Total Receptor Number Research Articles

Identification of muscarinic receptor subtypes in mouse parotid gland.

Immunoprecipitation of muscarinic receptors from mouse parotid membranes by specific subtype antisera showed that M3 and M1 receptors represented 75 and 15% of the total number of precipitable receptors, respectively. [N-methyl-3H]methylscopolamine (NMS) labeled a single class of high-affinity binding sites in membranes from parotid glands with a dissociation constant of 0.67 +/- 0.02 nM and a maximum binding capacity of 176 +/- 15 fmol/mg protein. Competition curves for NMS, atropine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and para-fluoro-hexahydro-sila-difenidol fit best to a one-site binding model, whereas pirenzepine and methoctramine fit best to a two-site binding model, indicating 76-90% M3 receptors. Results from the use of pirenzepine indicated that the second mouse parotid receptor subtype, unlike that of the submandibular gland, has atypical characteristics for an M1 receptor. The rank order of potency of muscarinic antagonists in inhibiting phosphoinositide turnover and biphasic effects of carbachol on isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was atropine > or = 4-DAMP >> pirenzepine > AF-DX 116. A specific M1 antagonist, m1-toxin, had no effect on carbachol augmentation or inhibition of isoproterenol responses. Results suggest that M3 receptors couple to both augmentation and inhibition of stimulated cAMP levels.

Regulation of β-adrenoceptor density and mRNA levels in the rat heart cell-line H9c2

The regulation of the expression of beta-adrenoceptor (beta-ARs) is not thoroughly understood. We demonstrate that the rat heart cell-line H9c2 expresses both beta 1- and beta 2-ARs. In radioligand-binding experiments, the maximal binding capacity of (-)-[125I]-iodocyanopindolol was determined as 18 +/- 0.6 fmol/mg of protein with a KD of 35.4 +/- 4.1 pM. Competitive radioligand-binding experiments with subtype-specific beta-antagonists reveal a subtype ratio of beta 1- to beta 2-ARs of 29%: 71%. With competitive reverse-transcriptase PCR we found beta 2-mRNA to be up to 1600 times more frequent than beta 1-mRNA. Treatment of the H9c2 cell-line with the beta-adrenergic agonist (-)-isoproterenol (10(-6) M), the antagonist (-)-propranolol (10(-6) M) and the glucocorticoid dexamethasone (500 nM) induces regulatory effects on both the beta-AR protein and mRNA level. Isoproterenol treatment leads to down-regulation of the total receptor number by 56 +/- 4%, due to a decrease in beta 2-ARs, while maintaining the beta 1-AR number constant. On the transcription level, both beta 1-and beta 2-mRNAs are decreased by 30% and 42% respectively. mRNA stability measurements reveal a reduced half-life of beta 2-mRNA from 9.3 h to 6.5 h after isoproterenol treatment. Incubation of cells with (-)-propranolol does not affect the amounts of beta-ARs and their mRNAs. Dexamethasone induces a 1.8 +/- 0.2-fold increase in beta-AR number over the basal level as well as a 1.9 +/- 0.2-fold increase in the amount of beta 2-mRNA. Because the half-life of beta 2-mRNA was unaffected by dexamethasone, the increased beta 2-mRNA level must be due to an enhanced transcription rate. The beta 1-mRNA levels are unchanged during dexamethasone-incubation of the cells. Our data clearly demonstrate that treatment of H9c2 rat heart cells with isoproterenol and dexamethasone induces alterations in the level of RNA stability as well as gene transcription, leading to altered receptor numbers.

EGF Receptor expression and growth of psoriatic and normal human keratinocytes are modulated by 1.25 (OH)2-vitamin D3 ex vivo

Calcitriol or 1.25 (OH)2-vitamin D3 is used in the treatment of psoriasis as an inhibitor of cell proliferation. We studied the action of calcitriol ex vivo on the growth of psoriatic and normal human keratinocytes, and on the expression of the EGF receptor. Third passaged normal and psoriatic keratinocytes were seeded (10(4)/cm2) in 24-well dishes in serum-free medium (MCDB supplemented with amino acids, with either 0.1 or 1.1 mM of calcium) and 10(-9) M calcitriol. When subconfluence was reached, cell counts and 125I-EGF binding studies were performed. Cell counts showed at least a 50% decrease in growth under all conditions studied (normal or psoriatic keratinocytes; 0.1 or 1.1 mM calcium) when calcitriol was added. 125I-EGF binding studies showed a decrease in total receptor numbers in the presence of calcitriol with acceleration of binding at low concentrations of 125I-EGF. Scatchard plot analysis showed only one type of high affinity receptor. Receptor sites were decreased (30% to 40% of controls) in the presence of calcitriol together with a decrease in the dissociation constant. In conclusion, at almost physiological concentrations ex vivo, calcitriol strongly decreased normal and psoriatic keratinocyte growth. This potent antiproliferative effect could in part be explained by the capacity of calcitriol to downregulate EGF receptor expression.

