Differences In Number Of Receptors Research Articles

The effect of genotypical and phenotypical variation in taste sensitivity on liking of ice cream and dietary fat intake

Emerging evidence suggests fat can be perceived as a taste. G-protein coupled receptors as well as CD36, a fatty acid translocase, have been proposed to be involved in fat perception. Therefore, differences in number of receptors and genotype of CD36 have both been proposed to influence inter-individual fat taste perception. Fungiform papillae density (FPD) and PROP taster status are phenotypes related to receptor number. Previous authors have proposed an association between such phenotypes and CA6 (gustin) genotype, because the latter influences receptor cell proliferation. The effect of these factors on fat perception, preference and intake, requires further investigation. Therefore, the main aim of this study was to investigate the effects of taste sensitivity, including both genotypic and phenotypic variation, on liking of ice cream and dietary fat intake. Participants (n=136) age 18–55years were recruited in the UK. Hedonic liking results demonstrated that liking for ice cream was significantly affected by the fat content of the sample, and by demographic factors (gender, ethnicity, age) but not by the consumers CD36 rs1761667 or CA6 rs2274333 genotype, PROP taster status nor FPD. However, categorising taste sensitivity from participant responses to salt taste alone (rather than to salt and PROP) found significant differences, with low salt perceivers liking the high fat (20%) ice cream substantially more than medium- and high salt perceivers. This indicated that increased taste sensitivity reduced liking of high fat. Cluster analysis highlighted that one group of consumers (18%) liked higher fat ice cream, whereas another (30%) liked lower fat ice cream compared to the 52% of consumers that liked ice cream regardless of fat content. There was a significant association between these groups and salt taste sensitivity. Concerning recorded dietary intake, the high-fat liker group were found to have substantially higher dairy product consumption compared to high-fat dislikers. Fat intake as a percentage of total energy intake was significantly related to CA6 genotype, however the minor allele frequency at rs2274333 is too low to draw firm conclusions within this study population.

Sex Differences in Osteoarthritis of the Knee

Osteoarthritis (OA) is a leading cause of disability in the United States. It is the most common form of arthritis and afflicts 13.9% of adults aged ≥25 years and 33.6% (12.4 million) of those aged >65 years—an estimated 26.9 million persons in the United States.1 Studies sponsored by the Centers for Disease Control and Prevention and the National Institutes of Health have identified differences in the incidence and severity of OA between men and women, as well as between racial and ethnic groups.2,3 The burden of OA is highest among women and African-Americans, who disproportionately develop knee and hand but not hip OA. The disproportionate number of women in the aging US population is of clinical concern because of the more severe knee OA and its impact on quality of life and independence. Based on these factors, there is a need for research focused on the effect sex differences have in the development and progression of OA as well as the impact on prevention and treatment strategies. However, most studies on the mechanisms underlying OA have not taken sex differences into account, whether in vitro cell culture or animal models were used. Although little is known about the mechanisms that contribute to disparities between men and women in disease incidence and severity, they likely involve mechanical and molecular events in the affected joint. Diagnosis of knee OA is based on evidence of joint pain and/or reduced space between articulating bone surfaces as a result of thinning of the opposing articular cartilages. However, multiple tissues that compose the knee joint appear to be compromised by the disease, including subchondral bone, articular cartilage, the meniscus, the anterior cruciate ligament, the synovium, and synovial fluid. A change in any of these tissues can influence the distribution of load across the joint, with corresponding adaptations in the other tissues and, ultimately, the cartilages. Such pathophysiologic changes may exacerbate age-related physiologic changes in joint function attributable to genetic characteristics, age, sex, and health status, leading to greater cartilage damage. To understand the expression of knee OA in males and females, it is important to view the knee as an organ rather than focusing only on the articular cartilage. Knee tissues are modulated by sex hormones during tissue development and throughout the life cycle in both males and females. Although menopause is associated with an increase in OA severity in women, systemic estrogen alone cannot explain the observed sex differences. Recent data, for example, show that sex-specific variations in the responses of chondrocytes to sex steroids are the result of differences in receptor number as well as mechanisms of hormone action.4 In addition to increased prevalence of knee OA, women often have greater pain and more substantial reduction in function and quality of life than do men.5 OA pain can be related to the sensory information that emerges from the knee joint. The pain does not always match the degree of injury, however, and can continue even after total joint arthroplasty. The neural and other mechanisms underlying these differences in pain between men and women with knee OA are unknown. By improving our understanding of the mechanisms responsible for sex differences in the perception of pain in OA, more effective and, possibly, sex-specific treatment strategies will emerge. Although the adaptations that accompany advancing age may be a major factor in its etiology in older patients, early-onset OA is becoming more common. Women with physically active lifestyles, such as athletes and workers in occupations that involve exposure to traumatic injury or to mechanical stress, are more subject to early onset OA. Anterior cruciate ligament injuries are particularly problematic in 16- to 20-year-old females. Approximately 50% of these young women will progress to OA in 10 to 15 years.6,7 The prevalence of obesity in children and young adults is escalating, and the impact of increased mechanical stress on the knee during bone growth and development is not yet fully understood. In addition, the role of sex differences, particularly hormonal regulation, and its influence on the onset and progression of OA is not yet known. In summary, epidemiologic studies have established that sex differences exist in the incidence and severity of knee OA. Therapeutic approaches to the management of OA, particularly regenerative medicine strategies, have not yet taken these sex differences into consideration. Effective interventions, however, will require a better understanding of the mechanisms involved in the disease and its differential expression in men and women.

