The alpha adrenergic binding properties of terazosin in the human prostate adenoma and canine brain

Clinical trials are currently underway to evaluate the efficacy of terazosin for the treatment of symptomatic benign prostatic hyperplasia (BPH). Terazosin is a potent and selective alpha 1 adrenergic blocking agent structurally similar to prazosin. The alpha adrenergic binding properties of terazosin were studied in human prostate adenomas and canine brains using radioligand receptor binding methods. Saturation analyses were performed at varying concentrations of [125I]-Heat and [3H]rauwolscine [( 3H]Ra) in human prostate adenomas and canine brains. The binding of [125I]-Heat and [3H]Ra in the human prostates and canine brains was consistently saturable and of high affinity. The equilibrium dissociation constant (Kd) for [125I]-Heat binding in the canine brains and human prostate adenomas was 84.4 +/- 4.3 pM and 65.4 +/- 19.2 pM, respectively (p greater than 0.05). The (Kd) for [3H]Ra binding in the human prostate adenomas and canine brains was 1.21 +/- 0.23 nM and 1.52 +/- 0.28 nM, respectively (p greater than 0.05). The density of alpha 1 (0.37 +/- 0.15 fmol/mg. wet wt.) and alpha 2 (0.29 +/- 0.09 fmol/mg. wet wt. adrenergic binding sites in the human adenomas were similar (p greater than 0.05). The IC50 corrected (IC50 corr) of terazosin for [125I]-Heat and [3H]Ra binding sites in the human prostate was 2.5 nM and 1.0 micron., respectively. The IC50 corr of terazosin for [125I]-Heat and [3H]Ra binding sites in the canine brain was 2.0 nM and 0.8 microM, respectively. The competitive binding assays indicate that terazosin binds selectively to alpha 1 adrenergic binding sites in the human prostate and canine brain.     

Density and localization of alpha 1-adrenoceptors in hypertrophied prostate.

Alpha-adrenoceptors have been considered to play an important role in the regulation of the voiding force. In order to determine the density and localization of the alpha-adrenoceptors in the prostate, binding assays for alpha-adrenoceptors were performed with [3H]prazosin and [3H] yohimbine in membrane preparations from enucleated hyperplastic prostate tissues. Furthermore, autoradiographic analysis of alpha-adrenoceptors in the sliced tissue specimens from benign prostatic hypertrophy was performed. Specific binding of both ligands were saturable and of high affinity in membrane preparations, and Scatchard analyses indicated Bmax = 104 fmole/mg. protein, Kd = 0.488 nM for [3H]prazosin, and Bmax = 41 fmole/mg, protein, Kd = 1.83 nM for [3H] yohimbine. Bmax and Kd for [3H]prazosin were greater in the adenoma than in the submucosal tissue of the prostatic urethra. No relationship was noted between the size of enucleated prostate and the density of alpha-adrenoceptors. Image analysis of autoradiograms using [3H]prazosin showed specific binding sites could not be clearly demonstrated, but only [125I]HEAT slightly exposed specific binding sites in the prostate.     

Characterization and localization of alpha 1-adrenoceptors in human prostate--with special reference to the zonal difference in receptor distribution]

