Downregulation of hepatocyte P2-purinoceptor binding capacity after trauma-hemorrhage/resuscitation

1994 ◽  
Vol 266 (6) ◽  
pp. R1804-R1809
Author(s):  
M. S. Mahmoud ◽  
P. Wang ◽  
S. R. Hootman ◽  
S. S. Reich ◽  
I. H. Chaudry
Keyword(s):  
Dissociation Constants ◽  
Binding Capacity ◽  
Isolated Hepatocytes ◽  
Scatchard Analysis ◽  
Midline Laparotomy ◽  
Binding Characteristics ◽  
Shed Blood ◽  
P2 Purinoceptors ◽  
Altered Glucose ◽  
Affinity Receptor

Although P2-purinoceptors play an important role in the regulation of liver metabolism under normal conditions, it is not known if trauma-hemorrhage and resuscitation have any effects on such receptors. To study this, we performed a 5-cm midline laparotomy (i.e., trauma induced) on rats and then bled them to and maintained them at a mean arterial pressure of 40 mmHg until 40% of maximum bleedout volume was returned in the form of Ringer lactate (RL). The animals were then resuscitated with 3x the volume of shed blood with RL over 45 min followed by 2x RL over 95 min. Hepatocytes were isolated at the time of maximum bleedout or at 0, 4, 17, and 27 h after the completion of crystalloid resuscitation. P2-purinoceptor binding characteristics were determined in the isolated hepatocytes by using [alpha-35S]ATP. Scatchard analysis revealed high- and low-affinity components of P2-purinoceptors in hepatocytes from sham-operated as well as hemorrhaged and resuscitated animals. The maximum binding capacity (Bmax) of the high-affinity receptor component decreased at the time of maximum bleedout and at 4, 17, and 27 h after resuscitation. In addition to this, the Bmax of low-affinity receptor components also decreased at 4-27 h after resuscitation. In contrast, the dissociation constants of both receptor components were not altered. Because hemorrhagic shock produces abnormalities in glucose metabolism, the downregulation of hepatocyte P2-purinoceptor Bmax may be responsible for the altered glucose homeostasis under such conditions.

ATP-MgCl2 treatment after trauma-hemorrhage/resuscitation increases hepatocyte P2-purinoceptor binding capacity

1994 ◽  
Vol 266 (6) ◽  
pp. R1810-R1815
Author(s):  
M. S. Mahmoud ◽  
P. Wang ◽  
S. R. Hootman ◽  
S. S. Reich ◽  
I. H. Chaudry
Keyword(s):  
Binding Capacity ◽  
Scatchard Analysis ◽  
Binding Characteristics ◽  
Beneficial Effects ◽  
High Affinity ◽  
Receptor Component ◽  
Receptor Dynamics ◽  
Maximum Binding Capacity ◽  
High Affinity Receptor ◽  
Affinity Receptor

Although our studies indicate that P2-purinoceptor binding capacity decreases after hemorrhage and resuscitation, it is not known whether ATP-MgCl2 administration after hemorrhage has any beneficial effects on the receptor dynamics. To study this, we performed laparotomy (i.e., trauma induced) on rats and bled them to and maintained them at a mean arterial pressure of 40 mmHg until 40% of maximum bleedout volume was returned in the form of Ringer lactate (RL). The animals were then resuscitated with 3 times the volume of maximum bleedout with RL over 45 min followed by 2 times RL along with ATP-MgCl2 (50 mumol/kg body wt) over 95 min. Hepatocytes were isolated at 4, 17, and 27 h after resuscitation. P2-purinoceptor binding characteristics were determined by using [alpha-35S]ATP. Scatchard analysis revealed high-affinity and low-affinity receptor components in the hepatocytes isolated from sham-operated or hemorrhaged animals with or without ATP-MgCl2 infusion. ATP-MgCl2 ameliorated and subsequently restored the decreased maximum binding capacity (Bmax) of the high-affinity receptor component and significantly improved Bmax of the low-affinity receptor component. ATP-MgCl2 administration also produced a progressive enhancement in the affinity of the low-affinity receptor component. Thus the beneficial effects of ATP-MgCl2 observed after trauma-hemorrhage and resuscitation may be, in part, due to the restoration of P2-purinoceptor binding capacity and the enhancement of the receptor affinity.

