Tissue Wet Wt Research Articles

Preparation of single smooth muscle cells from guinea pig taenia coli by combinations of purified collagenase and papain

Procedures for isolation of single smooth muscle cells from taenia coli of guinea pigs have been developed. The preparation was performed with a combination of highly purified collagenase prepared by Amano Pharmaceutical Co. (Japan) and papain obtained from Sigma Chemical Co. (Type III). This combination resulted in very high yield of the single cells (39.2 ± 4.5 × 10 3 cells mg tissue wet wt) and less cell debris. In the ordinary procedure, commercially available collagenase preparations contaminated with various peptidases have been used. With these enzyme preparations, however, the yield of single cells was dependent on the batch of the preparations, and a large amount of cell debris was contaminated. Combination of the highly purified collagenase and papain resulted in higher yields constantly. Cells, isolated with these enzymes in a medium consisting of 140 mM KCl, 1.0 mM MgCl 2, 4.2 mM Hepes, and 5.6 mM glucose (pH 7.4), were spindle shaped. The length of the cells was 185.9 ± 5.2 μm ( n = 90) and the diameter was approximately 12.6 μm. The diameter was not dependent on the cell length. More than 80% of the single cells were viable when examined by trypan blue exclusion technique. Under the depolarized condition, cells remained viable longer because of lower energy consumption, and these cells were contracted by Ca dose dependently. The dose-response relationship was similar to that obtained with intact tissue. Because the cells are constantly available with higher yield, the preparation might be applicable for biochemical research such as ion flux. Details of cell properties under the physiological conditions are under investigation.

Determination of polyamines in digestive and reproductive tissues of adult Asterias vulgaris (echinodermata: asteroidea)

1. 1. The polyamines spermine, spermidine and putrescine were extracted from tissues of Asterias vulgaris and quantitated using thin layer chromatography. 2. 2. In the pyloric caeca, mean (±SE) levels of spermine, spermidine and putrescine were 138(15), 86(24) and 415(77) nmol/g wet wt tissue, respectively. In the testes, levels were 95(12), 13(6) and <6 nmol/g wet wt. In the ovaries, levels were 105(9), 11(0.8) and 15(8) nmol/g wet wt. 3. 3. High levels of polyamines in the pyloric caeca may be related to secretion or excretion in this tissue. 4. 4. We hypothesize that polyamines will be important in the regulation of cellular activity in these tissues during the annual reproductive cycle.

Ultrastructural and ionic studies in global ischemic dog brain.

A time course of tissue ionic changes, and their relation to ultrastructural findings during reperfusion following a 15-min global ischemic brain insult was studied in a dog model. Parietal cortex was analyzed for Ca, Na, K, Mg and Fe in controls and after 10 min, 2, 4, and 8 h of reperfusion. After 8 h of reperfusion, the mean values (mumol/g tissue wet wt.) for Ca (control = 1.43, 8 h = 2.76) and Na (control 60.4, 8 h = 107.4) doubled and K (control = 90.4, 8 h = 48.5) decreased to half that of the control. Ultrastructural studies and subcellular localization of calcium in parietal cortex of in situ-fixed brains after 8 h showed cortical neurons with clumping of nuclear chromatin, dilatation of endoplasmic reticulum and disruption of plasma membranes. Large amounts of electron-dense precipitates of calcium were present within dilated astrocytic processes, synaptic vesicles, cytoplasm of edematous dendrites and mitochondria. Cortical neurons from postischemic dogs without reperfusion showed only slight chromatin clumping and edema of astrocytic processes, but no calcium accumulation. The large ionic shifts noted between 4 and 8 h of reperfusion, indicate a progressive inability of the cells to maintain normal transmembrane gradients of these ions and may reflect a membrane destructive process, as demonstrated ultrastructurally at 8 h. Enhanced calcium entry into the neuron during reperfusion appears to be a part of the cytotoxic mechanism leading to neuronal necrosis.