Radioligand binding assay and receptor autoradiography were undertaken to characterize alpha 1-adrenoceptors and to clarify their localization in human prostate. Eleven open prostatectomy specimens obtained from patients with benign prostatic hypertrophy were used for radioligand binding assay. The binding of [3H]-prazosin to prostatic crude membrane fraction was a saturable process of high affinity. Scatchard analysis of the specific [3H]-prazosin binding indicated the existence of a single population of receptor sites with an equilibrium dissociation constant of 1.00 +/- 0.19 nM (mean +/- SE) and a maximum binding capacity of 20.8 +/- 3.56 fmol/mg protein (mean +/- SE). alpha-Adrenergic antagonists competed for [3H]-prazosin binding in an order of potency: prazosin greater than or equal to bunazosin greater than phentolamine greater than yohimbine, suggesting that the binding sites for [3H]-prazosin have characteristics of alpha 1-adrenoceptors. To study the distribution of alpha 1-adrenoceptors in prostatic tissues obtained from total cystoprostatectomy specimens of six male bladder tumor patients, we used for autoradiography (+/-)-beta-[( 125I]Iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone [( 125I]-HEAT). Peripheral zone (PZ), central zone (CZ) and preprostatic region were dissected from the tissues and incubated with [125I]-HEAT, followed by an autoradiographic procedure. Distribution of alpha 1-adrenoceptors was quantitated by grain counting using a light microscope equipped with a micrometer. The specific binding sites for [125I]-HEAT were demonstrated predominantly in the fibromuscular stroma of CZ, that of PZ, preprostatic sphincter and subordinately in the glandular epithelium of CZ, but not in the glandular epithelium of PZ and periurethral glands.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-affinity specific [3H]tamsulosin binding to α1 in human prostates with benign prostatic hypertrophy

The binding of a novel radioligand, [³H]tamsulosin, to human prostatic membranes with benign prostatic hypertrophy (BPH) has been characterized. [³H]Tamsulosin rapidly associated with its binding sites in human prostatic membranes with BPH, and the binding reached steady state by 30 min at 25°C. The rate constants for association and dissociation of [³H]tamsulosin binding were calculated to be 0.21±0.05/nM per minute and 0.01±0.004/min, respectively. The specific binding of [³H]tamsulosin in human prostatic membranes was saturable and of high affinity (K _d=0.04±0.01 nM). The density of [³H]tamsulosin-binding sites (B _max) was 409±28 fmol/mg protein. The K _d and B _max values for [³H]tamsulosin binding in human prostates were significantly lower than those for [³H]prazosin binding. [³H]tamsulosin binding was remarkable for its significantly lower degree of nonspecific binding. Six -adrenoceptor antagonists competed with [³H]tamsulosin for the binding sites in the rank order: tamsulosin>WB4101>prazosin>S-(+)-isomer>naftopidil>yohimbine. The binding affinities (pK_i) of these antagonists for [³H]tamsulosin binding in human prostates closely correlated with their pharmacological potencies (pA₂) in prostates. In conclusion, [³H]tamsulosin selectively labels ₁-adrenoceptors in human prostates, and thus may become a useful radioligand for the further analysis of these receptors.     

Characterization of alpha1 adrenergic receptors in human benign prostatic hyperplasia

Bladder outlet obstruction in men with benign prostatic hyperplasia is decreased following administration of prazosin, a selective alpha1 adrenergic antagonist. Prazosin presumably binds and antagonizes alpha1 adrenergic receptors on the smooth muscle cells of the prostatic adenoma. This study represents the first identification and characterization of alpha1 adrenergic receptors in the prostate using radioligand receptor binding methods. The binding of [3H] prazosin in homogenates obtained from human prostatic adenomas was saturable and a single high affinity prazosin binding site was identified (Kd = 0.29 +/- 0.09 nM). The alpha1 adrenergic receptor concentration in these homogenates ranged between 0.28 to 2.05 fmol./ mg. wet wt. prostate. The equilibrium dissociation constant and density of prazosin binding sites were similar in different regions of an enucleated prostate suggesting homogeneity of receptor density and receptor binding sites within an adenoma. The receptor density was not directly proportional to the weight of the surgically removed adenoma. The pharmacology of the prazosin binding sites was characterized by competitive binding experiments using [3H] prazosin and several unlabelled adrenergic analogs. The IC50's determined from competitive binding experiments using [3H] prazosin and alpha-methylnorepinephrine, rauwolscine and corynanthine were characteristic of alpha1 adrenergic receptor binding.     