Juvenile hormone increases ouabain-binding capacity of microsomal preparations from vitellogenic follicle cells

1983 ◽  
Vol 61 (7) ◽  
pp. 826-831 ◽  
Author(s):  
T. T. Ilenchuk ◽  
K. G. Davey
Keyword(s):  
Juvenile Hormone ◽  
Binding Capacity ◽  
Follicle Cell ◽  
Follicle Cells ◽  
Follicular Epithelium ◽  
Curvilinear Relationship ◽  
Scatchard Analysis ◽  
Ouabain Binding ◽  
Binding Characteristics ◽  
Brain Microsomes

A comparison has been made of the effects of juvenile hormone (JH) on the binding characteristics for ouabain of microsomes prepared from brain and from cells of the follicular epithelium surrounding previtellogenic or vitellogenic oocytes in Rhodnius. JH has no effect on the binding of ouabain to brain microsomes and decreases the Kd, but does not alter the Bmax for previtellogenic follicle cells. For vitellogenic follicle cells, Scatchard analysis reveals a curvilinear relationship, which is interpreted as indicating that a new population of JH-sensitive ouabain-binding sites develops as the follicle cell enters vitellogenesis. These results are related to earlier data obtained on the effect of JH on ATPase activity, volume changes in isolated follicle cells, and the development of spaces between the cells of the follicular epithelium.

scholarly journals Influence of divalent cations on the detection of somatogenic and lactogenic binding sites in mouse liver cells

1985 ◽  
Vol 228 (3) ◽  
pp. 761-764 ◽  
Author(s):  
G N Ciccia-Torres ◽  
J M Dellacha
Keyword(s):  
Binding Sites ◽  
Mouse Liver ◽  
Binding Capacity ◽  
Specific Binding ◽  
Divalent Cations ◽  
Isolated Hepatocytes ◽  
Liver Cells ◽  
Scatchard Analysis ◽  
Male Mice ◽  
Ovine Prolactin

Specific binding of 125I-labelled human somatotropin was demonstrated in isolated hepatocytes from male mice. In the presence of divalent cations (Ca2+ and Mg2+) the binding of 125I-labelled human somatotropin was competitive with ovine prolactin. Scatchard analysis of competition data indicated a KD of 1.4 +/- 0.2 nM and a binding capacity of 13 000 +/- 2000 sites/cell. In the absence of divalent cations and in the presence of EDTA, human and bovine somatotropins were found to be equally effective to displace bound 125I-labelled human somatotropin, while ovine prolactin showed a weak competition. In this case, the binding capacity was 8400 +/- 1500 sites/cell and the KD was 1.1 +/- 0.1 nM.

Calcium-channel antagonist binding to isolated vascular smooth muscle membranes

1982 ◽  
Vol 60 (12) ◽  
pp. 1738-1741 ◽  
Author(s):  
C. R. Triggle ◽  
D. K. Agrawal ◽  
G. T. Bolger ◽  
E. E. Daniel ◽  
C. Y. Kwan ◽  
...  
Keyword(s):  
Calcium Channel ◽  
Calcium Channel Antagonist ◽  
Mesenteric Artery ◽  
Mechanical Response ◽  
Dissociation Constants ◽  
Binding Capacity ◽  
Microsomal Protein ◽  
High Potassium ◽  
Binding Characteristics ◽  
Rat Mesenteric Artery

The binding characteristics of the calcium-channel antagonist nitrendipine, an analog of nifedipine, have been measured in plasma membrane enriched microsomal fractions from the canine thoracic aorta, canine mesenteric artery, and rat mesenteric artery. The dissociation constants, KD, for the high-affinity binding sites were, respectively, 0.308, 0.254, and 0.101 nM and Bmax values for binding capacity were 20.3, 25.0, and 18.0 fmol/mg of microsomal protein. Studies with isolated tissues, in which the sensitivity of the high potassium mechanical response to nitrendipine was determined, indicate that the apparent KD for nitrendipine in the canine mesenteric artery, as reflected by the IC50 (mean inhibition constant) value, is 4.5 – 5.3 nM and for the rat mesenteric artery 2.5 – 6.6 nM.

