The effects of oestrogens and progesterone on oestrogen receptors in female rat liver.

The administration of oestradiol-17 beta or ethynyloestradiol as well as the synthetic progestogen norethisterone acetate resulted in translocation of the oestrogen receptor. Progesterone and the synthetic progestogen (+)-norgestrel were ineffective. The increases in nuclear oestrogen receptor content 1 h after injection of each steroid were similar but different subsequently. The increase with oestradiol-17 beta extended for 3--6 h and for at least 9 h with ethynyloestradiol. With norethisterone acetate, nuclear content was still increased after 24 h. Oestrogen injection resulted in cytosol receptor depletion and a 'deficit' in receptor content extending for 6 h, whereas norethisterone acetate-induced translocation was quantitative. With injections of norethisterone acetate + ethynyloestradiol the increase at 1 h and retention of the nuclear receptors were similar to that with norethisterone acetate alone. In contrast, the depletion of cytosol receptor and its restoration were similar to that seen with ethynyloestradiol alone, suggesting that norethisterone acetate did not interfere with the oestrogen receptor replenishment. Specific binding in vitro of [3H]oestradiol-17 beta in liver cytosols was inhibited by (+)-norgestrel and norethisterone acetate, but not progesterone, at concentrations of 10--100 microM. Nuclear receptors present after norethisterone acetate injection bound oestrogen with high affinity (Kd = 1.52 nM), similar to receptors of oestrogen-injected animals. In the uterus, differential retention of nuclear receptors in response to oestrogens is associated with different cellular responses. The differences in the response of the receptor system in liver to the various steroids suggests that the corresponding tissue responses may also be dissimilar. These results are discussed in relation to the problems of liver dysfunction in oral-contraceptive users.     

Non-opioid actions of opioid peptides

Beside the well known actions of opioid peptides on mu-, delta- and kappa-opioid receptors, increasing amount of pharmacological and biochemical evidence has recently been published about non-opioid actions of various opioid peptides. These effects are not abolished by naloxone treatments. Such non-opioid effects are observed both in nervous tissues and in the cellular elements of the immune system. Peptides exhibiting non-opioid effects include beta-endorphin, dynorphin A, nociceptin/OFQ, endomorphins, hemorphins and a number of Proenkephalin A derived peptides, such as Met-enkephalin, Met-enkephalin-Arg-Phe (MERF) and bovine adrenal medullary peptide (BAM22). Non-opioid actions are exerted through different neuronal receptors, e.g., dynorphin hyperalgesia through NMDA receptor, Met-enkephalin induced regulation of cell growth through zeta receptors, pain modulation by nociceptin through ORL-1 or NOP receptors, while BAM22 acts through sensory neuron specific G protein-coupled receptors (SNSR). We have investigated Met-enkephalin-Arg-Phe (MERF) and its analogues by the means of direct and indirect radioligand binding assays. It has been found that in addition to kappa(2) and delta-opioid receptors, MERF can act also through sigma(2)- or probably via FMRF-NH(2) receptors in rat cerebellum. A role of functionally assembling heterodimer receptors in mediating the non-conventional actions of these peptide ligands can not be excluded as well.     

beta-Carbolines, psychoactive compounds in the mammalian body. Part II: Effects

The biochemical and the pharmacological effects of beta-carbolines in animals and man are reviewed. Biochemical studies have revealed beta-carbolines' several actions, including inhibition of MAO-A, competitive inhibition of 5-HT uptake, general inhibition of Na+ dependent transports, binding to benzodiazepine and opiate receptors and probable action on dopamine receptors, which may all participate to a variable degree in the actions of different beta-carbolines. Many early in vivo studies, however, have concentrated on some harmala alkaloids, particularly harmaline or harmine. The effects of beta-carbolines in man are compared in this review with the symptoms of alcohol withdrawal. However, no human studies have been reported with those tetrahydro-beta-carbolines shown to occur in human body in normal conditions or after alcohol intake. To prove any connections of beta-carbolines with the withdrawal syndromes or other neurological and psychiatric diseases means that these compounds have to be shown to have abnormal central nervous system concentrations in these diseases. The physiological role of beta-carbolines has yet to be shown. They may act as neuromodulators and some, especially 6-methoxytetrahydro-beta-carboline, may have an endocrinological function. It has been suggested that some beta-carbolines act as the physiological ligands (agonists) of the benzodiazepine receptors, but the physiological beta-carbolines so far known seem to have other effects, such as the inhibition of MAO-A or 5-HT uptake in low concentrations.     