Sex Differences in Osteoarthritis of the Knee

Osteoarthritis (OA) is a leading cause of disability in the United States. It is the most common form of arthritis and afflicts 13.9% of adults aged ≥25 years and 33.6% (12.4 million) of those aged >65 years—an estimated 26.9 million persons in the United States.1 Studies sponsored by the Centers for Disease Control and Prevention and the National Institutes of Health have identified differences in the incidence and severity of OA between men and women, as well as between racial and ethnic groups.2,3 The burden of OA is highest among women and African-Americans, who disproportionately develop knee and hand but not hip OA. The disproportionate number of women in the aging US population is of clinical concern because of the more severe knee OA and its impact on quality of life and independence. Based on these factors, there is a need for research focused on the effect sex differences have in the development and progression of OA as well as the impact on prevention and treatment strategies. However, most studies on the mechanisms underlying OA have not taken sex differences into account, whether in vitro cell culture or animal models were used. Although little is known about the mechanisms that contribute to disparities between men and women in disease incidence and severity, they likely involve mechanical and molecular events in the affected joint. Diagnosis of knee OA is based on evidence of joint pain and/or reduced space between articulating bone surfaces as a result of thinning of the opposing articular cartilages. However, multiple tissues that compose the knee joint appear to be compromised by the disease, including subchondral bone, articular cartilage, the meniscus, the anterior cruciate ligament, the synovium, and synovial fluid. A change in any of these tissues can influence the distribution of load across the joint, with corresponding adaptations in the other tissues and, ultimately, the cartilages. Such pathophysiologic changes may exacerbate age-related physiologic changes in joint function attributable to genetic characteristics, age, sex, and health status, leading to greater cartilage damage. To understand the expression of knee OA in males and females, it is important to view the knee as an organ rather than focusing only on the articular cartilage. Knee tissues are modulated by sex hormones during tissue development and throughout the life cycle in both males and females. Although menopause is associated with an increase in OA severity in women, systemic estrogen alone cannot explain the observed sex differences. Recent data, for example, show that sex-specific variations in the responses of chondrocytes to sex steroids are the result of differences in receptor number as well as mechanisms of hormone action.4 In addition to increased prevalence of knee OA, women often have greater pain and more substantial reduction in function and quality of life than do men.5 OA pain can be related to the sensory information that emerges from the knee joint. The pain does not always match the degree of injury, however, and can continue even after total joint arthroplasty. The neural and other mechanisms underlying these differences in pain between men and women with knee OA are unknown. By improving our understanding of the mechanisms responsible for sex differences in the perception of pain in OA, more effective and, possibly, sex-specific treatment strategies will emerge. Although the adaptations that accompany advancing age may be a major factor in its etiology in older patients, early-onset OA is becoming more common. Women with physically active lifestyles, such as athletes and workers in occupations that involve exposure to traumatic injury or to mechanical stress, are more subject to early onset OA. Anterior cruciate ligament injuries are particularly problematic in 16- to 20-year-old females. Approximately 50% of these young women will progress to OA in 10 to 15 years.6,7 The prevalence of obesity in children and young adults is escalating, and the impact of increased mechanical stress on the knee during bone growth and development is not yet fully understood. In addition, the role of sex differences, particularly hormonal regulation, and its influence on the onset and progression of OA is not yet known. In summary, epidemiologic studies have established that sex differences exist in the incidence and severity of knee OA. Therapeutic approaches to the management of OA, particularly regenerative medicine strategies, have not yet taken these sex differences into consideration. Effective interventions, however, will require a better understanding of the mechanisms involved in the disease and its differential expression in men and women.

Considerations in the design and use of chimeric G protein-coupled receptors

This chapter describes methods that can be employed to generate hybrid G protein-coupled receptors (GPCRs) and that are useful for the proper analysis of such mutant receptors. All examples discussed in this chapter involve the mutational analysis of different members of the muscarinic acetylcholine receptor family (M 1 –M 5 ). These receptors are ideally suited for hybrid receptor studies, as the five muscarinic receptor subtypes show distinct pharmacological and functional properties, while sharing a high degree of sequence homology. A meaningful interpretation of hybrid receptor data requires that experimental conditions are carefully chosen, such that differences in receptor numbers or improper receptor folding or localization do not interfere with proper data analysis. Given the extraordinary size of the GPCR superfamily and the ease with which chimeric GPCRs can be created by the use of modern PCR technology, hybrid GPCRs will continue to serve as important tools for researches in the GPCR field for many years to come.

Transcriptional regulation of insulin-like growth factor-I receptor gene expression in prostate cancer cells.