Muscarinic acetylcholine receptor trafficking in streptolysin O-permeabilized MDCK cells.

We investigated the validity of streptolysin O (SLO)-permeabilized Madin-Darbin canine kidney (MDCK) cells which express muscarinic acetylcholine receptors (mAChRs) coupled to pertussis toxin-sensitive guanine nucleotide-binding proteins (G proteins) for the study of the molecular machinery that regulated mAChR internalization and recycling. Exposure of SLO-permeabilized cells to carbachol-reduced cell surface receptor number by up to 40% without changing total receptor number. The kinetics and maximal extent of receptor internalization as well as the potency of carbachol to induce receptor internalization were almost identical in SLO-permeabilized and non-permeabilized cells. Using this semi-intact cell system, we studied the effect of various agents affecting components potentially involved in receptor trafficking. Internalization was prevented by treatment of the SLO-permeabilized MDCK cells with (i) the stable ATP analogues, adenosine 5'-O-(3-thiotriphosphate) and adenylylimidodiphosphate, to block ATP-dependent processes, and (ii) heparin to block G protein-coupled receptor kinases. Inclusion of the stable GTP analogue, guanosine 5'-O-(3-thiotriphosphate), increased the rate but not the extent of receptor internalization. None of the membrane-impermeant agents affected receptor internalization in intact MDCK cells. This model system also allowed recycling of internalized receptors back to the plasma membrane. After removal of the agonist, cell surface receptor number in SLO-permeabilized cells returned to control values within 90 min with the same kinetics as seen in intact cells. Inclusion of guanosine 5'O-(3-thiotriphosphate) shortened the recovery time. These data suggest that both ATP-dependent kinases including G protein-coupled receptor kinases and G proteins participate in receptor internalization and recycling. In summary, the SLO-permeabilized MDCK cell is a feasible model system for the study of mAChR internalization and recycling and allows manipulation of the intracellular milieu with membrane-impermeable macromolecules.

Changes in the binding characteristics of the mu, delta and kappa subtypes of the opioid receptor in the hypothalamus of the normal cyclic ewe and in the ovariectomised ewe following treatment with ovarian steroids.

The mu, delta and kappa opioid receptor subtypes were measured across the oestrous cycle of the ewe and in ovariectomised (OVX) ewes treated with oestrogen and/or progesterone. We have used a subtype-specific opioid receptor binding assay, in which [3H]diprenorphine non-preferentially labelled each receptor subtype in the presence of blocking concentrations of site-specific opioid analogues. The density and affinity of each receptor subtype was measured in the preoptic area (POA) of the hypothalamus and the mediobasal hypothalamus (MBH). Normally cycling ewes were killed during the luteal phase of the oestrous cycle and at various times after an injection of a synthetic prostaglandin (cloprostenol) to synchronise the onset of the follicular phase. OVX ewes were either untreated as controls (n = 4) or treated with oestrogen (n = 4), progesterone (n = 4) or oestrogen and progesterone combined (n = 4). The total number of opioid receptors did not alter across the oestrous cycle or with steroid hormone treatment. In the POA, the mean (+/- S.E.M.) number of delta receptors was significantly (P < 0.05) greater during the luteal phase than 24 h into the follicular phase (133 +/- 45 vs 35 +/- 8 fmol/mg protein). A significantly (P < 0.05) greater number of delta receptors was also found in the OVX progesterone-treated ewes compared with the control animals (172 +/- 9 vs 39 +/- 4 fmol/mg protein). In the MBH, the number of delta receptors was significantly (P < 0.01) greater in ewes killed 56 h after prostaglandin than luteal-phase ewes (184 +/- 40 vs 51 +/- 7 fmol/mg protein). The number of mu receptors in both the POA and the MBH was also significantly (P < 0.05) higher in the 56-h group than in the 12-h group. A similar trend was also observed in the steroid-treated animals, although differences did not reach statistical significance. The delta:mu ratio in the POA was significantly (P < 0.05) higher in the luteal-phase animals than any of the other groups killed after a cloprostenol injection that causes luteolysis. Similarly the ratio of delta receptor density to mu receptor density was greater (P < 0.05) in the OVX progesterone-treated ewes than in the OVX control ewes. No differences were found in the kappa receptor density across the cycle or with different steroid treatments. These data suggest that the relative proportions of the delta and mu subtypes of the opioid receptor in the hypothalamus change during the oestrous cycle. Regulation appears to be due to the feedback effects of ovarian steroids with progesterone altering the delta:mu ratio. In the MBH, there was a general increase in both delta and mu subtypes during the follicular phase of the oestrous cycle. This may explain, in part, how the responsiveness of the GnRH/LH axis to opioid peptides and antagonists changes across the cycle.