Distribution of common peptide YY-neuropeptide Y receptor along rat intestinal villus-crypt axis

1990 ◽  
Vol 258 (5) ◽  
pp. G753-G759 ◽  
Author(s):  
T. Voisin ◽  
C. Rouyer-Fessard ◽  
M. Laburthe
Keyword(s):  
Neuropeptide Y ◽  
Peptide Yy ◽  
Dissociation Constants ◽  
Binding Capacity ◽  
Cell Populations ◽  
Scatchard Analysis ◽  
Binding Data ◽  
Active Concentration ◽  
Camp Levels ◽  
Crypt Cells

Rat small intestinal epithelium is equipped with peptide YY (PYY)-preferring receptors, which also recognize neuropeptide Y (NPY) with high affinity. We therefore examined the distribution of PYY-NPY receptors along the villus-crypt axis after separation of mature villus cells from proliferative crypt cells. Specific 125I-labeled PYY binding was nine times higher in crypt cells than in villus cells. This was not due to differential degradation of PYY or PYY binding sites by the two cell populations. Rather, Scatchard analysis of equilibrium binding data showed that binding capacity (Bmax) of receptors increased from villus to crypt. Bmax were 166 +/- 36 and 21 +/- 3 fmol/mg protein, and dissociation constants (Kd) were 0.10 +/- 0.02 and 0.05 +/- 0.02 nM in crude membranes prepared from crypt and villus cells, respectively. For all cell populations, NPY and rat pancreatic polypeptide were 8- and 1,800-fold less potent than PYY in inhibiting 125I-PYY binding, respectively. Therefore, receptors appear to be PYY preferring along the entire villus-crypt axis. Both peptides (at the maximally active concentration of 1 microM) reduced vasoactive intestinal peptide (VIP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) by 50% in crypt cells. PYY was four to six times more potent than NPY in agreement with the expression of a PYY-preferring receptor. By contrast, neither PYY nor NPY altered VIP-stimulated cAMP levels in villus cells. These results indicate that PYY-preferring receptors, negatively coupled to the cAMP production system, are preferentially expressed in crypt cells where intestinal ionic secretion is believed to take place.

scholarly journals A new unextracted-sample radioimmunoassay method for hepatic endogenous nuclear l-tri-iodothyronine content. Validity of its use in determining nuclear receptor binding characteristics

1982 ◽  
Vol 208 (3) ◽  
pp. 641-649 ◽  
Author(s):  
Toshihiro Yagura ◽  
Paul G. Walfish
Keyword(s):  
Receptor Binding ◽  
Binding Capacity ◽  
Nuclear Extract ◽  
Affinity Constant ◽  
Lower Sensitivity ◽  
Scatchard Analysis ◽  
Binding Characteristics ◽  
Radioimmunoassay Method ◽  
True Values ◽  
Good Agreement

Endogenous l-tri-iodothyronine content in an hepatic nuclear extract was measured by a new unextracted-sample radioimmunoassay method using 8-anilinonaphthalene-1-sulphonic acid to inhibit the l-[125I]tri-iodothyronine binding to the nuclear l-tri-iodothyronine receptor within the extract. For this method, the lower sensitivity limit was 3.125 pg/tube, the recovery of added l-tri-iodothyronine was 90–120%, and the between-assay coefficient of variation was 10%. The amount of endogenous l-tri-iodothyronine was 10–40 pg/0.2 ml of hepatic nuclear extract from euthyroid rats, compared with less than 3.125 pg/0.2 ml from thyroidectomized rats. The results obtained by this new method were compared with a Sephadex G-25 column extracted-sample radioimmunoassay method and showed a good agreement. The values for the endogenous l-tri-iodothyronine content were utilized to correct for the l-tri-iodothyronine concentration within the binding assay mixture in order to accurately determine by Scatchard analysis the binding characteristics of the nuclear l-tri-iodothyronine receptor. The validity of the correction for endogeneous l-tri-iodothyronine was demonstrated by using a nuclear extract from a thyroidectomized rat which was preincubated with a small known amount of l-tri-iodothyronine before determining the nuclear l-tri-iodothyronine receptor binding characteristics. When the Scatchard plots were corrected for the preincubated dose, the results obtained were similar to true values, but they were falsely lower when not corrected. It is concluded that the necessity and validity of using endogenous l-tri-iodothyronine corrections in the Scatchard analytical computations of the nuclear l-tri-iodothyronine receptor binding characteristics has been demonstrated, being particularly more important for affinity constant than maximum binding capacity.