Reproduction, juvenile growth, consumption and the effects of starvation upon the South China Sea whelk Hemifusus tuba (Gmelin) (Prosobranchia: Melongenidae)

The South China Sea whelk, Hemifusus tuba (Gmelin, 1791) (Melongenidae), is a predator of bivalves. This paper describes copulation, egg capsule laying, egg capsule structure, and hatchling morphology. Hatching took place 89 days post-laying at 21 ± 2°C. Hatchlings are born at a mean shell length of 5.53 mm and a total wet wt of 0.025 g and almost immediately commence feeding on living bivalve tissue. Growth of the juvenile has been followed for 62 wk post-hatching and can be divided into four phases. The first phase (Weeks 0–10) was a period of dramatic shell growth in length (from 5.53 to 28.68 mm) with some increase in wet tissue wt to 0.119 g after 8 wk and 0.750 g after 12. In Phase two (Weeks 10–20), growth proceeded at a slower rate to a shell length of 37.35 mm and the wet tissue increased to 1.489 g. In Phase three (Weeks 20–42), shell length and wet tissue weight remained relatively constant but total weight increased, suggesting that the shell was gaining weight but not length. This growth check at least in length and tissue weight appears to be endogenous. In Phase four (Weeks 42–62), growth in all parameters resumed and it is believed that following Phases 1–3 (essentially juvenile growth characteristics) adult growth will be a typical balance between somatic and gonadal growth. Adult consumption rates are ≈4% wet tissue wt · day −1. In the 4-wk old juvenile, daily consumption rates were calculated to be 36.51% wet tissue wt. Estimates of consumption ·g total wet wt. −1 for Week 2, however, were 26.89%. Using these data for an estimate of consumption/g wet tissue wt at Week 2, a figure of 92% is obtained, i.e., 2-wk-old H. tuba hatchlings ate virtually their body weight each day. Although this may be an over-estimate, it would help account for the dramatic increases in shell length up to Weeks 10–12. Following Week 2, consumption values progressively decreased to typical adult consumption rates of ≈3–4%. The effects of starvation upon H. tuba juveniles are typical, all parameters of growth declining proportionately. There was some evidence of sustained tissue weight increases but not shell growth, implying direct absorption of DOM. Surprisingly, moreover, no mortality took place until Week 27, and three animals survived 39 wk without food. In comparison with data from other predatory snails this is very long (especially for a juvenile). It is concluded that H. tuba is an opportunistic predator of shelf bivalves. The growth plan suggests urgent feeding to build up shell size, presumably to reduce predation pressure and that only later is the shell filled up and strengthened. Internal fertilization, the laying of egg capsules on the shells of others, rapid growth and an ability to survive long periods of starvation all can be used to explain the success of these whelks.

Production of pituitary protein 7B2 immunoreactivity by endocrine tumors and its possible diagnostic value.

7B2 is a protein originally isolated from pituitary, which has been shown to be present in the central nervous system and in certain peripheral tissues, with very high concentrations in pancreatic islets. Endocrine and nonendocrine tumors from 185 patients were investigated by RIA for the presence of immunoreactive pituitary protein 7B2. The highest mean concentration of 7B2 immunoreactivity was found in insulinomas [452 +/- 174 (+/- SEM) pmol/g wet wt tissue; n = 16], which was significantly higher than the concentration in normal adult pancreatic tissue (28.3 +/- 4.4 pmol/g; n = 7). High concentrations of 7B2 immunoreactivity also were found in other endocrine tumors. The cellular localization of 7B2 was studied in normal pancreas, pancreas with hyperplastic islets, and endocrine tumors. 7B2 immunoreactivity was localized to B-cells in the normal pancreas and to variable proportions of cells in islet cell hyperplasias, B-cell tumors, and pheochromocytomas. Plasma concentrations of 7B2 immunoreactivity also were determined in 255 patients with established diagnoses of endocrine or nonendocrine tumors. The proportion of patients with elevated plasma concentrations (arbitrarily set at more than 4 SD above the mean) were 42 of 72 with pancreatic islet cell tumors, 7 of 11 with midgut carcinoid tumors, and 5 of 13 with medullary carcinomas of the thyroid. Especially high values were found in patients with glucagonomas (14 of 20), vipomas (12 of 13), and pancreatic polypeptide-producing tumors (5 of 6). Thus, 7B2 immunoreactivity is produced by a variety of different tumors and may serve as a tumor marker, especially in patients with certain pancreatic islet tumors.