EFFECT OF MATERNAL MAGNESIUM SULFATE ADMINISTRATION ON BRAIN CELL FUNCTION IN THE COCAINE-EXPOSED FETAL GUINEA PIG. • 429

Previous studies from our laboratory have shown that prenatal cocaine exposure alters brain cell membrane function by decreasing membrane Na+,K+-ATPase activity and potentiates the glutamate/glycine-dependent activation of the NMDA receptor. Magnesium sulfate(MgSO4) attenuates neuronal membrane dysfunction in hypoxic newborn piglets by preserving N-methyl-D-aspartate (NMDA) receptor number and affinity, and Na+,K+-ATPase activity. Data describing the interaction of cocaine and magnesium on neuronal function are lacking. The present study tests the hypothesis that maternal MgSO4 administration will prevent cocaine-induced NMDA receptor/ion channel modification in the fetus. Studies were performed in 6 pregnant guinea pigs (58 days gestation) assigned to 3 groups: Cocaine-treated (COC, n= 2). Magnesium-treated (MG, n=2) and Magnesium/Cocaine-treated (MG-COC, n=2). Animals in the MG and MG-COC groups received an initial bolus of MgSO4 300 mg/kg i.p. followed by 50 mg/kg q 30 min for 5 hours. Mean maternal [Mg++] was 10.5 mg%. Cocaine HCl 30 mg i.p. was given to the COC and MG-COC groups one hour prior to delivery of the fetuses. 3[H]MK-801 binding studies, an index of NMDA receptor activation, were performed in a concentration range from 0.5 to 50 nM in a reaction medium containing 10mM HEPES (pH 7.0), 100 μM glutamate and glycine, and 75 μg protein. Total number of receptors (Bmax) in the COC, MG and MG-COC groups were 1.08 ± 0.13, 1.34 ± 0.07 and 1.19± 0.09 pmol/mg protein (mean±SD). Kd for the same groups were 2.64 ± 0.22, 5.18 ± 0.87 and 4.65 ± 0.01 nM. The data show that MgSO4 may prevent the cocaine-induced increase in the affinity (decreased Kd) of the NMDA receptor for 3[H]MK-801 in fetal brains. We speculate that cocaine-induced modification of the NMDA receptor ion/channel complex may alter NMDA-mediated developmental processes that are essential for fetal brain function. Furthermore, administration of MgSO4 to cocaine-exposed fetuses may attenuate neurotoxicity by the glutamate-type NMDA receptor in the developing fetal brain. (Funded by NIH-HD-20337, MOD#6- FY94-0135, UCPR 506-93)

Decrease in endothelin-1 renal receptors during the 1st month of life in the rat.

Endothelin-1 (Et1), like angiotensin II, is implicated in postnatal maturation and development. The present study was designed to identify Et1 receptors and subtype Et1 receptors present in rat kidney between 1 and 30 days of postnatal life. On day 1, high-affinity and high-density Et1 binding sites were identified in rat kidney. The dissociation constant and maximum binding for ET1 to membranes from whole kidney were 0.073 +/- 0.05 nM and 1,345.9 +/- 73 fmol/mg protein, respectively. On day 30, affinity and receptor density were markedly decreased. The dissociation constant and maximum binding were 0.147 +/- 0.021 nM (P < 0.01) and 633.2 +/- 56.4 fmol/mg protein (P < 0.001), respectively. Using BQ 123 (EtA-selective antagonist) and sarafotoxin S6c (EtB-selective agonist), the two Et1 receptor subtypes EtA and EtB were identified in 1- and 30-day-old rat kidney. BQ 123 selectively recognized EtA receptors with high affinity (2.9 +/- 0.44 on day 1 and 4.0 +/- 0.5 nM on day 30) and sarafotoxin S6c bound with higher affinity EtB receptors (0.871 +/- 0.14 on day 1 and 0.717 +/- 0.12 nM on day 30). Between birth and day 30, the EtA binding capacity was decreased (304 +/- 27 vs. 752 +/- 202 fmol/mg protein, P < 0.05), whereas EtB binding was not affected (514 +/- 87 vs. 656 +/- 171 fmol/mg protein, NS). The decrease in the total number of Et1 receptors during the 1st month of life may be due to the concomitant decrease in the number of EtA receptors. Increased Et1 receptor density in early postnatal life suggests an influence of Et1 on immature kidney circulation and/or kidney growth.