The angiotensin II receptor of rabbit liver: characterization in isolated hepatocytes and effect of GTP

1989 ◽  
Vol 123 (1) ◽  
pp. 131-136 ◽  
Author(s):  
A. Vandekerckhove ◽  
S. Keppens ◽  
H. De Wulf
Keyword(s):  
Angiotensin Ii ◽  
Rate Constant ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Isolated Hepatocytes ◽  
Dissociation Rate ◽  
Angiotensin Ii Receptors ◽  
Angiotensin Ii Receptor ◽  
Binding Characteristics ◽  
Binding Data

ABSTRACT A homologous population of specific angiotensin II receptors is present on the cell surface of isolated rabbit hepatocytes. The binding characteristics of [3H]angiotensin II to the cells were: association rate constant (K+1) 0·08 l/nmol per min and dissociation rate constant (K−1) 1·9/min, yielding a dissociation constant (Kd) of 24 nmol/l. A very similar Kd (32 nmol/l) has been derived from saturation binding data which indicate a maximal binding capacity of about 200 000 sites/cell. Analysis of the association binding data to purified liver plasma membranes indicated a Kd of 6 nmol/l in the absence and 30 nmol/l in the presence of GTP. Dissociation was clearly dependent upon the presence of the nucleotide, which shifted the K−1 from 0·12/min to 0·42/min. The studied binding sites are very likely to be involved in the glycogenolytic action of angiotensin II, since a highly significant correlation was established between the biological activity (activation of glycogen phosphorylase) and the binding affinity of a series of agonistic analogues. The reported characteristics of the rabbit hepatic angiotensin II receptors show much similarity with those of rat liver. Journal of Endocrinology (1989) 123, 131–136

Corticosteroid-binding studies in cytosol of colonic mucosa of the rat

1981 ◽  
Vol 240 (6) ◽  
pp. G417-G423
Author(s):  
E. T. Marusic ◽  
J. P. Hayslett ◽  
H. J. Binder
Keyword(s):  
Colonic Mucosa ◽  
Steroid Receptors ◽  
Binding Capacity ◽  
Potential Difference ◽  
Scatchard Analysis ◽  
Binding Studies ◽  
Minimal Dose ◽  
High Affinity Receptor ◽  
Affinity Receptor ◽  
Cytosolic Receptor

Cytosolic binding of [3H]dexamethasone was studied in the colon of the rat. [3H]dexamethasone binding was rapid and stable at 4 degrees C for 240 min. Scatchard analysis revealed a Kd of 6.2 +/- 0.5 X 10(-9) M and a binding capacity of 149 +/- 6.4 fmol/mg cytosolic protein (100,000-g fraction). Although dexamethasone inhibited [3H]dexamethasone binding more than that of aldosterone, [3H]aldosterone binding was inhibited equally by both aldosterone and dexamethasone. The relative order of potency of other steroids to inhibit [3H]dexamethasone binding was: dexamethasone greaterthan progesterone greater than spironolactone greater than aldosterone greater than corticosterone greater than cortexolone greater than estradiol. In other experiments, low doses of dexamethasone and aldosterone were infused into adrenalectomized animals to determine functional importance of these cytosolic steroid receptors. One-hour infusion of aldosterone at 2 micrograms/100 g body wt, which was the minimal dose of dexamethasone that increased transmural potential difference, did not alter the potential difference. These studies demonstrate the presence of a cytosolic receptor for dexamethasone and suggest that the action of dexamethasone on electrolyte transport in adrenalectomized animals is not mediated by the mineralocorticoid type of receptor and may be mediated by the specific high-affinity receptor for dexamethasone.