Alpha 2 adrenergic receptors in hyperplastic human prostate: identification and characterization using [3H] rauwolscine

[3H]Rauwolscine ([3H]Ra), a selective ligand for the alpha 2 adrenergic receptor, was used to identify and characterize alpha 2 adrenergic receptors in prostate glands of men with benign prostatic hyperplasia. Specific binding of [3H]Ra to prostatic tissue homogenates was rapid and readily reversible by addition of excess unlabelled phentolamine. Scatchard analysis of saturation experiments demonstrates a single, saturable class of high affinity binding sites (Bmax = 0.31 +/- 0.04 fmol./microgram. DNA, Kd = 0.9 +/- 0.11 nM.). The relative potency of alpha adrenergic drugs (clonidine, alpha-methylnorepinephrine and prazosin) in competing for [3H]Ra binding sites was consistent with the order predicted for an alpha 2 subtype. The role of alpha 2 adrenergic receptors in normal prostatic function and in men with bladder outlet obstruction secondary to BPH requires further investigation.     

Binding and functional properties of doxazosin in the human prostate adenoma and canine brain

The binding and functional properties of doxazosin were characterized in the canine brain and human prostate. 3H-Doxazosin binding sites were characterized in canine brain and human prostate homogenates using saturation experiments. The binding of 3H-doxazosin in the canine brain was consistently saturable and of high affinity. The mean equilibrium dissociation constant (Kd) and density (Bmax) of 3H-doxazosin binding sites in the canine brain were 0.19 nM and 2.17 fmol/mg wet wt, respectively. The binding of 3H-doxazosin in human prostate homogenates was not consistently linear owing to a relatively high proportion of nonspecific doxazosin binding sites. The mean Kd and Bmax of 3H-doxazosin binding sites in the prostate determined from the saturation experiments yielding linear Scatchard plots were 0.2 nM and 0.51 fmol/mg wet wt. The pharmacology of doxazosin binding sites was further characterized in the canine brain using competitive binding experiments. The rank order of IC50corr values for norepinephrine, clonidine, yohimbine, terazosin, and prazosin indicated that doxazosin binds selectively to alpha 1 and alpha 2 adrenergic binding sites. The relative affinity of unlabeled doxazosin for alpha 1 and alpha 2 binding sites in the human prostate was determined by displacing 125I-Heat or 3H-rauwolscine with varying concentrations of unlabeled doxazosin. The affinity of doxazosin for alpha 1 binding sites in the prostate adenoma was approximately 100-fold greater than its affinity for alpha 2 binding sites. The potency of doxazosin for inhibiting phenylephrine-induced contractions in the prostate indicated that prostate smooth muscle contraction is mediated by alpha 1 adrenoceptors.     

Characterization and localization of the muscarinic cholinergic receptor in human prostatic tissue

Radioligand receptor binding and autoradiography were used to characterize and localize the muscarinic cholinergic receptor in human benign prostatic hyperplastic tissue. These methods have not been used previously to investigate the autonomic innervation of the human prostate. The binding of [3H]N-methylscopolamine ([3H]NMS), a muscarinic cholinergic antagonist, to homogenates of human prostate was saturable and of high affinity. The equilibrium dissociation constant, (Kd), for [3H]NMS binding to human prostate homogenates was 0.10 +/- 0.03 nM (mean +/- SEM). The values of the Kd's for [3H]NMS binding to prostates of man (0.10 nM), dog (0.20 nM), pig (0.11 nM), rat (0.07 nM) and rabbit (0.15 nM) were similar, suggesting homogeneity of muscarinic cholinergic receptors in varying species. The mean density, B(max), of muscarinic cholinergic receptors identified in the human prostate was 2.1 fmol./mg. prostate wet weight. The relative density of receptors in the human prostates were similar in the homogenates and slide-mounted tissue sections. The pharmacology of NMS binding sites on slide-mounted tissue sections was evaluated by competitive binding experiments using [3H]NMS and atropine. The IC50 corrected of atropine on slide-mounted tissue sections (0.42 nM) was similar to values obtained in prostate homogenates (1.16 nM). Autoradiography on slide-mounted tissue sections demonstrated that the muscarinic cholinergic receptors were localized to the epithelium of the prostate. The ratio of specific NMS binding in the epithelial and stromal components of the prostate, expressed as autoradiographic grains/unit area and autoradiographic grains/cell, was 71:1 and 33:1 respectively. Because prostatic secretion is dramatically enhanced by muscarinic cholinergic agonists, localization of muscarinic cholinergic receptors to the epithelium is consistent with the neuropharmacology of prostatic secretion. These studies have provided basic insight into the neuropharmacology of the prostate. Future studies will be necessary to characterize and localize other neurotransmitters in the human prostate in order to further enhance our understanding of prostatic function.     