The development of vasopressin antagonists

Antagonists of the physiological actions of vasopressin can be useful probes for detecting the influences of endogenous vasopressin on cardiovascular regulation. Antagonists that block vascular receptors of the V1 type have been widely used for this purpose. Recently effective antagonists of renal antidiuretic receptors of the V2 type have become available, and we have made progress toward improving their specificity for V2 receptors. Vasopressin antagonists of both types of responses could become useful in the diagnosis and treatment of pathophysiological states in which elevated levels of circulating vasopressin may disturb cardiovascular and renal functions.     

Glutamate receptors and pain

Pain is an important survival and protection mechanism for animals. However, chronic/persistent pain may be differentiated from normal physiological pain in that it confers no obvious advantage. An accumulating body of pharmacological, electrophysiological, and behavioral evidence is emerging in support of the notion that glutamate receptors play a crucial role in pain pathways and that modulation of glutamate receptors may have potential for therapeutic utility in several categories of persistent pain, including neuropathic pain resulting from injury and/or disease of central (e.g., spinal cord injury) or peripheral nerves (e.g., diabetic neuropathy, radiculopathy) and inflammatory or joint-related pain (e.g., rheumatoid arthritis, osteoarthritis). This review focuses on the role of glutamate receptors, including both ionotropic (AMPA, NMDA and kainate) and metabotropic (mGlu1-8) receptors in persistent pain states with particular emphasis on their expression patterns in nociceptive pathways and their potential as targets for pharmacological intervention strategies.     

Zinc modulation of glycine receptors in acutely isolated rat CA3 neurons

Glycine and GABA are the primary inhibitory neurotransmitters in the spinal cord and brain stem, with glycine exerting its physiological roles by activating strychnine-sensitive ionotropic receptors. Glycine receptors are also expressed in the brain, including the cortex and hippocampus, but their physiological roles and pharmacological properties are largely unknown. Here, we report the pharmacological properties of functional glycine receptors in acutely isolated rat CA3 neurons using conventional whole-cell patch clamp techniques. Both glycine and taurine, which are endogenous agonists of glycine receptors, elicited Cl(-) currents in a concentration-dependent manner. The glycine-induced current (I(Gly)) was inhibited by strychnine, picrotoxin or cyclothiazide in a concentration-dependent manner. At lower concentrations (0.01-1 microM), ICS-205,930 potentiated I(Gly), but at higher concentrations (>10 microM) it inhibited I(Gly). These pharmacological properties strongly suggest that CA3 neurons express functional strychnine-sensitive glycine receptors containing alpha2 subunits. Furthermore, at lower concentrations (1-30 microM), Zn(2+) potentiated I(Gly), but at higher concentrations (>100 microM) it inhibited I(Gly). Considering that Zn(2+) is synaptically co-released with glutamate from mossy fiber terminals that make excitatory synapses onto CA3 neurons, these results suggest that endogenous Zn(2+) modulation of these glycine receptors may have an important role in the excitability of CA3 neurons.     

Molecular mechanisms of steroid receptor-mediated actions by synthetic progestins used in HRT and contraception

Synthetic progestins are used by millions of women as contraceptives and in hormone replacement therapy (HRT), although their molecular mechanisms of action are not well understood. The importance of investigating these mechanisms, as compared to those of progesterone, has been highlighted by clinical evidence showing that medroxyprogesterone acetate (MPA), a first generation progestin, increases the risk of breast cancer and coronary heart disease in HRT users. A diverse range of later generation progestins with varying structures and pharmacological properties is available for therapeutic use and it is becoming clear that different progestins elicit beneficial and adverse effects to different extents. These differences in biological activity are likely to be due to many factors including variations in dose, metabolism, pharmacokinetics, bioavailability, and regulation of, and/or binding, to serum-binding proteins and steroidogenic enzymes. Since the intracellular effects on gene expression and cell signaling of steroids are mediated via intracellular steroid receptors, differential actions via the progesterone and other steroid receptors and their isoforms, are likely to be the major cause of differential intracellular actions of progestins. Since many progestins bind not only to the progesterone receptor, but also to the glucocorticoid, androgen, mineralocorticoid, and possibly the estrogen receptors, it is plausible that synthetic progestins exert therapeutic actions as well as side-effects via some of these receptors. Here we review the molecular mechanisms of intracellular actions of old (MPA, norethisterone, levonorgestrel, gestodene) vs. new (drospirenone, dienogest, trimegestone) generation progestins, via steroid receptors.     

Hyaluronan.