Modulation by 5-hydroxytryptamine of the somatostatin receptor-effector system and somatostatin levels in rat brain.

The role of 5-hydroxytryptamine (5-HT) in the acute regulation of the rat brain somatostatin (SS) receptor-effector system and somatostatin-like immunoreactivity (SSLI) content was examined. 5-HT administered i.c.v. in a volume of 10 microliters at a dose of 0.5 microgram (pH 3.4) increased the SSLI concentration at 60 min in the Wistar rat frontoparietal cortex and hippocampus (60%, P < 0.05; 72%, P < 0.01; respectively). These changes were associated with a significant increase in the total number of specific SS receptors in the frontoparietal cortex (24%, P < 0.05) and hippocampus (20%, P < 0.05), without changes in the affinity constant as compared with the control group. No significant differences were seen in the basal and forskolin (FK)-stimulated adenylate cyclase (AC) activities in both brain areas of 5-HT-treated rats when compared to the control group. The capacity of SS to inhibit the FK-stimulated AC activity in the frontoparietal cortex and hippocampus of 5-HT-treated rats was lower than in the control groups. The ability of the stable GTP analogue 5'-guanylylimidodiphosphate (Gpp(NH)p) to inhibit FK-stimulated AC activity in frontoparietal cortical and hippocampal membranes was markedly decreased in 5-HT-treated rats. To determine if the above-mentioned changes were related to the 5-HT activation of central 5-HT1 and 5-HT2 receptors, a non-selective 5-HT1 and 5-HT2 receptor antagonist, methysergide, was administered 60 min before the 5-HT injection. Pretreatment with methysergide (5 mg/kg i.p. in a volume of 400 microliters) prevented the 5-HT-induced changes in the SS receptor-effector system and in SSLI levels in both brain areas. Methysergide alone had no observable effect on the somatostatinergic system. These results suggest that the frontoparietal cortical and hippocampal somatostatinergic system can be regulated by 5-HT receptors.

Differentiation of NG108‐15 Neuroblastoma Cells by Serum Starvation or Dimethyl Sulfoxide Results in Marked Differences in Angiotensin II Receptor Subtype Expression

Differentiation of NG108-15 neuroblastoma cells following exposure to either 1.5% dimethyl sulfoxide (DMSO)/0.5% fetal bovine serum (FBS) or serum starvation resulted in significant differences in angiotensin (AT) receptor levels and the AT1/AT2 receptor ratio. When NG108 cells were differentiated for 4 days with DMSO/low serum, the number of AT binding sites increased 30-fold compared with the binding levels on undifferentiated (blast) cells. However, cells differentiated by serum starvation for 4 or 14 days resulted in only a modest 2.5- and fivefold increase in AT receptor levels, respectively, over the levels seen with the undifferentiated cells. KD values for all treatment conditions were not significantly different (0.71 +/- 0.11 nM, p = 0.06). Using the AT1 and AT2 isoform-specific receptor antagonists losartan and PD123319, the relative numbers of AT receptor subtypes on undifferentiated and differentiated cells were determined by competitive inhibition against 125I-[Sar1,Ile8]-angiotensin II (sarile). A majority of the AT receptors on undifferentiated NG108 cells were the AT1 subtype (AT1/AT2 receptor ratio of 8:3). Differentiation by serum starvation and DMSO/low serum treatment resulted in fivefold and 30-fold increases in AT receptor levels, respectively, compared with the levels seen with the undifferentiated cells. Although serum starvation increased the total number of AT1 and AT2 receptors, it did not significantly alter the AT1/AT2 receptor ratio. In contrast, differentiation with DMSO/low serum both increased the total number of AT1 and AT2 receptors and reversed the AT1/AT2 receptor ratio (1:3). The increase in AT receptors following differentiation with DMSO/low serum for 4 days was largely accounted for by an 80-fold increase in the AT2 receptor level. Previous studies by Tallant at al. (1991) and Bryson et al. (1992) reported increased AT2 receptor expression following neuroblastoma differentiation with dibutyryl cyclic AMP and DMSO/low serum, respectively, and suggested a role for the AT2 receptor in neuronal differentiation. In the present study, we have extended these earlier observations by demonstrating that the method of differentiation significantly affects both the AT receptor level and the ratio of AT1 to AT2 receptor expression. Finally, our findings indicate that the AT2 receptor is expressed as a consequence of neuronal maturation and dose not mediate morphological differentiation.