Identification and characterization of alpha 1 adrenergic receptors in the canine prostate using [125I]-Heat

We have recently utilized radioligand receptor binding methods to characterize muscarinic cholinergic and alpha adrenergic receptors in human prostate adenomas. The primary advantages of radioligand receptor binding methods are that neurotransmitter receptor density is quantitated, the affinity of unlabelled drugs for receptor sites is determined, and receptors can be localized using autoradiography on slide-mounted tissue sections. Recently, [125I]-Heat, a selective and high affinity ligand with high specific activity (2200 Ci/mmole) has been used to characterize alpha 1 adrenergic receptors in the brain. In this study alpha 1 adrenergic receptors in the dog prostate were characterized using [125I]-Heat. The Scatchard plots were linear indicating homogeneity of [125I]-Heat binding sites. The mean alpha 1 adrenergic receptor density determined from these Scatchard plots was 0.61 +/- 0.07 fmol/mg. wet wt. +/- S.E.M. The binding of [125I]-Heat to canine prostate alpha 1 adrenergic binding sites was of high affinity (Kd = 86 +/- 19 pM). Steady state conditions were reached following an incubation interval of 30 minutes and specific binding and tissue concentration were linear within the range of tissue concentrations assayed. The specificity of [125I]-Heat for alpha 1 adrenergic binding sites was confirmed by competitive displacement assays using unlabelled clonidine and prazosin. Retrospective analysis of the saturation experiments demonstrated that Bmax can be accurately calculated by determining specific [125I]-Heat binding at a single ligand concentration. [125I]-Heat is an ideal ligand for studying alpha 1 adrenergic receptors in the prostate and its favorable properties should facilitate the autoradiographic localization of alpha 1 adrenergic receptors in the prostate.     

Changes in alpha-adrenergic receptors in dog livers during endotoxic shock

The effects of endotoxin administration on alpha-adrenergic receptors in dog liver plasma membranes were studied using [3H]dihydroergocryptine as a radioactive ligand. The Scatchard analysis revealed a two-component binding characteristic both in control and endotoxin-injected dogs. The Kd (dissociation constant) of the high-affinity component was increased by 32.5% (0.4 +/- 0.04 nM for control vs 0.53 +/- 0.06 nM for endotoxic; P less than 0.05) with no significant change in the Kd for the low-affinity component (3.0 +/- 0.44 nM for control vs 3.4 +/- 0.44 nM for endotoxic) 2 hr following endotoxin administration. The maximum binding capacity of the high-affinity component was decreased by 38.1% (460 +/- 19.3 and 285 +/- 14.8 fmole/mg protein for control and endotoxic, respectively; P less than 0.01) and that of the low-affinity component was decreased by 34.2% (1050 +/- 66.4 and 690 +/- 44.6 fmole/mg protein for control and endotoxic, respectively; P less than 0.05) 2 hr after endotoxin injection. The competitive inhibition studies show that the apparent Kd values for (-)-epinephrine, (-)-norepinephrine, and prazosin were increased 15, 13, and 25 times, respectively, with no significant change in the apparent Kd values for yohimbine or phentolamine 2 hr postendotoxin. These data demonstrate that the binding affinity of the high-affinity component and the number of alpha-adrenergic receptor binding sites were decreased in endotoxic shock. A modification of the alpha-adrenergic receptors in dog livers induced by endotoxin administration may play an important role in the development of hepatic glucose dyshomeostasis during shock.     