Hyaluronan (hyaluronic acid) is a high-molecular-mass polysaccharide found in the extracellular matrix, especially of soft connective tissues. It is synthesized in the plasma membrane of fibroblasts and other cells by addition of sugars to the reducing end of the polymer, whereas the nonreducing end protrudes into the pericellular space. The polysaccharide is catabolized locally or carried by lymph to lymph nodes or the general circulation, from where it is cleared by the endothelial cells of the liver sinusoids. The overall turnover rate is surprisingly rapid for a connective tissue matrix component (t1/2 0.5 to a few days). Hyaluronan has been assigned various physiological functions in the intercellular matrix, e.g., in water and plasma protein homeostasis. Hyaluronan production increases in proliferating cells and the polymer may play a role in mitosis. Extensive hyaluronidase-sensitive coats have been identified around mesenchymal cells. They are either anchored firmly in the plasma membrane or bound via hyaluronan-specific binding proteins (receptors). Such receptors have now been identified on many different cells, e.g., the lymphocyte homing receptor CD 44. Interaction between a hyaluronan receptor and extracellular polysaccharide has been connected with locomotion and cell migration. Hyaluronan seems to play an important role during development and differentiation and has other cell regulatory activities. Hyaluronan has also been recognized in clinical medicine. A concentrated solution of hyaluronan (10 mg/ml) has, through its tissue protective and rheological properties, become a device in ophthalmic surgery. Analysis of serum hyaluronan is promising in the diagnosis of liver disease and various inflammatory conditions, e.g., rheumatoid arthritis. Interstitial edema caused by accumulation of hyaluronan may cause dysfunction in various organs.     

On the specificity of naloxone as an opiate antagonist.

Since the discovery of endogenous opioid peptides in brain (68,69,97,113, 128) and the pituitary gland (26,81,105,125) there has been considerable interest in their possible roles in a variety of physiological and pharmacological processes. Many studies have used antagonism by naloxone as a criterion for implicating endogenous opiates in a process, assuming that naloxene has no pharmacological actions other than those related to blockade of opiate receptors. The doses of naloxene used are often higher than those required to antagonize the analgesic and other effects of morphine. However, multiple forms of opiate receptors are present in nervous tissue and higher concentrations of naloxene are required to antagonize effects mediated by some of these receptors (83). Although the earlier literature supports the assumption that the effects of naloxene are due to the blockade of opiate receptors (87), there are an increasing number of reports which indicate that naloxene may have pharmacological actions unrelated to opiate receptor blockade. The subsequent review serves to emphasize that antagonism by naloxene is a necessary but not sufficient criterion for invoking the mediation of a response by an endogenous opiate (61). Additional lines of evidence which serve to strengthen the conclusion that endogenous opiates mediate a process will be considered.     

The use of brain slices in central nervous system pharmacology

Brain slice preparations have most frequently been employed to answer questions of a biochemical or physiological nature. Nevertheless, in vitro brain slices also have considerable value as pharmacological tools with which to study the physiological actions of neurotransmitters and drugs on the central nervous system. Several aspects of the slice preparation facilitate this type of analysis. Because drugs can be applied and tested in a relatively quantitative manner, many classical pharmacological techniques (e.g., dose-response curves, tests for competitive vs. noncompetitive antagonism) can be used to examine drug responses. At the simplest level, these techniques facilitate the differentiation of specific (primarily receptor-mediated) and nonspecific actions of drugs and neurotransmitters. As another consequence, the electrophysiological actions of drugs can be directly compared to their biochemical effects in vitro (receptor binding, activation or inhibition of adenylate cyclase, etc.). Ultimately, parallel studies of this type can be used to establish mechanisms of action for various neurotransmitters, particularly those that may employ biochemical substrates as second messengers. In this paper we describe in general terms many of the advantages of the slice preparation as a neuropharmacological tool. Some of the criteria useful in determining the involvement of various receptors in drug-induced changes in electrophysiological responses are discussed in detail. Finally, the responses of hippocampal slices to various adrenergic agents are used to illustrate the way in which various features of this preparation can be exploited pharmacologically. The results of these experiments constitute a significant advance in terms of our understanding of the neuropharmacology of catecholamine responses in this brain region.     

Effect of Oestrogens and Progestogens on Liver Function in the Puerperium

A modified bromsulphthalein test has been used to detect alterations in liver function in puerperal women taking either a synthetic oestrogen, stilboestrol, or a pure progestogen, megestrol acetate. The oestrogen appreciably reduced the ability of the liver to excrete dye into the bile. It also reduced significantly the equivalent liver volume and the plasma clearance of dye. The progestogen did not have this effect. In so far as these results represent liver dysfunction they are due to the oestrogen component of the combined preparation.     