A highly sensitive assay for phenylethanolamine N-methyltransferase in human brain.

A rapid, highly sensitive assay for phenylethanolamine N-methyltransferase in brain using the natural substrate, norepinephrine, is described. The method is based on the selective adsorption and elution of the reaction product, epinephrine, from alumina. A small but important further lowering of blanks and increase in sensitivity is attained by removal of the radiolabeled substrate, [methyl-3H]-S-adenosylmethionine by precipitation as the reineckate prior to adsorption of norepinephrine to alumina. The assay has a sensitivity of 30 fmole and the PNMT activity could be measured in as little as 1 mg (wet wt) of human locus coeruleus tissue. The sensitivity is enhanced by homogenizing tissue in small volumes and removing potential inhibitors by dialysis. We report for the first time PNMT activity in specific regions of the human cerebral and cerebellar cortex.

The isolation and purification of a proteinase with chymotrypsin-like properties from ovine mucosal mast cells

1. 1. A mast cell granule proteinase was purified from isolated ovine mucosal mast cells by cation exchange chromatography, which defined the conditions for enzyme purification from sheep gastric mucosae. 2. 2. Antibodies raised against the proteinase were used in subsequent purification procedures which yielded 78 μg of enzyme per 5 g wet wt of abomasal tissue. 3. 3. Immuno-histochemistry confirmed that mucosal mast cells were the source of the enzyme. 4. 4. The proteinase had chymotrypsin-like esterase activity, with a molecular weight between 19,000 and 25,000.

Passive calcium-buffering capacity of a rabbit ventricular homogenate preparation.

This study characterizes the passive Ca2+-binding capacity of a crude homogenate fraction prepared from rabbit ventricular tissue. The soluble and particulate fractions of the homogenate were separated by centrifugation, and the Ca2+-binding capacity of each fraction was measured separately with Ca2+-selective electrodes and then summed to obtain the Ca2+-binding capacity of the total tissue homogenate. Qualitatively, the Ca2+ bound to the homogenate exhibited both relatively high- (Km less than 1 microM) and low-affinity (Km greater than 10 microM) sites over the physiological range of free Ca2+ concentrations. The total homogenate bound 72, 128, and 308 mumol Ca2+/kg wet wt tissue at 1, 10, and 50 microM free Ca2+ concentrations, respectively. The Ca2+ bound to the homogenate was mostly La3+ displaceable, which suggests that it represents a largely noncompartmentalized, rapidly exchangeable fraction of Ca2+. Myofibrillar ATPase activity was examined as a function of free Ca2+ concentration in an ionic medium similar to that used in the Ca2+-binding experiments. At free Ca2+ concentrations that stimulated myofibrillar ATPase activity half maximally, the measured rise in Ca2+ bound to the homogenate above resting values was greater than 100 mumol/kg wet wt. These data provide base-line values for the passive Ca2+-buffering capacity of the myocardium, although relevance to the physiological setting must be interpreted with caution.

Elevation of monoacetylated polyamines in human breast cancers

Normal tissues contain only trace amounts of monoacetylated polyamines. N 1-Acetylpersmidine is present in high concentrations in mouse liver cells damaged by hepatotoxins and is also found in specialised cells of the hamster epididymis. In the present study human breast cancers were analysed for the presence of monoacetylated polyamines because the N 1-acetylspermidine is selectively elevated in human colorectal cancers. Free and monoacetylated polyamines ( N 1-acetylspermidine, N 8-acetylspermidine and N 1-acetylspermine), measured by high-performance liquid chromatography, were expressed as nmol/g wet wt of breast cancers ( n = 54) or normal breast tissue ( n = 15). Putrescine and monoacetylated polyamines were absent from normal breast tissue. Mean total content of monoacetylated polyamines in breast cancers 14.9 ± 5.3 (S.E.) exceeded the mean total content of free polyamines (8.3 ± 1.0) in normal breast tissue. Detectable levels of at least two of the monoacetylated polyamines were found in all brest cancers: N 1-acetylspermidine was present in 51 (13.1 ± 6.3), N 8-acetylspermidin in 32 (0.6 ± 0.1) and N 1-acetylspermine in 28 tumours (1.2 ± 0.3). There was no correlation between monoacetylated polyamine content of breast cancers and factors known to affect survival, i.e. tumour size, histological grade, oestrogen receptors status and node status. Monoacetylated polyamines are present in human breast cancers but not in normal breast tissue, implying that polyamine catabolism in breast cancers differs from that in normal breast tissue.