A marked decrease in the type 1 insulin-like growth factor (IGF) receptor (IGF-IR) occurs in prostate epithelial cells during transformation from the benign to the metastatic state. One of the principal regulators of IGF-IR gene expression, the WT1 tumor suppressor, is expressed in prostate cancer and in prostate cancer cell lines. The purpose of this study was to determine whether the decrease in IGF-IR expression was transcriptionally regulated, and whether WT1 action may be involved in the repression of the IGF-IR gene in prostate cancer cells. The P69 cell line was derived by immortalization of human primary prostate epithelial cells with simian virus-40 T antigen and is rarely tumorigenic. The M12 line was derived from the P69 line by selection for tumor formation in nude mice and is tumorigeneic and metastatic. P69 cells express 20,000 IGF-IR/cell, whereas M12 cells express 3,500 IGF-IR/cell. These differences in receptor number are reflected in proportional differences in IGF-IR mRNA levels. To assess IGF-IR promoter activity in these cell lines, each was transiently transfected with luciferase reporter vectors containing the IGF-IR gene transcription start site and 476 bp of 5'-flanking sequence, 640 bp of 5'-untranslated region sequence, or both regions. The promoter activity of the full-length construct was 50% lower (P < 0.01) in M12 cells compared with P69 cells, the activity of the 5'-flanking region construct was 53% lower (P < 0.0001), and that of the 5'-untranslated region construct was 36% lower (P = 0.01). P69 clones stably transfected with a WT1 expression vector exhibited decreased expression of the endogenous IGF-IR gene and decreased promoter activity in transient transfection assays with IGF-IR promoter constructs containing multiple WT1 binding sites. The observed reduction in endogenous IGF-IR expression was sufficient to inhibit IGF-I-stimulated cell proliferation. These data suggest that most of the decreased expression of the IGF-IR seen in malignant prostate epithelium is the result of transcriptional repression of the IGF-IR gene, and that this repression may be due in part to the increased expression of the WT1 tumor suppressor in metastatic prostate cancer.

Transcriptional Regulation of Insulin-Like Growth Factor-I Receptor Gene Expression in Prostate Cancer Cells

A marked decrease in the type 1 insulin-like growth factor (IGF) receptor (IGF-IR) occurs in prostate epithelial cells during transformation from the benign to the metastatic state. One of the principal regulators of IGF-IR gene expression, the WT1 tumor suppressor, is expressed in prostate cancer and in prostate cancer cell lines. The purpose of this study was to determine whether the decrease in IGF-IR expression was transcriptionally regulated, and whether WT1 action may be involved in the repression of the IGF-IR gene in prostate cancer cells. The P69 cell line was derived by immortalization of human primary prostate epithelial cells with simian virus-40 T antigen and is rarely tumorigenic. The M12 line was derived from the P69 line by selection for tumor formation in nude mice and is tumorigeneic and metastatic. P69 cells express 20,000 IGF-IR/cell, whereas M12 cells express 3,500 IGF-IR/cell. These differences in receptor number are reflected in proportional differences in IGF-IR mRNA levels. To assess IGF-IR promoter activity in these cell lines, each was transiently transfected with luciferase reporter vectors containing the IGF-IR gene transcription start site and 476 bp of 5′-flanking sequence, 640 bp of 5′-untranslated region sequence, or both regions. The promoter activity of the full-length construct was 50% lower (P < 0.01) in M12 cells compared with P69 cells, the activity of the 5′-flanking region construct was 53% lower (P < 0.0001), and that of the 5′-untranslated region construct was 36% lower (P = 0.01). P69 clones stably transfected with a WT1 expression vector exhibited decreased expression of the endogenous IGF-IR gene and decreased promoter activity in transient transfection assays with IGF-IR promoter constructs containing multiple WT1 binding sites. The observed reduction in endogenous IGF-IR expression was sufficient to inhibit IGF-I-stimulated cell proliferation. These data suggest that most of the decreased expression of the IGF-IR seen in malignant prostate epithelium is the result of transcriptional repression of the IGF-IR gene, and that this repression may be due in part to the increased expression of the WT1 tumor suppressor in metastatic prostate cancer.

Contribution of single-channel properties to the time course and amplitude variance of quantal glycine currents recorded in rat motoneurons.

The amplitude of spontaneous, glycinergic miniature inhibitory postsynaptic currents (mIPSCs) recorded in hypoglossal motoneurons (HMs) in an in vitro brain stem slice preparation increased over the first 3 postnatal weeks, from 42 +/- 6 pA in neonate (P0-3) to 77 +/- 11 pA in juvenile (P11-18) HMs. Additionally, mIPSC amplitude distributions were highly variable: CV 0.68 +/- 0.05 (means +/- SE) for neonates and 0.83 +/- 0.06 for juveniles. We wished to ascertain the contribution of glycine receptor (GlyR)-channel properties to this change in quantal amplitude and to the amplitude variability and time course of mIPSCs. To determine whether a postnatal increase in GlyR-channel conductance accounted for the postnatal change in quantal amplitude, the conductance of synaptic GlyR channels was determined by nonstationary variance analysis of mIPSCs. It was 48 +/- 8 pS in neonate and 46 +/- 10 pS in juvenile HMs, suggesting that developmental changes in mIPSC amplitude do not result from a postnatal alteration of GlyR-channel conductance. Next we determined the open probability (Popen) of GlyR channels in outside-out patches excised from HMs to estimate the contribution of stochastic channel behavior to quantal amplitude variability. Brief (1 ms) pulses of glycine (1 mM) elicited patch currents that closely resembled mIPSCs. The GlyR channels' Popen, calculated by nonstationary variance analysis of these currents, was approximately 0.70 (0.66 +/- 0.09 in neonates and 0.72 +/- 0.05 in juveniles). The decay rate of patch currents elicited by brief application of saturating concentrations of glycine (10 mM) increased postnatally, mimicking previously documented changes in mIPSC time course. Paired pulses of glycine (10 mM) were used to determine if rapid GlyR-channel desensitization contributed to either patch current time course or quantal amplitude variability. Because we did not observe any fast desensitization of patch currents, we believe that fast desensitization of GlyRs underlies neither phenomenon. From our analysis of glycinergic patch currents and mIPSCs, we draw three conclusions. First, channel deactivation is the primary determinant of glycinergic mIPSC time course, and postnatal changes in channel deactivation rate account for observed developmental changes in mIPSC decay rate. Second, because GlyR-channel Popen is high, differences in receptor number between synapses rather than stochastic channel behavior are likely to underlie the majority of quantal variability seen at glycinergic synapses throughout postnatal development. We estimate the number of GlyRs available at a synapse to be on average 27 in neonate neurons and 39 in juvenile neurons. Third, this change in the calculated number of GlyRs at each synapse may account for the postnatal increase in mIPSC amplitude.