Molybdate modulates mitogen and cyclosporin responses of human peripheral blood lymphocytes

The trace element molybdenum (Mo) is an essential component of key physiological systems in animals, plants and microorganisms. The molybdate oxoanion MoO(4)(2-) has been demonstrated to cause diverse yet poorly understood biochemical and pharmacological effects, such as non-specific inhibition of phosphatases and stabilization of steroid receptors. This study aimed to investigate the effects of molybdate on the activation of human peripheral blood lymphocytes (hPBLs) ex vivo and its potential interaction with the widely used immunosuppressant drug cyclosporin A (CsA). Lymphocyte activation was evaluated by performing multiple experiments determining blastogenesis in cultured peripheral blood lymphocytes obtained from 5 healthy volunteers, following stimulation induced by phytohemagglutinin (PHA), in the absence or presence of 0.05-10 mM sodium molybdate or/and 2.5-30 μg/mL CsA. Blastogenesis was assessed by a morphometric assay based on the relative proportions of unactivated lymphocytes, activated lymphoblasts and cells with aberrant morphology after PHA-induced activation. Molybdate concentrations up to 1 mM showed no effect on lymphocyte blastogenesis, while higher concentrations exerted immunosuppressive actions on cultured hPBLs. Co-administration of 0.1 mM sodium molybdate with CsA, at doses up to 20 μg/mL, induced no alteration in the response of cultured hPBLs to CsA. However, molybdate potentiated the immunosuppressive action of higher CsA concentrations, implying a likely dose-related synergistic interaction of the two agents in PHA-stimulated blood lymphocytes. These observations are indicative of the possible biological importance of molybdate oxoanions in the modulation of hPBL activation that may have pharmacological consequences during the therapeutic application of immunomodulatory drugs.     

The therapeutic potential of hydrogen sulfide: separating hype from hope

Hydrogen sulfide (H(2)S) has become the hot new signaling molecule that seemingly affects all organ systems and biological processes in which it has been investigated. It has also been shown to have both proinflammatory and anti-inflammatory actions and proapoptotic and anti-apoptotic effects and has even been reported to induce a hypometabolic state (suspended animation) in a few vertebrates. The exuberance over potential clinical applications of natural and synthetic H(2)S-"donating" compounds is understandable and a number of these function-targeted drugs have been developed and show clinical promise. However, the concentration of H(2)S in tissues and blood, as well as the intrinsic factors that affect these levels, has not been resolved, and it is imperative to address these points to distinguish between the physiological, pharmacological, and toxicological effects of this molecule. This review will provide an overview of H(2)S metabolism, a summary of many of its reported "physiological" actions, and it will discuss the recent development of a number of H(2)S-donating drugs that show clinical potential. It will also examine some of the misconceptions of H(2)S chemistry that have appeared in the literature and attempt to realign the definition of "physiological" H(2)S concentrations upon which much of this exuberance has been established.     

Multiple opiate receptors: emerging concepts

Increasing biochemical evidence indicates that the wide spectrum of opiate pharmacological actions are mediated via heterogeneous classes of receptors. μ receptors have been identified as the high affinity sites where morphine-like opiates exert their analgesic effects. δ receptors have a somewhat different CNS distribution and have been identified as sites relatively selective for the naturally occuring enkephalins. Recent biochemical studies provide evidence for two additional classes of opiate receptor sites which were originally proposed on the basis of physiological studies. Ketocyclazocine-like opiates produce their unique ataxic and sedative effects via interaction with K receptors, and SKF-10,047 (N-allylnorcyclazocine) and related opiates produce stimulant and psychotomimetic effects via interactions with σ receptors.Many opiate drugs interact at multiple receptor sites. Thus, the constellation of neuropharmacological actions of a particular opioid ligand may reflect its various potencies at a combination of μ, δ, K, and σ receptors.     