Stochastic model of receptor-mediated cytomechanics and dynamic morphology of leukocytes

The proposed mathematical model investigates the simplified cytomechanics of cell shape change driven by stochastic stimulation from chemosensory receptors. The cytomechanical component of our model describes the dynamical distribution of F-actin and associated forces in an idealized cortical actin network around the cell periphery. The chemosensory component describes the distribution of chemotactic receptors in the cell membrane surrounding the cortex, where bound receptors give rise to an intracellular signal which modulates some property of the cortical network. As in our earlier models, an account is made for (1) the reactive, contractive properties of cortical actin, but here also for a stress induced by curvature of the cortex-membrane complex which carries an effective surface tension, and (2) statistical fluctuations in receptor binding, but generalized here to include statistical fluctuations in the spatial distribution of receptors, entirely determined by the additional prescription of membrane diffusion coefficients along with total receptor number, receptor binding rate constants and the local concentration field of chemotactic factor. We simplify the analysis by restricting the model to a prototype in which viscous stresses in the cortical network are negligible and the radial extension of the cell cortex is a prescribed function of the cortical actin concentration. We assume in particular that the assembly rate of cortical actin depends on the local density of bound receptors. These assumptions lead to a 4th-order parabolic differential equation on the unit circle coupled to a system of stochastic differential equations. We characterize via bifurcation analysis, stochastic simulations, and analytical correlation functions the spatial-temporal pattern of cell morphology under the influence of fluctuations in the bound receptor distribution for the case of a uniform concentration field of chemotactic factor. In addition to addressing the biological significance of our model, we remark on its relevance to the generic problem of the influence of correlated stochastic perturbations on spatial patterns in morphogenetic media.

Modulation of macrophage scavenger receptor transport by protein phosphorylation

The identification of three highly conserved phosphorylation sites in the cytoplasmic domain of each of the monomeric subunits of the macrophage scavenger receptor suggests that protein phosphorylation may regulate this receptor pathway. To investigate this, mouse peritoneal macrophages were pretreated with either the protein phosphatase inhibitor okadaic acid or the protein kinase inhibitor staurosporine to modulate cellular protein phosphorylation and their effects on the metabolism of acetyl-LDL were measured. Both okadaic acid and staurosporine inhibited the degradation of acetyl-low density lipoprotein (LDL) without affecting cellular lactic dehydrogenase (LDH) levels. The inhibition by okadaic acid was due to a 70% decrease in acetyl-LDL binding whereas post-receptor processing was minimally affected. Calyculin A, another serine/threonine phosphatase inhibitor, also reduced acetyl-LDL binding, whereas lithium chloride, an inositol phosphatase inhibitor, did not. Okadaic acid did not decrease steady state receptor mRNA levels nor decrease the number of total cellular receptors, consistent with a posttranslational mechanism of action. Interestingly, protease sensitivity studies showed that the receptors were still located on the cell surface. These studies suggest that okadaic acid inhibits acetyl-LDL binding by causing the redistribution of surface receptors into a sequestered compartment or inactivating the receptors. In contrast, staurosporine produced a paradoxical increase in receptor expression (30%) but slowed post-receptor processing (2.3-fold decrease). The latter was due to an inhibition of ligand internalization (2.9-fold decrease) via a protein kinase C-independent mechanism. Macrophage pinocytosis was also slowed by staurosporine (38% decrease); however, this does not appear to account for the inhibition of scavenger receptor internalization. Direct receptor phosphorylation was also slowed by staurosporine (38% decrease); however, this does not appear to account for the inhibition of scavenger receptor internalization. Direct receptor phosphorylation was also investigated and it was established that the receptor can be phosphorylated; however, changes in receptor function did not correlate with changes in the degree of receptor phosphorylation. Together these studies demonstrate that changes in cellular protein phosphorylation affect the expression, surface transport, and internalization of the macrophage scavenger receptor and suggest that the regulated phosphorylation/dephosphorylation of cellular proteins may be an important biochemical mechanism that controls normal processing of ligands by this receptor pathway.