Size distribution of ribosomes in biopsy specimens of human skeletal muscle during starvation

The changes in the poly- and monoribosome distribution and in the total ribosome concentration in muscle during short term starvation were investigated. Transcutaneous muscle biopsies of 50 mg wet wt were taken from healthy human subjects, nonstarved and after one, two, and three days of total starvation. The percentage amount of polyribosomes was significantly lower ( P < 0.02) on days 2 and 3 of starvation than on day 0 (nonstarved). No significant sex-dependent differences were observed between the group of five females and six males. Ribosome concentration per g wet wt of muscle tissue was significantly lower on day 3 than on each preceding day ( P < 0.05). The reproducibility of the polyribosome analyses, together with the changes observed, suggest a future application of this method for evaluation of the effects of nutritional support in patients with posttraumatic and septic conditions.

Bombesin-like peptides in human endocrine tumors: quantitation, biochemical characterization, and secretion.

Bombesin-like immunoreactivity (BLI) in 20 human endocrine tumors was studied using an antiserum directed toward the C-terminal region of bombesin. Additionally, plasma BLI was assayed in normal subjects and patients with known BLI-containing tumors. In 7 tumors (medullary carcinoma of the thyroid, n = 2; carcinoid of the lung, n = 3; hepatic carcinoid, n = 1; pheochromocytoma, n = 1), the BLI content ranged from 6-2000 pg/mg wet wt of tissue. Sephadex G-50 gel chromatography of tumor extracts under acid-dissociating conditions revealed 2 peaks of BLI: 1 coeluting with porcine gastrin-releasing peptide (GRP) and 1 with bombesin. Reverse phase ODS silica HPLC analysis of the G-50 peaks using a methanol-trifluoroacetic acid gradient showed that human tumor BLI more closely resembled porcine GRP and its C-terminal fragment GRP-(14-27) than bombesin itself. Partial tryptic digestion of the tumor GRP-like peptide generated a product which, on HPLC, was similar to GRP-(14-27). Elevated plasma BLI was detected in the peripheral circulation of three subjects and in the vessels draining the tumor metastases of one of these patients. BLI was undetectable in normal subjects. These results indicate 1) that BLI is present in and may be secreted by various human endocrine tumors, and 2) that human tumor BLI closely resembles porcine GRP and its C-terminal fragment GRP-(14-27).

β-Endorphinlike immunoreactivity and somatostatinlike immunoreactivity in normal gastric mucosa, muscle layer, and adenocarcinoma

β-Endorphinlike immunoreactivity and somatostatinlike immunoreactivity were detected in the mucosa and muscle layer of normal gastric antrum and corpus and in moderately differentiated adenocarcinoma derived from the antral mucosa. The concentration of β-endorphinlike immunoreactivity in the normal gastric tissues was 4–15 pmol/g wet wt tissue; this value varied from 9.64 to 241.39 pmol/g wet wt tissue (81.38 ± 37.82 pmol/g wet wt tissue) in adenocarcinomas. The concentration of somatostatinlike immunoreactivity was 18–25 pmol/g wet wt tissue in normal gastric mucosa, whereas it was 1–2 pmol/g wet wt tissue in adenocarcinomas. Gel exclusion chromatography of β-endorphinlike immunoreactivity revealed two peaks corresponding to β-endorphin and β-lipotropin. In normal mucosa and in the whole layer of antrum, the major peak was eluted near the position of β-lipotropin, and the minor broad peak was eluted near the position of β-endorphin. In contrast, in adenocarcinoma, β-endorphinlike immunoreactivity was eluted broadly at the position of β-endorphin and the other smaller peak was at the position of β-lipotropin. Gel exclusion chromatography of somatostatinlike immunoreactivity also showed different patterns between antral mucosa and adenocarcinoma. This study revealed the presence of the opioid peptide, β-endorphinlike immunoreactivity, not only in normal gastric tissue but also in adenocarcinomas with highly increased concentration and different elution patterns in combination with decreased concentration of somatostatinlike immunoreactivity.

