Gordon F. Anderson

Thrombin-Elicited Contractile Responses of Aortic Smooth Muscle:

Abstract Human α-thrombin at physiologically relevant concentrations of 0.75 to 225 nM (0.01 to 22 clotting units/ml) caused rabbit thoracic aorta to slowly contract in isolated organ baths. Near maximum contractile tension (10% below the irreversible contraction caused by 22 units/ml) was slowly generated by 75 nM α-thrombin (8 units/ml) over 18 min and was 50% that of the norepinephrine (NE) control. The initial tonus could only be regained by repeated washing and 60 min equilibration. Tissues were refractory to a second 75 nM α-thrombin challenge but responded fully to 1.0 nM NE. Conversion of human α-thrombin to nonclotting but estero/amidolytically active 7-thrombin (<0.1% clotting activity) or nitration of the enzyme (1% clotting activity) did not interfere with the contractile activity, whereas chemical conjugates of the parent enzyme at the catalytic site were inactive. Neither atropine, phentolamine, nor indomethacin blocked the thrombin-induced contractions, whereas D-600 was markedly inhibitory. Preincubation of the tissue with inactive forms of thrombin did not prevent the α-thrombin-induced response. Removal of the vascular endothelium did not prevent contraction. Aorta preparations with intact endothelium relaxed in the presence of very low concentrations (0.75 to 750 pM) of α-thrombin prior to contracting in response to higher concentrations during cumulative dose-response experiments. Our data suggest that catalytically active thrombin forms (α- or nonclotting β- and 7-thrombins) may have hemostatic functions at the vascular levels in hemostasis.

Evidence for presynaptic adenosine A2a receptors associated with norepinephrine release and their desensitization in the rat nucleus tractus solitarius

Abstract: Rat medullary brain segments containing primarily nucleus tractus solitarius (NTS) were used for superfusion studies of evoked transmitter release and for isotherm receptor binding assays. Isotherm binding assays with [3H]CGS‐21680 on membranes prepared from NTS tissue blocks indicated a single high‐affinity binding site with a KD of 5.1 ± 1.4 nM and a Bmax of 20.6 ± 2.4 fmol/mg of protein. The binding density for [3H]CGS‐21680 on NTS membranes was 23 times less than comparable binding on membranes from striatal tissue. Electrically stimulated (1 min at 25 mA, 2 ms, 3 Hz) release of [3H]norepinephrine ([3H]NE) from 400‐µm‐thick NTS tissue slices resulted in an S2/S1 ratio of 0.96 ± 0.02. Superfusion of single tissue slices with 0.1–100 nM CGS‐21680, a selective adenosine A2a receptor agonist, for 5 min before the S2 stimulus produced a significant concentration‐dependent increase in the S2/S1 fractional release ratio that was maximal (31.3% increase) at 1.0 nM. However, superfusion of tissue slices with CGS‐21680 over the same concentration range for 20 min before the S2 stimulus did not alter the S2/S1 ratio significantly from control release ratios. The augmented release of [3H]NE mediated by 1.0 nM CGS‐21680 with a 5‐min tissue exposure was abolished by 1.0 and 10 nM CGS‐15943 as well as by 100 nM 8‐(3‐chlorostyryl)caffeine, both A2a receptor antagonists, but not by 1.0 nM 8‐cyclopentyl‐1,3‐dipropylxanthine, the A1 receptor antagonist. Taken together, these results suggest that CGS‐21680 augmented the evoked release of [3H]NE in the NTS via activation of presynaptic A2a receptors within the same concentration range as the binding affinity observed for [3H]CGS‐21680. It was also apparent that this population of presynaptic adenosine A2a receptors in the NTS desensitized within 20 min because the augmenting action of CGS‐21680 on evoked transmitter release was not evident at the longer interval.

The Response of Autonomic Receptors to Castration and Testosterone in the Urinary Bladder of the Rabbit

The effects of castration and testosterone on the autonomic receptor density and contractility in the urinary bladder smooth muscle of male rabbits were compared to untreated animals. Four groups of rabbits were studied over a similar time span with Group 1 animals serving as the untreated controls. Two groups (Groups 2 and 3) were castrated 28 days prior to sacrifice, Group 2 animals received corn oil for 14 days, and Group 3 animals received testosterone, 10 mg./day, for 14 days. The Group 4 animals were non-operated and received testosterone 10 mg./day for 14 days. Ligand saturation binding studies for alpha adrenoceptors in the bladder base and proximal urethra were performed with [3H]dihydroergocryptine ([3H]DHE). Muscarinic cholinergic receptors (MChR) were assayed with [3H]quinuclidinyl benzilate ([3H]QNB) and beta adrenoceptors with [125I]iodocyanopindolol ([125I]CYP) on the detrusor smooth muscle. Castrated Group 2 animals showed no significant change in receptor density with either [3H]QNB or [125I]CYP in detrusor muscle, but did exhibit a significant reduction (59%) of alpha adrenoceptors in the bladder base-urethra. The testosterone treated castrate and testosterone treated non-operated animals had significant increases in the MChR density, but no change in the alpha adrenergic, or beta adrenergic receptor density as compared to untreated controls. Cumulative dose response contractile studies were performed with carbachol on detrusor muscle strips and with phenylephrine on bladder base strips in isolated organ baths. The contractile studies on muscles from Groups 1, 2 and 3 showed no change in the ED50 or maximal contractile strength between control, castration or testosterone treated castrated animals. The ratio of wet bladder weight as compared to total body weight between each of the treatment groups showed a slight increase in both of the testosterone treatment groups. It was concluded that castration down regulates the alpha adrenergic receptors of the bladder base, while testosterone treatment increases the density of MChRs, and increases the ratio of the bladder to total body weight. Although no contractile changes were observed in the bladder base tissue it is conceivable that longer chronic testosterone deficits might ultimately affect the bladder outlet resistance in the male because of the reduced alpha adrenergic receptor density.