ATP-MgCl2 administration normalizes macrophage cAMP and beta-adrenergic receptors after hemorrhage and resuscitation

1994 ◽  
Vol 267 (1) ◽  
pp. G52-G58 ◽  
Author(s):  
P. Wang ◽  
S. M. Tait ◽  
Z. F. Ba ◽  
I. H. Chaudry
Keyword(s):  
Receptor Binding ◽  
Kupffer Cells ◽  
Adrenergic Receptors ◽  
Binding Capacity ◽  
Beta Adrenergic Receptors ◽  
Beta Adrenergic ◽  
Midline Laparotomy ◽  
Binding Characteristics ◽  
Beta Receptor ◽  
Camp Levels

Although ATP-MgCl2 attenuates the release of inflammatory cytokines and restores the defective macrophage (M phi) antigen presentation function after hemorrhage and resuscitation, it is not known whether administration of this agent after hemorrhage affects M phi adenosine 3',5'-cyclic monophosphate (cAMP) levels and beta-adrenergic receptors. To determine this, rats underwent a midline laparotomy (i.e., induction of trauma) and were then bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of maximum bleedout volume was returned in the form of Ringer lactate (RL). Animals were resuscitated with four times the volume of shed blood with RL, during and after which ATP-MgCl2 (50 mumol/kg) or saline was administered over 95 min. At 1.5 h postresuscitation (i.e., 10 min after completion of ATP-MgCl2 infusion), peritoneal M phi and Kupffer cells were isolated, and cAMP levels were measured by radioimmunoassay. beta-Receptor binding characteristics were also determined in isolated Kupffer cells. The results indicate that cAMP levels increased significantly in both peritoneal M phi and Kupffer cells after hemorrhage and resuscitation. Maximum binding capacity (Bmax) of beta-receptors increased in Kupffer cells, suggesting that the elevated cAMP may be due to the increased beta-receptor Bmax under such conditions. ATP-MgCl2 treatment, however, markedly decreased beta-receptor Bmax in Kupffer cells and cAMP in both peritoneal M phi and Kupffer cells, and the values were similar to shams. Thus normalization of M phi cAMP levels and beta-receptor binding capacity by ATP-MgCl2 may contribute to the immunoenhancing effects of this agent observed after trauma-hemorrhage and fluid resuscitation.

Fasting alters somatostatin binding to liver membranes of rainbow trout (Oncorhynchus mykiss)

1996 ◽  
Vol 150 (2) ◽  
pp. 179-186 ◽  
Author(s):  
M J Pesek ◽  
M A Sheridan
Keyword(s):  
Rainbow Trout ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Binding Characteristics ◽  
High Affinity ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
C Terminus ◽  
Liver Membranes

Abstract Somatostatins are a diverse family of peptides that influence various aspects of animal growth, development, and metabolism. Recent work in our laboratory has shown that somatostatins stimulate hepatic lipolysis in rainbow trout. In this study we characterized somatostatin-binding sites in trout hepatic membrane preparations. We also examined changes in binding characteristics brought about by food deprivation. Binding of [Tyr11]-somatostatin-14 (SS-14) was saturable, reversible, and time- and temperature-dependent. Under optimal conditions, [Tyr11]-SS-14 specific binding averaged 5·7 ± 0·3%. While SS-14 and SS-28 (an N-terminally extended form of SS-14 and derived from the same gene as SS-14) displaced [Tyr11]-SS-14 specific binding (ED50 values of approximately 50 nm and 100 nm respectively), salmon SS-25 (containing [Tyr7,Gly10]-SS-14 at its C terminus and presumably derived from a gene different from that giving rise to SS-14/SS-28), except at pharmacological concentrations, did not. Significant specific binding was also detected in brain, esophagus, stomach, upper and lower intestine, pancreas, and adipose tissue. Scatchard analysis suggested the existence of two classes of hepatic somatostatin-binding sites: a high-affinity site with a Kd of 23 nm and Bmax of 1·4 pmol/mg protein and a low-affinity site with a Kd of 379 nm and Bmax of 4·9 pmol/mg protein. Fasting resulted in reduced growth and elevated plasma levels of SS-14 compared with fed animals. SS-14 binding capacity of the high-affinity class in liver membranes isolated from fasted fish increased by 120% over that from fed counter-parts. No difference in Kd for the high-affinity binding class or in either Kd or Bmax of the low-affinity class was noted between fasted and fed animals. These data support the role of the liver as a target of somatostatin and suggest that fasting enhances hepatic sensitivity to SS-14 binding. Journal of Endocrinology (1996) 150, 179–186

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