other pattern regulators may be involved in initiation, branching, and differentiation of the respiratory system.