Identification and characterization of parathyroid hormone receptors in rat renal cortical plasma membranes using radioligand binding

Parathyroid hormone (PTH) receptors have been described in renal tissue from several species, but not in the rat. In this study, radioligand binding techniques were used to identify and characterize PTH receptors in rat kidney cortical membranes. The sulfur-free PTH analog [Nle8,18Tyr34]bovine PTH-(1-34)amide was iodinated using the iodogen method. This ligand was suitable for use in identifying PTH receptors in canine renal membranes, but not rat renal membranes. Synthetic, unsubstituted rat PTH-(1-34) was iodinated using the milder, lactoperoxidase technique and was purified by HPLC on a C8 column. [125I]rat PTH-(1-34) bound rapidly to both rat and dog renal membranes. At 22 degrees C reaction reached steady state within 20 minutes, and this level was maintained for at least 3 h. Specific binding was routinely greater than 90% for rat kidney and greater than 95% for dog kidney. Similar results were obtained at 4 degrees C with a longer time required to attain steady state (approximately 45 minutes). Binding was reversible as demonstrated by dissociation of bound ligand after either infinite dilution or displacement with excess nonradioactive PTH. Binding was saturable and of high affinity (rat kidney: Bmax = 2.3 pmol/mg protein, Kd = 3.1 nM, dog kidney: Bmax = 2.1 pmol/mg protein, Kd = 3.7 nM). Rat renal cortical adenylate cyclase activity was stimulated by rat PTH in a dose-dependent manner with an EC50 of 4 nM, a value in good agreement with the binding data. This study demonstrates the feasibility of identifying and characterizing parathyroid hormone receptors in rat renal cortical plasma membranes using radioligand binding techniques.     

Direct evidence of alpha-adrenoceptor binding in rat and human adrenal glands

In order to determine whether or not alpha-adrenoceptors are present in adrenal glands, radioligand receptor binding assay was performed in both Sprague-Dawley (SD) rat and human adrenal gland membranes. Radioligand binding assay using 3H-prazosin as an alpha 1-adrenoceptor ligand and 3H-yohimbine as an alpha 2-adrenoceptor ligand, clearly demonstrated alpha 1 and alpha 2 receptors present in both rat and human adrenal gland membranes. Maximal binding capacity (Bmax) and dissociation constant (Kd) of 3H-prazosin binding to the rat adrenal gland were 12.5 fmol/mg protein, and 0.11 nM, respectively. Those for the membrane preparations from adrenal cortex and medulla of the normal human were 16.3 fmol/mg protein, 0.34 nM and 16.3 fmol/mg protein, 0.27 nM, respectively. And those of the human pheochromocytoma were 25.6 fmol/mg protein, 0.15 nM, respectively. On the other hand, Bmax and Kd of 3H-yohimbine binding in the rat adrenal gland to were 22.9 fmol/mg protein, and 4.28 nM, respectively. Those for the membrane preparations from adrenal cortex and medulla of the normal human were 40.4 fmol/mg protein, 5.15 nM and 12.2 fmol/mg protein, 5.39 nM, respectively. And those of the human pheochromocytoma were 35.8 fmol/mg protein, and 1.08 nM, respectively. Bmax (35.8 fmol/mg protein) of 3H-yohimbine binding in the pheochromocytoma was significantly (p less than 0.01) greater than that (12.2 fmol/mg protein) in the human normal adrenal medulla, while Kd (1.08 nM) of this binding in the human pheochromocytoma was significantly (p less than 0.01) lower than that (5.39 nM) in the human normal adrenal medulla. Our data suggest that the alpha 2 receptor had greater affinity and binding site density to its agonist in the human pheochromocytoma than in the human normal adrenal medulla.     