Ontogeny of immunoreactive insulin in the fetal bovine pancreas.

The aim of this study was to characterize the development of immunoreactive insulin (IRI) in the fetal bovine pancreas. Pancreatic IRI was acid extracted, and both pancreatic and serum IRI were quantitated by RIA. The amount of pancreatic IRI per wet tissue wt in first trimester fetuses was similar to that in the adult animal (8.2 +/- 0.7 and 5.9 +/- 1.7 U/g pancreas, respectively). IRI increased progressively during gestation, attaining 39.2 +/- 6.5 U/g pancreas in the third trimester, 7-fold higher than that in the adult. When pancreatic IRI concentrations were standardized for protein content of the extracts, a decrease was noted between the midsecond and third trimesters. This is most likely the result of dilution of the endocrine portion of the pancreas by the rapidly growing exocrine pancreas. IRI was also detectable in fetal sera from all three trimesters. In contrast to the profile for pancreatic concentrations of IRI, serum concentrations remained constant throughout gestation at approximately 20 microU/ml. Poly(A+)RNA was isolated from adult and fetal pancreata, and the relative levels of preproinsulin mRNA were assessed by DNA/RNA filter hybridization. There was a 2- to 3-fold increase in the relative level of preproinsulin mRNA in fetal pancreata between the first and second trimesters which was maintained through the third trimester. In the adult pancreas, preproinsulin mRNA levels were similar to those in the first trimester fetus. This profile for the ontogeny of pancreatic preproinsulin mRNA was similar to that for pancreatic IRI (units per pancreas) during fetal maturation. We conclude that in the bovine fetus: the endocrine pancreas synthesizes IRI during all three trimesters of development; pancreatic (units per g pancreas), but not serum, concentrations of IRI increase progressively as development proceeds; and the ontogeny of preproinsulin mRNA is paralleled by that of pancreatic IRI (units per pancreas).

Effect of pentagastrin on gastric mucosal histamine in dogs.

We studied the effect of pentagastrin on histamine content of gastric mucosa obtained from dogs with gastric fistulas to determine whether pentagastrin mobilizes cellular stores of histamine. Experiments were also performed on canine gastric mucosa in vitro to investigate the effects of pentagastrin on histamine release, per se. During in vivo studies basal histamine content averaged 0.9 nmol/mg wet wt tissue or 18.8 nmol/mg tissue prot. No significant difference in gastric mucosal histamine content occurred during intravenous administration of pentagastrin (6 or 16 micrograms . kg-1 . h-1) or saline (control), even though acid output from the gastric fistula increased significantly above control during pentagastrin infusion. Moreover, pentagastrin (10(-5) and 10(-8)M) did not release more histamine from gastric mucosa in vitro than the buffer (control), whereas Triton X-100 and the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, alone and in combination with calcium ionophore A23187, released significant amounts of histamine above control values. From these experiments we conclude that pentagastrin did not alter histamine content of canine gastric mucosa in vivo, even though acid secretion was stimulated maximally, nor did pentagastrin release histamine in vitro.