Glycine increases the number of somatostatin receptors and somatostatin-mediated inhibition of the adenylate cyclase system in the rat hippocampus

The glycine and somatostatin (SS) neurotransmission systems in the brain have been implicated in the function of sensory, motor, and nociceptive pathways. To investigate a possible relationship between these two components, we studied the influence of glycine on the binding of 125I-Tyr11-SS to its receptors and on SS-like immunoreactivity (SSLI) levels in the rat hippocampus and frontoparietal cortex. An intracerebroventricular (i.c.v.) dose of 16 or 160 nmol of glycine induced an increase in the total number of specific SS receptors in the hippocampus but not in the frontoparietal cortex at 15 min following injection, with no changes in the affinity constant. This effect seems to be mediated by inhibitory strychnine-sensitive glycine receptors since pretreatment with the antagonist strychnine (80 micrograms/100 g body weight, intravenously) abolished this response. No significant changes in SSLI content were detected in either brain region of glycine- and strychnine plus glycine-treated rats as compared to control values. Since SS receptors are coupled via guanine nucleotide-binding G proteins to the adenylyl cyclase (AC) system, we also examined the inhibitory effects of SS and the guanine nucleotide Gpp(NH)p on AC activity in hippocampal membranes of control, glycine- and strychnine plus glycine-treated rats since the increase in SS receptors was observed only in this brain area. No significant differences were observed for the forskolin (FK)-stimulated AC enzyme activities in hippocampal membranes from all the experimental groups studied. In the hippocampus of the glycine- (160 nmol) treated group, however, basal AC activity was significantly lower, and the capacity of SS to inhibit FK-stimulated AC activity was increased as compared to the control group. Pretreatment with strychnine prevented the increase in SS-mediated inhibition of AC activity. The functional activity of the inhibitory guanine nucleotide-binding protein Gi, as determined by the inhibitory effect of the stable GTP analogue Gpp(NH)p on FK-stimulated AC activity, was significantly higher in hippocampal membranes of glycine- (160 nmol) treated rats as compared to controls. This suggests that the increased inhibition of AC activity by SS in the glycine-treated group may be due to the increase in Gi activity and/or the increase in the number of SS receptors observed. Alternatively, the greater Gi activity may be responsible for the increased binding of 125I-Tyr11-SS to its receptors observed after glycine administration. Altogether, these data suggest that the hippocampal somatostatinergic system can be regulated by strychnine-sensitive glycine receptors in the rat.

Chronic increases in blood flow upregulate endothelin-B receptors in arterial smooth muscle.

Experiments were designed to characterize endothelin receptors in arteries after chronic increases in blood flow. A fistula was created between the femoral artery and vein in one hindlimb of dogs; contralateral blood vessels were sham operated. Sham- and fistula-operated arteries were removed 6 wk postoperatively. Some arteries were prepared for measurement of isometric force or for isolation of membrane proteins. Other arteries were used for histological staining with an endothelin-B (ETB) receptor antibody. In arteries suspended for the measurement of isometric force, endothelin-1 produced concentration-dependent increases in tension that were significantly greater in fistula- than in sham-operated arteries without endothelium. The ETB-receptor-selective peptide sarafotoxin S6c produced concentration-dependent increases in tension only in fistula-operated arteries. In receptor-binding studies of membrane proteins, Scatchard analysis of saturation binding with 125I-labeled endothelin-1 (125I-endothelin-1) indicated that the total number of receptors was greater in fistula-operated arteries; affinity was threefold less in fistula- than in sham-operated arteries. Competitive displacement of 125I-endothelin-1 by endothelin-3 was significant for a two-site model in membranes prepared from sham-and fistula-operated arteries. Competitive inhibition of 125I-endothelin-1 binding by sarafotoxin S6c was significant for a one-site binding model in all arteries. Sarafotoxin S6c binding sites were elevated significantly in fistula-operated arteries. Immunohistochemical staining for the ETB receptor was significantly greater in both the endothelium and smooth muscle of fistula- than in sham-operated arteries. These results suggest that chronic increases in blood flow upregulate endothelin receptors, including ETB receptors in arterial smooth muscle.

Cholecystokinin and morphine pharmacological intervention during 99mTc-HIDA cholescintigraphy: A rational approach

Pharmacological intervention with either cholecystokinin-8 (CCK-8) or morphine during 99mTc- hepatoiminodiacetic acid (HIDA) cholescintigraphy is required primarily for the assessment of the diseases affecting the gallbladder, the common bile duct, or the sphincter of Oddi. For imaging, the patient should be prepared by an overnight fast, or with 4 hours of minimum fast. Pre-emptying with CCK-8 is probably undesirable and should either be avoided or one should wait for at least 4 hours after CCK-8 to begin the 99mTc-HIDA study to achieve higher specificity of the test for acute cholecystitis. When he gallbladder is not observed by 60 mins in a clinical setting of acute cholecystitis, a dose of 0.04 mg/kg of morphine is administered intravenously and imaging continued for an additional 30 mins. Nonvisualization of the gallbladder by 90 mins with morphine in an appropriate clinical setting is diagnostic for acute cholecystitis. When the gallbladder is not observed by 60 min but is seen with morphine administered after 60 mins, a positive diagnosis of abnormal gallbladder function can be made. When the gallbladder is observed in a clinical setting of biliary pain or chronic calculous or acalculous cholecystitis, CCK-8 at a dose rate of 3.3 ng/kg/min is infused intravenously for 3 mins (10 ng/kg/3 min) for the measurement of the ejection fraction. An ejection fraction value of less than 35% is indicative of calculous or acalculous chronic cholecystitis. The gallbladder emptying is directly related to the total number of cholecystokinin receptors in the smooth muscle. The ejection fraction can be controlled to any desired level simply by controlling the dose rate or the duration of infusion of CCK-8. Morphine and other opiate metabolites circulate for many hours in blood and act on the sphincter of Oddi and decrease the gallbladder ejection fraction. Careful drug history, especially that of opiates, is very critical in all subjects with a low ejection fraction before assigning an abnormality to the gallbladder motor function.