Signaling via A2A adenosine receptor in four PC12 cell clones.

PC12 cells are genetically labile and so-called wild-type cells comprise multiple subclones. We have examined the A2A adenosine receptor signal transduction pathways in four such clones (denoted clones 1, 19, 21 and 27) of PC12 cells. Adenosine A2A, A2B and A1 receptor mRNAs were detected in all four clones by RT-PCR, whereas no A3 receptor mRNA was found. A2A receptors were quantitated by radioligand binding using the antagonist radioligand [3H]SCH 58261 ([3H]-5-amino-7(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo [1,5-c] pyrimidine). The Bmax was highest in clone 1 followed by clones 21, 19 and 27. Whereas the amount of G(i) protein appeared similar in all four clones, the amount of G(s) protein was higher in clones 21 and 27 than in the other two clones. Maximal responses to the non-selective adenosine analogue NECA (5'-N-ethylcarboxamidoadenosine) were similar to those observed with the selective adenosine A2A receptor agonist CGS 21680 (2-[p-(2-carbonylethyl) phenylethylamino]-5'-N-ethylcarboxamidoadenosine), and were approximately equal in clones I and 21, but lower in clone 19 and very low in clone 27. For both compounds EC50 was significantly higher in clone 27 than in clone 1. In both clones the response to NECA could be competitively antagonized by a selective adenosine A2A antagonist, SCH 58261. The present results show that different clones of PC 12 cells differ widely in the cAMP increase induced by adenosine analogues and that this is due to differences in the amount of adenosine A2A receptor, G protein and effector. A large difference in receptor number resulted in differences in potency of an agonist.

Structure and function of the ovine type 1 corticotropin releasing factor receptor (CRF 1) and a carboxyl-terminal variant

Corticotropin releasing factor (CRF) is the major neuropeptide regulating the hypothalamo–pituitary–adrenocortical axis in most species. A pituitary receptor for CRF (designated CRF 1) belonging to the seven-transmembrane helix, G-protein-coupled receptor superfamily has been cloned for human, rat, mouse and xenopus. Since ovine CRF shares only 84% identity to human/rat CRF (h/rCRF) we postulated that the sheep pituitary CRF 1 receptor may have similarly diverged from the rodent and human CRF 1. We report the molecular cloning of an ovine pituitary cDNA containing a 1245 bp open reading frame encoding a 415 amino acid sheep CRF 1 receptor 78, 86, 94, and 95% homologous to xenopus, chicken, rat, mouse, and human CRF 1, respectively. The divergence in primary structure between the sheep CRF 1 and the other mammalian CRF 1s is primarily localized to the extracellular amino terminal domain of the receptor (18 of 22 divergent residues, ovine vs human CRF 1). A variant of the oCRF 1 was also isolated (oCRF 1var) with 133 bp deleted from nucleotide (nt) 1080 to nt 1213 of the open reading frame (ORF) resulting in a new ORF of 1176 nt predicting a 392 residue CRF 1 variant receptor. The 133 bp deletion would cause a frame-shift at residue 358 within the carboxyl-third of the seventh transmembrane domain (TM7) resulting in a shortened cytoplasmic tail with a new amino acid sequence from residue 358 to 392. Scatchard analysis of saturation curves using membrane prepared from Cos 7 cells transfected with oCRF 1 or oCRF 1var indicated that both wild-type and variant receptors were expressed similarly (number of CRF binding sites) and both bound oCRF with high affinity [oCRF 1 ( K d): 2.5+1.6 nM; oCRF 1var: 5.1+2.3 nM]. The non-hydrolyzable GTP analogue (GTP γS) lowered the affinity of both wild-type and variant oCRF 1 receptors to a similar extent (oCRF 1: 18.2 nM; oCRF 1var: 22.4 nM). Both wild-type and variant oCRF 1 receptors exhibited ≈10-fold greater selectivity for oCRF and sauvagine compared to h/rCRF or α-helical [9–41]oCRF. CRF effectively stimulated the accumulation of cAMP (EC 50=51 pM) in Cos 7 cells transiently transfected with wild-type but not variant oCRF 1 receptor. In Cos 7 cells transfected with oCRF 1var, cAMP accumulation was only observed at the highest concentration of oCRF utilized (100 nM). Basal (unstimulated) levels of cAMP in Cos 7 cells transfected with oCRF 1var (in the presence of 2 mM IBMX) were ≈50% lower than for the wild-type oCRF 1. Differences in cAMP accumulation could not be attributed to differences in receptor number since total binding sites in the transfected cells were not different between wild-type or variant oCRF 1 receptors. Agonist-induced receptor internalization, determined as the percent of total [ 125I] Tyr 0-oCRF bound located in the acid-resistant fraction of transfected Cos 7 cells, increased with time (0–60 min at 37°C) for both wild-type and variant oCRF 1. Wild-type CRF 1 internalized ≈2-fold greater percent of total [ 125I] Tyr 0-oCRF bound compared to the variant receptor. In summary, an ovine CRF 1 and a CRF 1 cytoplasmic tail receptor variant displaying high affinity binding to oCRF as well as selectivity for oCRF vs h/rCRF, were cloned from an adult sheep pituitary cDNA library. GTP γS studies indicate that both variant and wild-type receptors couple efficiently to G αs however, only the wild-type oCRF 1 is capable of stimulating cAMP production at physiological levels of CRF. Agonist-induced internalization of the ovine CRF 1var is also reduced compared to the wild-type CRF 1 receptor. We suggest that the oCRF 1var interacts efficiently with G αs but is unable (post-hormonal binding) to effectively stimulate G-protein activation of adenylate cyclase, indicating that the cytoplasmic tail of the CRF 1 can modulate receptor function related to signal transduction. The cytoplasmic carboxyl terminus of the CRF 1 also appears to play a role in agonist-induced internalization of this member of the class II subfamily of G-protein coupled receptors (GPCRs).