COMPARATIVE IN VITRO EFFECTS OF IMIPRAMINE, OXYBUTYNIN, AND FLAVOXATE ON RABBIT DETRUSOR*

The ability of imipramine, oxybutynin, and flavoxate to antagonize carbamylcholine and barium chloride (BaCl2)-induced contractions of rabbit detrusor, and to block impulse conduction in desheathed frog sciatic nerves has been assessed in vitro. Impiramine exerts noncompetitive carbamylcholine and BaCl2 blockade by 10(-5) M and a local anesthetic effect equipotent with that of tetracaine. Oxybutynin exerts a strong competitive antagonism of carbamylcholine by 10(-8) M, a noncompetitive antagonism of BaCl2 equivalent to that of imipramine, and moderate local anesthetic activity. Flavoxate, under these experimental conditions exerts little anticholinergic, antispasmodis, or local anesthetic activity. Imipramine and oxybutynin thus demonstrate a number of smooth muscle effects by which their therapeutic actions may be exerted. The mechanisms of action of flavoxate remain obscure.

Y2 receptors for neuropeptide Y in the nucleus of the solitary tract mediate depressor responses

In anesthetized, spontaneously breathing rats, microinjections of selective agonists of neuropeptide Y (NPY) receptor subtypes were made into the medial region of the caudal nucleus of the solitary tract (NTS) at the level of the area postrema. This region of the rat NTS exhibits very high densities of NPY binding sites. Microinjections of the long C-terminal NPY fragment, NPY(13-36), a selective agonist at Y2 receptors, into the caudal NTS elicited pronounced, dose-related reductions in blood pressure and respiratory minute volume. Moreover, the specific pattern of cardiorespiratory responses elicited by NPY(13-36) was remarkably similar, over approximately the same dosage range, with the cardiorespiratory response pattern elicited by intact NPY. In contrast to the potent NTS-mediated responses evoked by NPY(13-36), similar microinjections conducted with either NPY(26-36), an inactive C-terminal NPY fragment, or [Leu31,Pro34]NPY, a NPY analog with specific agonist properties at Y1 receptors, into the same caudal NTS sites did not appreciably affect cardiorespiratory parameters even at 10-20-fold higher dosages. The present results with selective agonists for NPY receptor subtypes suggest that the depressor responses and reductions in minute volume elicited by microinjections of intact NPY and NPY(13-36) were mediated by Y2 receptors in the caudal NTS, likely distributed at presynaptic sites in the medial region of the subpostremal NTS.

Interactions of Calcium, Prostaglandins and Indomethacin on the Smooth Muscle of the Bladder

The effects of prostaglandin (PG) E2, PGF2alpha and indomethacin (Indo) were studied on isolated detrusor smooth muscle strips in balanced salt solution and in 80 mMK+ depolarizing solution. The addition of Indo to the smooth muscle preparation at concentrations of 0.1 to 1.0 micronM produced depression of spontaneous motility that was partially antagonized by PGE2 or by elevating the extracellular Ca2+ level. Alone, both PGE2 and Ca2+ caused a marked increase in motility, increasing both frequency and amplitude. In low Ca2+, K+ depolarized bathing medium with 0.1 mM EGTA added PGE2 or PGF2alpha augmented Ca2+ contractures both in velocity and amplitude while either PG without Ca2+ had no effect on the smooth muscle. Indo produced a noncompetitive antagonism of the Ca2+ dose response curve in 80 mM K+ depolarized preparations suggesting a direct effect on Ca2+ flux. Also Indo depressed both PG and Ca2+ contractures in terms of velocity and magnitude, suggesting that Indo may act at Ca2+ channels in addition to its action on PG synthetase. These data support the work of others who suggest that PGs may augment Ca2+ permeability, acting at the Ca2+ channel or as a carrier for Ca2+ across smooth muscle cell membranes.

Intracellular Localization of Fibrinogen.