Calcitonin gene-related peptide immunoreactive sensory and motor nerves of the rat, cat, and monkey esophagus

In the mammalian esophagus calcitonin gene-related peptide (CGRP)-immunoreactive nerves form abundant subepithelial plexuses and penetrate the mucosa. The levels of extractable CGRP in separated epithehlial layers are 15.8 ± 2.4 pmol/g wet wt of tissue (n = 8, mean ± SEM). Treatment of neonatal rats with capsaicin and ablation of the central portion of the feline nodose ganglion led to a marked reduction in the numbers of CGRP-immunoreactive nerve fibers. The loss of CGRP nerves demonstrated by immunocytochemistry was accompanied by a parallel reduction in the tissue content of CGRP, as measured by radioimmunoassay (1.5 ± 0.5 pmol/g in capsaicin-treated animals compared with 9.4 ± 1.9 pmol/g in vehicle-treated controls; p < 0.0025). These findings indicate the sensory nature of the CGRP-immunoreactive nerves. Substance P-immunoreactive nerve fibers innervated in particular the blood vessels of the lamina propria; very few penetrated the esophageal epithelium and these were only partially depleted after removal of the central portion of the nodose ganglion. The esophageal muscle contained nerves immunoreactive for substance P and, in particular, for CGRP which was also found in the motor end plates of the striated muscle. No changes in the CGRP-containing motor end plates were observed either after treatment of neonatal rats with capsaicin or ablation of cell bodies from the central portion of the nodose ganglion. These nerve fibers may originate from rostral areas of the nucleus ambiguus, where CGRP-immunoreactive motor neurons have previously been described. Thus, our findings reveal dual components, motor and sensory, of the CGRP-containing innervation of the esophagus.

Peritoneal absorption of macromolecules studied by quantitative autoradiography.

Transport experiments of 125I-human serum albumin from the peritoneal cavity to the plasma were conducted in 200-g female rats. Blood and peritoneal samples were collected at intervals over 2-3 h. After death and rapid freezing of the animal, transverse sections were cut in a cryomicrotome from several tissues surrounding the peritoneal cavity, and the distribution of the labeled albumin was measured by computerized quantitative macroautoradiography. Tissue concentrations (counts/min per wet tissue wt) in parietal tissues (anterior abdominal wall and the diaphragm) were relatively constant versus distance from the peritoneum and represented a large fraction (0.5-1.0) of the concentration in the peritoneal cavity. Fractional concentrations in visceral tissues (liver, stomach, intestine) decreased from 0.20-0.35 at the peritoneal surface to 0.03-0.06 at a distance of 900 micron from the peritoneum. Uterine tissue concentrations lay between those of the parietal tissues and those of the viscera. The data are related to mechanisms of interstitial and lymphatic transport in these tissues.

Relationship between type L hormone-sensitive lipase and endogenous triacylglycerol in rat heart.

The purpose of this study was to determine whether, under physiological conditions, intracellular lipoprotein lipase [type L hormone-sensitive lipase (HSL)] activity varied inversely with triacylglycerol (TG) content of the heart. The results show that fasting from 7:30 A.M. to 7:30 P.M. increased type L HSL activity from 72 +/- 1 to 96 +/- 1 U/g wet wt tissue (P less than 0.001) in the myocardium. At the same time, cardiac TG stores decreased from 1.83 +/- 0.03 to 1.37 +/- 0.01 mumol/g tissue (P less than 0.001). In a separate experiment, one night of eating a fat-rich diet (60% of calories from fat) caused a 42% increase in type L HSL activity, which was accompanied by a 42% reduction in the TG content of the heart. Likewise cold exposure (overnight) activated type L HSL (73%) and, at the same time, decreased cardiac TG stores (44%). When data from the fasting, fat-feeding, and cold-exposure experiments were combined, a significant correlation coefficient of -0.81 was obtained between type L HSL activity and TG content of the heart (P less than 0.001). These data provide evidence for a physiological relationship between intracellular lipoprotein lipase activity and cardiac TG content.

Inactivation of atrial natriuretic substance by kallikrein.