Characterization of endothelin receptors in human varicose veins

Experiments were designed to characterize endothelin receptors in human varicose veins. Three groups of veins were studied: (1) varicose vein (VV) tributaries of the greater saphenous vein from patients who were undergoing vein stripping for primary varicosity; (2) greater saphenous veins (SVs) from the same patients; and (3) greater saphenous veins from patients without varicosity who were undergoing arterial reconstruction (control). Veins were either cut into rings and suspended in organ chambers for measurement of isometric force, prepared for receptor binding of membrane proteins, or were prepared for measurement of preproendothelin mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). Endothelin-1 (10(-11) to 10(-7) mol/L) produced similar concentration-dependent contractions in rings with or without endothelium. Maximal tensions were significantly greater in control veins compared with either SVs or VVs. Sarafotoxin S6c (10(-11) to 3 x 10(-7) mol/L), which is selective for the endothelin-B receptor, also produced concentration-dependent increases in tension in all veins. Sarafotoxin S6c responses in VVs were shifted significantly rightward compared with either SVs or control. Maximal tensions to sarafotoxin S6c also were significantly greater in control veins compared with either SVs or VVs. In receptor binding studies, the number of binding sites as defined by competitive inhibition of 125I-endothelin-1 by endothelin-1 was less in VVs than control veins. Competitive inhibition of 125I-endothelin-1 with endothelin-3 (both A and B receptors) or sarafotoxin S6c (B receptors only) suggests that the difference in receptor number between varicose and nonvaricose veins is attributable to differences in the endothelin-B receptor subtype. Binding affinities were not significantly different for either of the receptor subtypes in all veins studied. Preproendothelin mRNA as quantitated by RT-PCR tended to be higher in VVs compared with either SVs or control veins. Decreased contractions to endothelin-1 in both varicose and saphenous veins of patients with primary varicosity may be associated with a decrease in the number of receptors. These receptors may be downregulated in response to increased production of endothelin-1, which is regulated at the transcriptional level.

Changes in reactivity towards 5-hydroxytryptamine in the renal vasculature of the diabetic spontaneously hypertensive rat.

Diabetes and hypertension are both associated with an increased risk of renal disease. The combination of these diseases produces marked acceleration of the problems. To examine the reactivity in the renal vasculature of diabetic hypertensive rats. We investigated the reactivity towards 5-hydroxytryptamine (5-HT) in Krebs solution-perfused kidneys of diabetic normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). In addition, the interaction between the thromboxane A2 mimetic U46619 and 5-HT was examined. Discrete dose-response curves were obtained for the response to 5-HT (0.1-30 micrograms/g kidney) in Krebs solution-perfused kidneys of control and diabetic WKY rats and SHR. The following order of reactivity was determined: control SHR > diabetic SHR > control WKY rats = diabetic WKY rats. The thromboxane A2 mimetic U46619 (10 ng/ml) potentiated responses to 5-HT significantly in kidneys of diabetic WKY rats and control SHR, but not in kidneys from control WKY rats and diabetic SHR. The differential affected reactivity towards 5-HT kidneys from diabetic and hypertensive rats might be due to previously documented differences in receptor number. The marked effect of U46619 on the reactivity towards 5-HT in kidneys of diabetic and control rats indicates that this interaction might be important given the increased levels of thromboxane A2 reported to occur in these diseases. The reason for the lack of effect of thromboxane/prostaglandin receptor stimulation on responses to 5-HT in the combined model (i.e. diabetic hypertensive rats) needs to be determined.

Spinal nicotinic receptor expression in spontaneously hypertensive rats.

Intrathecal administration of nicotinic agonists previously has been shown to result in exaggerated pressor and heart rate responses as well as greater nociceptive behavior in adult (12-week-old) spontaneously hypertensive rats (SHR) than in age-matched normotensive Wistar-Kyoto rats (WKY). Paradoxical to these augmented responses to nicotinic agonists in SHR, nicotinic receptor number in the spinal cord as measured by cytisine binding sites is lower in adult SHR than normotensive WKY and Sprague-Dawley rats. Using the high-affinity agonist epibatidine, we found similar differences in receptor number between strains in both in vitro ligand binding experiments with spinal cord membranes and in situ autoradiographic analyses. Spinal nicotinic receptor number did not differ in 5-week-old prehypertensive SHR and age-matched WKY; however, receptor numbers were higher in young rats of both strains than in their adult counterparts. Antihypertensive treatment (25 mg/kg per day hydralazine PO) in 6-week-old SHR from 6 to 12 weeks of age markedly reduced the progressive rise in blood pressure yet did not alter nicotinic receptor number compared with untreated rats. Similar treatment of WKY with hydralazine produced a slight fall in blood pressure but no change in receptor number. Thus, normalization of blood pressure by hydralazine in SHR does not result in a return of receptor expression to levels seen in normotensive rats. Higher centrally mediated pressor activity or augmented postcoupling events after neuronal nicotinic receptor stimulation may slowly downregulate expression of spinal nicotinic receptors in this genetically hypertensive rat strain.