Pharmacological examination of cholecystokinin (CCK-8)-induced contractile activity in the rat isolated pylorus

The actions of cholecystokinin (CCK) in the production of a satiety-like state have been suggested to be mediated via receptors for CCK which are located in the pylorus. We investigated the actions of CCK and other pharmacological agents upon the isolated rat pylorus in vitro. We used the change in isometric tension of the tissue preparation (contraction amplitude) as the measure of the effects of the pharmacological agents. Cholecystokinin COOH-terminal octapeptide (CCK-8) was observed to elicit contraction in a dose-dependent manner, with the half-maximal dose (ED50) in the vicinity of 1 nM. Rapid desensitization to CCK was observed. The contraction amplitude was atropine-independent, and was not significantly antagonized by a wide variety of other pharmacological agents. The Na+-channel blocker tetrodotoxin was without effect upon contractile amplitude, as was the K+-channel blocker 4-aminopyridine, except at very high concentrations. Neurotensin, bombesin, and the substance P and bombesin antagonist spantide all elicited contraction in the isolated tissue; neurotensin had a similar potency to CCK-8 and bombesin was 10-15-fold less potent than CCK-8. Unsulfated CCK-8 was at least 170-fold less potent than sulfated CCK-8 and tetragastrin was at least 500-fold less potent than CCK-8. These results suggest that pyloric CCK receptors, which appear to have a pharmacological profile typical of peripheral CCK receptors, may have a physiological role in the peptidergic control of gastric emptying in the rat.     

Enantiomers of 11-hydroxy-10-methylaporphine having opposing pharmacological effects at 5-HT1A receptors

The two enantiomers of the title compound have been prepared by different synthetic routes. Both bind strongly to 5-HT1A receptors from rat forebrain membrane tissue. However, in a guinea pig ileum preparation, the (R)-enantiomer exhibits properties consistent with its being an agonist, whereas the (S)-enantiomer shows no agonist effect, but it blocks the actions of the (R)-enantiomer and of 8-hydroxy-2-di-n-propylaminotetralin (8-OH-DPAT), a 5-HT1A agonist. These data are presented as a rare example of enantiomers which demonstrate opposite pharmacological effects at the same receptor.     

Biochemistry, physiology and pathophysiology of the extracellular calcium-sensing receptor

Calcium (Ca(2+)) has long been recognized as a physiologically indispensable ion owing to its numerous intra- and extracellular roles. More recently, it has become apparent that extracellular calcium (Ca(2+)(o)) also serves as an extracellular first messenger following the cloning of a Ca(2+)(o)-sensing receptor (CaR) that belongs to the superfamily of G protein-coupled receptors (GPCR). The CaR probably functions as a dimer in performing its central role of "sensing" minute alterations in Ca(2+)(o) and adjusting the secretion of parathyroid hormone (PTH) so as to normalize Ca(2+)(o) through the actions of PTH on the effector elements of the mineral ion homeostatic system (e.g., kidney, bone and intestine). Several inherited human conditions are caused by inactivating or activating mutations of this receptor, and mice have been generated with targeted disruption of the CaR gene. Characteristic changes in the functions of parathyroid and kidney in patients with these conditions and in CaR-deficient mice have proven the physiological importance of the CaR in mineral ion homeostasis. An accumulating body of evidence, however, suggests that the CaR also plays numerous roles outside the realm of systemic mineral ion homeostasis. The receptor regulates processes such as cellular proliferation and differentiation, secretion, membrane polarization and apoptosis in a variety of tissues/cells. Finally, the availability of specific "calcimimetic", allosteric CaR activators - which are currently in clinical trials - will probably have therapeutic implications for diseases caused by malfunction of the CaR in tissues not only within, but also outside, the mineral ion homeostatic system.     

Nuclear receptors, mitochondria and lipid metabolism

Lipid metabolism is a continuum from emulsification and uptake of lipids in the intestine to cellular uptake and transport to compartments such as mitochondria. Whether fats are shuttled into lipid droplets in adipose tissue or oxidized in mitochondria and peroxisomes depends on metabolic substrate availability, energy balance and endocrine signaling of the organism. Several members of the nuclear hormone receptor superfamily are lipid-sensing factors that affect all aspects of lipid metabolism. The physiologic actions of glandular hormones (e.g. thyroid, mineralocorticoid and glucocorticoid), vitamins (e.g. vitamins A and D) and reproductive hormones (e.g. progesterone, estrogen and testosterone) and their cognate receptors are well established. The peroxisome-proliferator activated receptors (PPARs) and liver X receptors (LXRs), acting in concert with PPARgamma Coactivator 1alpha (PGC-1alpha), have been shown to regulate insulin sensitivity and lipid handling. These receptors are the focus of intense pharmacologic studies to expand the armamentarium of small molecule ligands to treat diabetes and the metabolic syndrome (hypertension, insulin resistance, hyperglycemia, dyslipidemia and obesity). Recently, additional partners of PGC-1alpha have moved to the forefront of metabolic research, the estrogen-related receptors (ERRs). Although no endogenous ligands for these receptors have been identified, phenotypic analyses of knockout mouse models demonstrate an important role for these molecules in substrate sensing and handling as well as mitochondrial function.