To further characterize the properties of the potent natriuretic and diuretic substance that can be extracted from atrial tissue, we investigated its susceptibility to inactivation by kallikrein and other proteolytic enzymes. Extracts of rat atrial tissue (tissue wet wt 100 mg/ml) were incubated with enzymes under standard conditions and tested by injection into nondiuretic anesthetized rats. One hour of incubation at 37 degrees C with pure porcine pancreatic kallikrein at concentrations of 250 micrograms/ml or greater significantly reduced the activity of atrial natriuretic substance. The reduction in activity was dependent on both enzyme concentration and time of incubation. The kallikrein-catalyzed degradation was completely blocked by aprotinin but was only partially retarded by soybean trypsin inhibitor. Trypsin reduced natriuretic and diuretic activity of extracts at concentrations of 400 micrograms/ml or greater, with nearly complete inactivation at a concentration of 1,000 micrograms/ml. Carboxypeptidase B also caused a concentration-dependent inactivation of the natriuretic material. Last, alpha-chymotrypsin (1,000 micrograms/ml) and elastase (1,000 micrograms/ml) were found to destroy the natriuretic activity. In a separate set of experiments natriuretic activity was observed to be retained by a 1,000 mol wt cutoff membrane. Inactivation of the natriuretic peptide by renal kallikrein is a possible mechanism for in vivo regulation of natriuretic activity.

Opioid binding sites of the κ-type in guinea-pig cerebellum

(−)-[ 3H]Bremazocine interacts almost equally well with the μ-, δ- and κ-types of opioid binding sites. In homogenates of guinea-pig cerebellum, it was bound with high affinity (K D = 0.046 nM) and the maximum binding capacity was 7.04 pmol/g wet wt of tissue. When the μ- and δ-binding of (−)-[ 3H]bremazocine was prevented with unlabelled ligands, aK D of 0.034 nM and a capacity of 5.94 pmol/g tissue was found for the κ-binding site, which therefore comprised 84% of the opioid binding sites in the cerebellum. Autoradiographic analysis showed that the binding of (−)-[ 3H]bremazocine was relatively low in lobules IX and X and that it was predominantly located in the molecular and to a lesser extent in the granular layers. The addition of unlabelled μ- and δ-ligands did not alter the distribution. Thus, the guinea-pig cerebellum contains opioid binding sites of which almost all are of the κ-type and is therefore an ideal tissue for the isolation of κ-receptors and for the investigation of their biochemical and pharmacological properties.

High affinity binding of [3H]R5020 and [3H]progesterone by putative progesterone receptors in cytosol and nuclear extract of turtle oviduct.

The purpose of this investigation was to identify and characterize putative cytosolic and nuclear forms of progesterone receptor in the female reproductive tract of a turtle, Chrysemys picta. A dextran-coated charcoal adsorption assay and DNA-cellulose affinity chromatography were used as the primary methodologies with [3H]R5020 [3H-labeled 17 alpha-dimethyl-19-norpregna-4,9-diene-3,20-dione) and [3H]progesterone (P4) as the ligands. The receptor was of high affinity (Kd = 4.7 X 10(-10) M for [3H]P4; 2.2 X 10(-10) M for [3H]R5020) and limited capacity (500-6000 fmol/g tissue wet wt). Association was rapid (apparent equilibrium being reached in 30-40 min), as was dissociation (t1/2 = 45 min for [3H]P4 and 180 min for [3H] R5020). The putative receptor demonstrated strict steroid specificity, binding progestins but not estrogens, androgens, or glucocorticoids. Heterogeneity of the cytosolic receptor was demonstrated as two forms eluting off DNA-cellulose columns at 0.2- and 0.3-M salt concentrations. Binding of cytosolic receptor to DNA-cellulose was not increased by preexposure of cytosol to 25 C for 30 min. Some variations in cytosolic, but not nuclear, receptor were associated with different stages of the reproductive cycle and were positively correlated with body weight. Preliminary studies using an explant culture system suggest that the progesterone receptor in turtle oviduct may be maintained by estrogen and translocated from the cytosol to the nucleus by P4. In summary, we have partially characterized a putative P4 receptor in the oviduct of the turtle that is similar to mammalian and avian P4 receptors in specificity, affinity, and other physicochemical properties, supporting the idea that steroid receptor proteins have been highly conserved in vertebrate evolution. However, temperature sensitivity of activation and DNA affinity are different in the turtle and suggest modifications that may be related to physiological adaptation in such a poikilothermic species.