Sex differences in estrogen receptor and progestin receptor induction in the guinea pig hypothalamus and preoptic area

Quantitative in vitro autoradiography was used to determine if regional sex differences in estrogen receptor (ER) content and/or estrogen responsiveness, as indicated by an increase in progestin receptor (PR), are present in the adult guinea pig brain. Adult male and female guinea pigs were gonadectomized 1 week before subcutaneous injection of 25 μg estradiol benzoate (EB)/kg body wt or the sesame oil vehicle. Animals were killed by decapitation 44 h after injection. Unoccupied PRs, and unoccupied and occupied ERs, were measured in discrete brain regions by quantitative in vitro autoradiography using [ 3H]R5020 and [ 3H]estradiol as ligands, respectively. In vehicle-injected controls, a higher level of ER was found in the arcuate nucleus (ARC), dorsal medial nucleus (DMN) and ventrolateral nucleus (VLN) of females as compared to males. At 44 h after EB injection, 32–55% of the ERs were occupied; however, EB treatment caused a marked down-regulation of total receptor (calculated as occupied + unoccupied receptor) in most of the brain regions examined, including the periventricular preoptic area (PVP), medial preoptic area (WO), bed nucleus of the stria terminalis, paraventricular nucleus, ARC, ventrolateral hypothalamus (VLH), VLN, and DMN. In EB-treated animals, PR binding was detectable in the PVP, MPO, ARC, VLH, and VLN, with higher levels of binding observed in the PVP, MPO, and VLN of the female as compared to the male. No PR binding was observed in oil-injected control animals. These results demonstrate region-specific sex differences in ER as well as estrogen-induced regulation of progestin and ERs in the guinea pig brain. The discordance between the regional distributions of sex differences in ER and estrogen-induced PR implies that sex differences in ER and estrogen-induced PR implies that sex differences in estrogen response may not be clearly linked to a sex difference in receptor number. Instead, sex differences in response may involve differences in receptor number within specific subpopulations of estrogen target cells or may involve differences in ER dynamics.

Analysis of the antinociceptive actions of the κ-opioid agonist enadoline (CI-977) in neonatal and adult rats: comparison to κ-opioid receptor mRNA ontogeny

Earlier reports indicate that κ-opioid agonists may be especially potent in the formalin test of tonic nociception, and that neonatal rat pups are more sensitive to μ-agonists, when compared to adults. We have assessed the potency of enadoline (CI-977), a novel and selective κ-opioid agonist, in the formalin and tail-flick nociceptive tests in 3-day-old rat pups and compared their responses to adults in the same tests. In addition, the recent cloning of the κ-opioid receptor has allowed us to make the first evaluation of the ontogeny of κ-opioid receptor mRNA in an effort to elucidate a possible mechanism for differences in sensitivity to κ-opioid agonists. Enadoline was found to be a potent antinociceptive agent in the formalin test; the neonates were eight times more sensitive than the adults. κ-Opioid receptor mRNA, however, is present in whole brain at adult levels as early as postnatal day 3. Previous studies have shown κ-opioid receptor levels, as measured by radioligand binding and receptor autoradiography, to be present at postnatal day 3 as well. Consequently, it is unlikely that gross differences in receptor number subserved the modest increase in sensitivity to enadoline observed in the neonates in the formalin test. Enadoline was less potent and less effective in the tail-flick test in the neonates. The adults were similarly insensitive to the antinociceptive effects of enadoline in the tail-flick test. Additional studies indicated that enadoline significantly increased locomotor activity, as assessed by the open-field test, in neonates, while decreasing activity in the adults. Studies with U50.488 (U50), and nor-binaltorphamine ( nor-BNI) suggest that these increases in activity observed in the neonates may be κ-mediated. Although the open-field studies do not relate directly to κ-opioid antinociception, the observation of behavioral activation in the neonates may limit the use of κ-agonists in pediatric populations.

Specific [3H]spiperone binding sites in the pituitary of rainbow trout (Oncorhynchus mykiss) and goldfish (Carassius auratus)