Comparison of selective alpha-1 blockades for alpha-receptors in human hypertrophied prostatic adenomas]

The adrenergic alpha-1 and -2 adrenoceptors in six human hypertrophied prostatic adenomas were measured in the saturation experiment using 3H-prazosin and 3H-yohimbine. Not only alpha-1 adrenoceptons, but also alpha-2 adrenoceptors were found to exist in large amounts in prostatic adenomas. In the inhibition experiment selective alpha-1 antagonists inhibited the 3H-prozosin or 3H-yohimbine bindings to adenomas. The potency of alpha-1 antagonists in the order prazosin greater than bunazosin greater than alfuzosin greater than urapidil greater than terazosin and that of alpha-2 antagonists is urapidil greater than alfuzosin greater than terazosin greater than bunazosin greater than prazosin. These data suggest that urapidil, alfuzosin and terazosin may affect the human hypertrophied prostatic adenoma like phenoxybenzamine, nonselective alpha-1 antagonist, which was used for benign prostatic hypertrophy.     

Binding properties of a selective tritiated vasopressin V2 receptor antagonist, [H]-SR 121463

BACKGROUND: [3H]-SR 121463 is the first radiolabeled selective nonpeptide vasopressin V2 receptor antagonist ligand that has been reported to date. In the present work, we studied the binding properties of [3H]-SR 121463 for renal V2 receptors from animal and human origins. METHODS: Binding studies were performed with [3H]-SR 121463 in Chinese hamster ovary (CHO) cells transfected with the human V2 receptor and in various kidney preparations expressing the native V2 receptors (rat, rabbit, dog, pig, monkey, and human). Autoradiographies were performed in rat and human kidney sections. RESULTS: [3H]-SR 121463 binding to CHO cells stably transfected with the cloned human renal V2 receptor was specific, highly stable, time dependent, saturable, and reversible. A single population of high-affinity binding sites was identified (Kd = 0.94 +/- 0.34 nmol/L, Bmax = 9876 +/- 317 fmol/mg protein). Of note, [3H]-SR 121463 revealed a higher number (about 40%) of V2 sites than [3H]-AVP in the same preparation. Displacement of [3H]-SR 121463 binding by reference peptide and nonpeptide vasopressin/oxytocin compounds exhibited a typical AVP V2 profile. [3H]-SR 121463 also displayed a high affinity for native V2 receptors in several kidney preparations from rat, pig, dog, rabbit, bovine, monkey, and human. The autoradiographic experiments using rat and human kidney sections showed intense labeling in the medullopapillary region and lower intensity in the cortex, consistent with a main localization of V2 receptors on collecting tubules. CONCLUSION: [3H]-SR 121463 is a useful ligand for the specific labeling of animal and human V2 receptors and could be a suitable probe for the search and in situ localization of V2 sites.     

Depression of calcium channel blocker binding to rat brain membranes by halothane.

The present study evaluates the action of volatile anesthetics on the voltage-dependent Ca2+ channels in isolated rat brain membranes, measured as changes in binding of the Ca2+ channel blocker [3H]isradipine to these membranes. Equilibrium binding studies with increasing concentrations of [3H]isradipine (0.01-1 nM) in the presence of halothane (1.9%), isoflurane (2.3%), and enflurane (4.8%) at 25 degrees C were performed. Only halothane produced a significant depression in the specific binding of isradipine to the brain membranes at 0.5 and 1.0 nM [3H]isradipine (P = 0.028 and 0.018, respectively). Isoflurane and enflurane had such inconsistent effects that the data were inconclusive. Halothane produced a significant dose-dependent inhibition of binding, the maximum inhibition being 44% (P less than 0.005). Nonlinear regression analysis fit of the binding data indicates halothane produced a 48% decrease (P less than 0.05) in the maximal number of binding sites (Bmax) with no effect on the dissociation constant (Kd). As voltage-dependent Ca2+ channels are important in mediating neurotransmission, the marked decrease in channel number (Bmax) associated with halothane exposure suggests that this phenomenon might be related to the mechanism of general anesthesia.     

Characterization of cholinergic receptors in Madin-Darby canine kidney cells.