Permanent alteration of muscarinic acetylcholine receptor binding in rat striatum after circling training during development

We evaluated the effect of circling training (CT) in the expression of muscarinic acetylcholine receptor (mAchR) in developing rat striatum. For this, male and female rats were subjected to CT at 20, 30, 40 and 60 days of age during 7 days. Animals trained at 30 days but not at other ages showed an average decreased binding to mAchR of 33% in males and 24% in females, representing a significant difference with respect to control non-trained animals (males P < 0.001, females P < 0.005), and showing also a differential response between sex ( P < 0.01). mAchR drop was found invariably either 2 months or 1 year after training indicating a long term plastic change due to circling training. Scatchard analysis showed that altered binding represents a variation of the total receptor number instead of its binding affinity, with no significant differences found among K d ( P > 0.1). mAchR variation was correlated with the motor performance accomplished in the test. Regarding total distance run, male rats trained for 3 days (300 meters run), for 5 days (600 meters) and for 7 days (900 meters) showed a drop of 19, 28 and 33% respectively ( r 2 = 0.91, P < 0.001), while female changes were of 21, 23 and 24% ( r 2 = 0.78, P < 0.001). Nevertheless, no correlation with running speed was found ( r 2 = 0.13 male, r 2 = 0.02 female; P > 0.1). In summary, these results demonstrate the presence of a limited sensitivity period during striatum development where mAchR expression may be affected by the activity performed during CT, representing a permanent alteration of the receptor levels.

Characterization of Insulin Resistance and NIDDM in Transgenic Mice With Reduced Brown Fat

We recently created a new model of murine obesity through transgenic ablation of brown adipose tissue (BAT) using a tissue-specific toxigene (6). The goal of the present study was to further define the altered glucose homeostasis and insulin resistance in these transgenic animals. Despite an approximately 30% increase in total body lipid, no abnormalities were observed in 6-week-old transgenic animals. At the age of 22-26 weeks, marked obesity in transgenic mice was associated with significant increases in blood glucose and plasma insulin levels and an abnormal response to both intraperitoneal glucose and insulin tolerance tests. Glucose transport in soleus muscle was reduced, with the response to insulin stimulation blunted by up to 85% in males and 55% in females. The total number of insulin receptors was decreased by 36% in muscle and 59% in adipose tissue of transgenic animals. Insulin receptor tyrosine kinase activity, which was assessed following maximal insulin stimulation in vivo, was reduced in transgenic animals by 59% in muscle and 56% in fat. GLUT4 mRNA and protein was unchanged in muscle of transgenic animals compared with in that of controls but was significantly reduced in adipose tissue. In conclusion, primary BAT deficiency results in the development of glucose intolerance or diabetes and severe insulin resistance with both receptor and postreceptor components. These animals should be a useful model for studies of obesity-linked diabetes and insulin resistance and related complications.

Decrease in β1-adrenergic and M2-muscarinic receptor mRNA levels and unchanged accumulation of mRNAs coding for Gαi‒2 and Gαs proteins in rat cardiac hypertrophy

During compensatory cardiac hypertrophy in the rat, hemodynamic overload induces a parallel decrease in the densities of both beta 1-adrenergic (beta 1-AR) and M2-muscarinic (M2-MR) receptors in the left ventricle, but the total number of receptors remains unchanged. It is not known whether this reduction is transcriptionally or translationally regulated, or if the functionally closely linked alpha-subunits of G protein (G alpha s and G alpha i-2) partake in this regulation. In order to resolve these questions, the absolute concentrations of mRNAs for both receptors and for G alpha s and G alpha i-2 were quantified by slot blot analysis of the left ventricles of adult rats 5 weeks after aortic banding. The results showed a significant decrease of both receptor mRNA levels in hypertrophied left ventricle (beta 1-AR: -48%; M2-MR: -42%) that paralleled the reduction in receptor protein densities and was negatively correlated with the left ventricular weight/body weight ratio (LVW/BW). By contrast, the relative levels of G alpha s and G alpha i-2 mRNAs remained unchanged, and both accumulated proportionally to the increase in LVW/BW. These results show that the beta 1-AR and the M2-MR were pretranslationally regulated. This suggests the hypothesis that the corresponding genes do not follow the general increase in transcriptional activity. By contrast, the genes coding for G alpha s and G alpha i-2 may follow the general pattern of activation during hypertrophy. Receptors and coupling proteins belong to two different groups of genes that are controlled by distinct mechanisms of regulation.