Dopamine, a catecholamine neurohormone, modulates pituitary hormone release in teleost fishes and other vertebrates. The existence and binding parameters of a pituitary dopamine-neuroleptic receptor from trout were examined and compared with those from goldfish. Pituitary homogenate was incubated with [3H]spiperone (D2 antagonist) under several experimental paradigms; incubations were terminated by filtration and bound 3H radioactivity was assessed by liquid scintillation spectroscopy. Specific binding of [3H]spiperone was tissue dependent. Equilibrium displacement analyses using domperidone (D2 antagonist) indicated a single class of binding site (LIGAND) with Kd = 2.49 +/- 0.89 microM and a capacity of 3.10 +/- 0.45 nmol/mg protein; the goldfish Kd and capacity were both significantly (p < 0.05) larger: Kd = 4.63 +/- 0.30 microM and capacity = 20.66 +/- 2.03 nmol/mg protein. The Kd and capacity for the trout pars distalis (2.45 +/- 0.33 microM and 3.27 +/- 0.24 nmol/mg protein, respectively) did not differ significantly (p < 0.05) from that of the neurointermediate lobe (2.50 +/- 0.08 microM and 3.58 +/- 0.56 nmol/mg protein, respectively). Dopamine D2 receptor ligands differentially displaced [3H]spiperone from the trout pituitary, while D1 ligands, a D4 ligand, and a 5-hydroxytryptamine (5HT2) receptor antagonist had only small nonspecific effects. Comparison of the trout and goldfish pituitary dopamine-neuroleptic receptor indicates conservation of receptor affinity (Kd); however, differences in receptor numbers and in the distribution of receptors between the pars distalis and neurointermediate lobe in the two species may be due in part to species or developmental differences, and may reflect differences in the role(s) and degrees of influence of dopamine in these fishes.

Islet Amyloid Polypeptide Stimulates Cyclic AMP Accumulation via the Porcine Calcitonin Receptor

CHO-cells stably transfected with an expression vector for the porcine calcitonin receptor were exposed to various concentrations of IAPP, CGRP or calcitonin from different species. In these, but not in untransfected cells, rat IAPP mediated cAMP accumulation at concentrations above 25 nM. This potency was three orders of magnitude lower than that of porcine calcitonin and two and four orders of magnitude lower than those of human and salmon calcitonin, respectively. Human β-CGRP had an effect similar to rat IAPP whereas human α-CGRP was at least one order of magnitude less potent than rat IAPP. COS cells expressing recombinant porcine calcitonin receptors transiently or stably showed a different pattern of responses to calcitonin, IAPP and the CGRPs. In these cells, rat IAPP was as potent an inducer of cAMP as was salmon or porcine calcitonin and more potent than human calcitonin or the CGRPs. The dissociation constants for salmon calcitonin binding to the porcine calcitonin receptors on CHO and COS cells were 0.2 nM and 2.0 nM and the corresponding number of binding sites per cell were approximately 7 × 10 4 and 2 × 10 6, respectively. These results demonstrate that IAPP and CGRP can mediate signal transduction via the porcine calcitonin receptor and provide a possible explanation for the calcitonin-like effects of pharmacological levels of IAPP and CGRP administrated in vivo. The difference between CHO and COS cells in their relative response to the various ligands may relate to the difference in receptor number, post-transcriptional processing, or to dissimilarities in the signal transduction pathways between the two cell types.

Distribution of angiotensin II receptors in rat and human adipocytes.

Angiotensin II (AII) receptor binding assays were performed in rat adipocytes from three separate anatomic depots. Fat cells were isolated by collagenase digestion, and plasma membranes were prepared from the epididymal, mesenteric, and retroperitoneal fat depots of male Sprague-Dawley rats at 100 days of age. Binding of 125I-labeled [Sar1,Ile8]AII was rapid, saturable, and specific in membranes from all depots, identifying a receptor with a similar affinity of approximately 1 nM. Site-associated differences in receptor number were observed, with epididymal and mesenteric fat cell membranes exhibiting significantly more receptors than retroperitoneal fat cells when binding was expressed per unit of membrane protein. When corrected for cell volume, the number of receptors per cell ranked epididymal > retroperitoneal > mesenteric. Inhibitory constants for the peptide agonists AII and AIII and the peptide antagonist [Sar1,Ala8]AII indicated similar affinities in all three depots. Because the receptor has been classified pharmacologically into two subtypes, the AT1 selective antagonist losartan, and the AT2 selective antagonist PD 123,319 were used to classify the adipocyte receptor, indicating an AT1 subtype with an affinity for losartan in the mesenteric and retroperitoneal adipocytes that was significantly greater than the epididymal. Similar studies were performed in adipocyte membranes obtained from human omental and subcutaneous adipose tissue, revealing the presence of an AII receptor in both depots with an affinity of approximately 10 nM for losartan. These data indicate site-specific differences in AII receptor number in fat cell membranes from rats and the existence of human adipocyte AII receptors, suggesting that the adipocyte is significant for the peripheral metabolism of components of the renin-angiotensin system.