Summary Hepatic parenchymal cells contained fibrinogen when immunologic technics and isolated, functional, parenchymal cell concentrates were applied in this study. With cellular fractions the greatest concentrations of fibrinogen, intracellularly, were in the soluble and microsomal fractions. These data are compatible with the view that fibrinogen is synthesized in the microsomes, released to the soluble part of the parenchymal cell and stored there until additional fibrinogen is required in the blood.

Characterization of Cocaine Binding Sites in the Rat Testes

PURPOSE An estimated 29 million individuals use cocaine in the United States. Studies have shown a high affinity for dose dependent binding of cocaine in the testes. Recent work done in our laboratory has shown that chronic administration of cocaine to male rats has an adverse effect on fertility and spermatogenesis by producing extensive morphological changes in the testes, leading to reduction in sperm production. As a first step toward understanding this process, we characterized and identified the pharmacological properties of [3H]cocaine binding sites in the testes. MATERIALS AND METHODS Crude membranes from the testes were prepared from 35 days old male Sprague-Dawley rats. [3H]cocaine binding was measured by using the method of Madras et al. (1989) with modifications. The data from saturation binding assays were analyzed by Inplot (GraphPad Software, San Diego, CA) to determine the Kd and Bmax. RESULTS Specific binding of [3H]cocaine was linearly dependent on membrane protein concentrations ranging from 0.2 to 8 mg./ml. The pooled data from three independent experiments revealed a mean affinity of 36 +/- 2.0 nM and Bmax of 1.84 +/- 0.13 pmol/mg. The present study demonstrates that testicular tissue has receptor protein that binds [3H]cocaine saturably and specifically. Competition displacement experiments revealed a shallow displacement curve for (-)cocaine and Win 35,428 with r2 = 0.96, indicative of multiple binding components. Computer analysis confirmed that a two component binding model was preferred statistically over a one component model in all three experiments (p < 0.001). CONCLUSION The results from these studies suggest that the testicular tissue contains a protein that binds [3H]cocaine in a saturable and specific manner. It has a different sensitivity from the [3H]cocaine binding protein in the brain and placenta. Further clarification of the relationship between cocaine and its recognition site is necessary to understand the mechanism of testicular damage after cocaine exposure.

Quantitation and Stability of Cholinergic Receptors In Human Bladder Tissue from Post Surgical and Postmortem Sources

Ligand binding for cholinergic muscarinic receptors with (3H) quinuclidinyl benzilate was performed on human detrusor smooth muscle from post surgical and postmortem sources. Following cystectomy, detrusor smooth muscle was serially sampled over an interval of 0 to 9 hours. (3H) quinuclidinyl benzilate binding on individual samples indicated that the total receptor density remained relatively unchanged. Postmortem specimens obtained from patients ranging in age from 1 day to 82 years were also assayed and the cholinergic receptor density was found to range between 18.4 to 82.1 fmoles/mg. of protein. All of the affinity constants from both sources were also in a relatively narrow range of 20.0 to 99.0 pmolar. The results of this study provide evidence for the usefulness of postmortem human tissue for evaluating cholinergic receptors in normal and dysfunctional bladder states.

INTRACELLULAR LOCALIZATION OF PROTHROMBIN.

Prothrombin formation and storage are clearly functions of liver parenchymal cells according to information obtained with the fluorescent antibody technique (1,2). The present report extends the information on prothrombin localization to the intracellular level in dogs and cattle. Materials and methods. Isolated parenchymal cell concentrates were obtained with the technique of St. George (3) on Dowex 50 W resin columns. Evaluation of the homogeneity of the fractions was made by differential cell counts on Leishman stained smears. A typical cell concentrate contained 96.5% parenchymal cells, 3% free nuclei and 0.5% red blood cells. In cell counts up to 600 cells neither Kupffer cells nor platelets were found. Subcellular fractions were prepared in 0.25 m sucrose by the procedures of Schneider and Hogeboom(4) and de Duve and associates (5). The following fractions were studied: total cell homogenate (TCH), cytoplasmic extract (CE), nuclei (N), mitochondria (M), lysosomes (L), microsomes (Mic) and soluble fraction (S). Further purification of the nuclear fraction was achieved by centrifugation at 345 g for 5 minutes to sediment the associated whole cells and other subcellular particulates. The resulting supernatant containing nuclei was recentrifuged at 750 g for 5 minutes. The nuclei in this precipitate were then resuspended in 0.25 m sucrose and washed twice. All fractions were finally suspended in sucrose so that the stock solutions were 10% by volume. (TCH) and soluble (S) fraction from a normal dog. B. Identical antigenic substances exist in mierosomes (Mic) and soluble (S) fraction of normal dogs. C. Note the preeipitin band indicative of prothrombin localization in soluble fraction of dicumarolized dogs (SD) as well as in soluble fraction from Vit. K1 stimulated (SK) animals, D. Microsomes obtained from a dicumarolized dog (MicD) did not react with antiprothrombin.