Tissue- and subtype-specific modulation of angiotensin II receptors by chronic treatment with cyclosporin A, angiotensin-converting enzyme inhibitors and AT1 antagonists.

We wished to determine whether chronic treatment of rats with cyclosporin A (CSA), an angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor antagonists modulates the angiotensin receptor density. In rats treated chronically with CSA, the vasoconstrictor response to angiotensin II (AII) is increased and this increase is modulated by ACEI and angiotensin receptor antagonists. Rats were treated for 6 weeks orally with CSA (15 mg/kg/day), the ACE inhibitor lisinopril (10 mg/kg/day), the angiotensin receptor antagonists DUP 753 (10 mg/kg/day), and D 8731 (10 mg/kg/day) and the combinations CSA + lisinopril, CSA + DUP 753, and CSA + D 8731. Olive oil was used as a control. The number of total AII receptors (Bmax) was determined by Scatchard analysis of [125I]Sar1 Ile8 AII binding in kidney, liver, adrenal cortex, and adrenal medulla. The receptor subtypes were analyzed with the specific antagonists DUP 753 (subtype 1) and PD 123319 (subtype 2). CSA upregulated angiotensin receptors in all organs studied. Lisinopril alone downregulated angiotensin receptors and abolished the effect of CSA in liver and adrenal cortex and medulla, but not in the kidney, where it had no effect. DUP 753 alone downregulated the angiotensin receptor subtype 1 in kidney, liver and adrenal cortex; its effect on the adrenal medulla in which 89% of angiotensin receptors are subtype 2, did not quite reach significance. The combination of DUP 753 and cyclosporin CSA abolished the CSA-induced increase in angiotensin receptor density in all four organs. The angiotensin receptor antagonists D 8731 downregulated the angiotensin receptors (subtype 1) in liver and kidney and upregulated angiotensin receptors (subtype 2) in the adrenal medulla.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo binding of a biologically active oligopeptide in vegetative buds of the scyphozoan Cassiopea andromeda: demonstration of receptor-mediated induction of metamorphosis

Vegetative buds of Cassiopea andromeda (Cnidaria, Scyphozoa) metamorphose into polyps in the presence of short synthetic peptides containing proline as the preterminal amino acid at the carboxyl terminus. In an in vivo assay, the binding of the biologically active hexapeptide 14C-dansyl-GPGGPA in buds was shown to be specific and saturable. Scatchard plot analysis of the specific binding data revealed a dissociation constant (KD) of about 7 μM. The total number of receptors was calculated to be approximately 1×1010 bud-1 under saturation conditions. In correlation with the results of prior investigations, our finding of receptor-mediated induction of metamorphosis strongly supports the hypothesis that binding of biologically active peptides to receptors leads to initiation of phosphatidylinositol breakdown and activation of protein kinase C.

Dopamine D1 receptor distribution in Sf9 cells imaged by confocal microscopy: a quantitative evaluation.

A c-myc epitope-tagged human dopamine D1 receptor (c-myc D1 receptor) was expressed in Sf9 cells and its cellular distribution under basal conditions and after exposure to the agonist dopamine was examined. In the basal state, immunofluorescently labeled c-myc D1 receptors imaged by confocal microscopy appeared as a bright ring of label predominantly on the cell surface, and to a lesser extent as intracellular clusters of label. This pattern of receptor distribution was confirmed by radioligand-binding assays on plasma membrane and light membrane fractions using the D1 receptor-antagonist [3H]-SCH-23390. After exposure to dopamine, c-myc D1 receptors were redistributed on the cell surface, changing from a continuous ring to a discontinuous pattern of label. Analysis of fluorescence intensity and three-dimensional computer reconstruction of labeled receptors revealed a 30% decrease in surface labeling with no decrease in total number of receptors confirmed by radioligand-binding analysis. These findings constituted the first direct evidence of agonist-induced D1 receptor internalization. The results showed that the combination of confocal microscopy and three-dimensional reconstruction can be used to visualize and assess receptor distribution in Sf9 cells.