Association between neutrophil functions and periparturient disorders in cows

Summary Neutrophil functions were examined in healthy periparturient dairy cows (n = 46) and in cows with retained placenta and metritis complex (n = 20); metritis (n = 18); or mastitis (n = 13). Blood samples (50 ml) were collected from each cow via jugular vein twice weekly from 1.5 weeks before to 4 weeks after parturition. Neutrophil function was evaluated, using 6 tests: random migration, chemotaxis, ingestion, myeloperoxidase activity (iodination), superoxide production (cytochrome C reduction), and antibodydependent cell-mediated cytotoxicity. Ability to ingest bacteria and random migration activity of neutrophils from clinically normal cows were high around parturition and increased immeidately after parturition, whereas myeloperoxidase activity and antibody-dependent cell-mediated cytotoxicity ability of neutrophils from these cows decreased after parturition. Measurement of neutrophil function in 4 over-iectomized cows revealed significant (P &lt; 0.0005) seasonal changes in results of all 6 functional assays. We observed various defects of neutrophil function in all cows with abnormal conditions after parturition. Before parturition, superoxide production activity by neutrophils from cows with metritis and chemotaxis by neutrophils from cows with mastitis were significantly (P &lt; 0.001 and P &lt; 0.05, respectively) lower, indicating that a defect of neutrophil function may be a predisposing factor in the development of these disorders. In conclusion, the host defense role of neutrophils in periparturient cows was impaired, principally because of a defect in killing capacity, which may increase susceptibility to infections. We also investigated the in vitro effects of arachidonic acid metabolites and recombinant human colonystimulating factors (rhcsf) on functions of neutrophils from clinically normal and postparturient cows with abnormalities, including retained placenta, metritis, or mastitis (n = 5/group). Each abnormal cow was matched for postpartum period with a clinically normal cow. Neutrophils from individual cows were preincubated with arachidonic acid metabolites (prostaglandin F2a, 10−7M; prostaglandin E2, 10−6M; leukotriene B4, 10−8M; and lipoxin B, 10−8M) and rhcsf (rh-granulocyte-csf, 1,000 or 6,000 U/ml; rh-granulocyte-macrophage-csf, 5 or 15 ng/ml) in a 37 C water bath for 30 minutes before submitting them to function assays. There was no response by neutrophils from either clinically normal or abnormal postparturient cows to treatment with either arachidonic acid metabolites or rhcsf in any of the 6 functional assays. However, preincubation of neutrophils alone in a 37 C water bath for 30 minutes resulted in some alteration of neutrophil function. This may indicate translocation of receptors between an intracellular pool and the cell surface, as well as differences in receptor numbers or signal transduction mechanisms in these abnormal cows.

Regulation of intracellular free Ca 2+ by the LH/CG receptor in an established cell line 293 expressing transfected rat receptor

Luteinizing hormone (LH)/chorionic gonadotropin (CG) receptor (LHR) regulation of intracellular free Ca 2+, [Ca 2+] i, was studied by spectrofluorometric analysis and digital imaging of intracellularly trapped fura-2 fluorescence in a stable cell line 293 expressing transfected rat LHR. Exposure of the suspensions of 293-LHR cells to human CG (hCG) resulted in single, dose-dependent burst of elevated [Ca 2+] i, the maximum being obtained at 0.1–1 μg hCG/ml. The subconfluent individual 293-LHR cells responded to 1 /gmg hCG/ml with either a single or oscillating [Ca 2+] i bursts, the appearance of the oscillation being dependent upon the presence of extracellular Ca 2+ ([Ca 2+] e). 72% of the cells produced oscillation in response to hCG in the presence of [Ca 2+] c and only 33% in the absence. Moreover, removal of [Ca 2+] e from the incubation medium lowered the elevated basal [Ca 2+] i level to or below the prestimulatory level and concomitantly damped out the oscillation, while its readdition without hCG was capable of re-elevating [Ca 2+] i level and of gradually restoring the oscillation. The 293-LHR cells exposed to increasing doses of hCG also developed a dose-dependent desensitization of [Ca 2+] i increase to renewed hormonal stimulation. The [Ca 2+] i bursts within individual 293-LHR cells appeared in particular regions at the cell peripheries rather than distributed uniformly throughout the cytoplasm, pointing to compartmentation of the Ca 2+ stores and to a local differences in receptor number in most cells. Similarly, the second [Ca 2+] i burst in the partially desensitized cells initiated from the same peripheral locations as the first one, this finding being consistent with the fact that no massive receptor redistribution on the cell surface occurs as a consequence of the first hormone challenge. These results are compatible with the concept that the LH/CG receptor is capable of activating the Ca 2+ intracellular signalling pathway, and that rapid intracellular Ca 2+ increase may represent one of the initial steps in the mechanism of LH/CG action.

Evidence for receptors specific for 17 beta-estradiol and testosterone in chondrocyte cultures.

Recently, sex hormones were shown to stimulate chondrocyte differentiation and matrix protein synthesis in vitro in a sex-specific and maturation-dependent manner. The aim of the present study was to determine whether cytosolic receptors in these cells would specifically bind 17 beta-estradiol and testosterone, and if so, whether binding was gender- and maturation-dependent. Confluent, fourth passage cultures of cells derived from male or female rat costochondral growth zone and resting zone cartilage were homogenized and specific binding of 17 beta-estradiol or testosterone measured in the cytosolic fraction. Scatchard analysis indicated the presence of a high-affinity 17 beta-estradiol receptor (Kd = 4.5 to 8.7 x 10(-11) M), with low binding capacity (3.9 to 11.2 fmol/mg protein). Chondrocytes from female rats were found to have a significantly greater binding capacity for 17 beta-estradiol than chondrocytes from male rats. However, cells from both sexes had binding capacities that were independent of cell maturation. A high-affinity testosterone receptor (Kd = 4.3 to 6.3 x 10(-11) M) with low binding capacity (4.1 to 5.9 fmol/mg protein) was found in both males and females, but no difference in binding capacity was noted, either as a function of gender or stage of cell maturation. Immunohistochemistry using antibodies against 17 beta-estradiol and testosterone and the 17 beta-estradiol nuclear receptor (D-75) confirmed that 17 beta-estradiol and testosterone receptors were present in chondrocytes from both male and female rats. These data demonstrate that chondrocytes from growth zone and resting zone cartilage are capable of binding both 17 beta-estradiol and testosterone. This suggests that these hormones mediate their direct effects on chondrocytes via receptors specific for their appropriate ligand. The sex-specific effects of 17 beta-estradiol may be due to differences in receptor number between chondrocytes derived from female and male rats. In contrast, the sex-specific effects of testosterone may be regulated at the post receptor level since no differences in binding capacity were found between males and females.