Muscarinic-type cholinergic receptors coupled to the phosphoinositide (PI) second messenger system are reported to be present in the inner medullary collecting duct cells. Madin-Darby canine kidney (MDCK) cells have several characteristics of collecting duct cells and have been shown to respond to muscarinic agonists. To determine if MDCK cells have PI-coupled muscarinic receptors, the radioligand binding and the effects of cholinergic agonists and antagonists on PI hydrolysis in MDCK cells were studied. The specific binding of [3H]1-quinuclidinyl benzilate ([3H]QNB), a muscarinic antagonist, to MDCK cell membranes had a Kd = 88 +/- 7 pM and a Bmax = 1464 +/- 88 fmol/mg of protein. The displacement of [3H]QNB from MDCK cell membranes by various cholinergic antagonists and agonists showed the order of potency: atropine greater than 4-diphenylacetoxy N-methylpiperidine (4-DAMP) greater than p-fluorohexahydrosiladifenidol greater than pirenzepine greater than metoctramine greater than arecoline greater than carbachol. The cholinergic agonists carbachol and arecoline stimulated PI hydrolysis in a concentration-dependent manner with an EC50 of 3.7 and 1.3 microM, respectively. Muscarinic antagonists abolished carbachol-stimulated PI hydrolysis in the following order of potency: atropine greater than 4-DAMP greater than pirenzepine much greater than methoctramine. The order of potency of muscarinic antagonists is consistent with the characteristics of the M3 subtype of muscarinic receptors. It is concluded that: (1) muscarinic receptor density in MDCK cells is 50 times higher than that in inner medullary collecting duct cells; (2) muscarinic receptors in MDCK cells are putative M3 subtype; and (3) muscarinic receptors in MDCK cells are functionally coupled to the PI second messenger system. This intracellular messenger system may, at least, be partially responsible for the action of cholinergic agonists in these cells and in the kidney.     

The identification of adrenergic receptors in human pial membranes

Recent experimental work has suggested that the adrenergic nervous system is important in regulating cerebral blood flow under conditions of hypoxia and systemic arterial hypertension. Although previous investigations have demonstrated the presence of adrenergic neurons adjacent to human cerebral vessels, the nature of adrenergic receptors on human cerebral blood vessels remains poorly defined. The present study was performed to characterize adrenergic reception on membranes prepared from human pia, a rich source of small blood vessels, using radioligand binding techniques. Adrenergic membrane receptors were characterized using the binding of [3H]dihydroalprenolol for the beta subtypes and [3H]prazosin and [3H]yohimbine for the alpha subtypes. Displaceable binding was demonstrated with each agent. A small series of adrenergic agents competed for the [3H]dihydroalprenolol, [3H]yohimbine, and [3H]praz binding sites in a fashion suggesting the presence of alpha 1-, alpha 2-, beta 1-, and beta 2-receptors on the vessels within human pia.     

Evidence of functional gastric inhibitory polypeptide (GIP) receptors in human insulinoma. Binding of synthetic human GIP 1-31 and activation of adenylate cyclase

Specific gastric inhibitory polypeptide (GIP) receptors were characterized in human benign insulinoma plasma membranes employing [mono-[125I]iodo-Tyr10]-GIP (125I-GIP) as the radioligand. GIP 1-42 inhibited 125I-GIP binding with an IC50 value of 10(-9) M. Scatchard analysis showed two classes of binding sites: a high-affinity site (Kd = 2.23 x 10(-10) M; Bmax = 24 fmol/mg protein) and a low-affinity site (Kd = 8.39 x 10(-9) M; Bmax = 118 fmol/mg protein). A synthetic replicate of human GIP 1-31 inhibited 125I-GIP binding with an IC50 value of 10(-8) M. The GIP binding sites of human insulinoma were coupled to adenylate cyclase stimulation. GIP 1-31 regulated the adenylate cyclase activity to the same extent as GIP 1-42. The concentrations of GIP required for maximal activity ranged from 10(-9) to 10(-8) M for either GIP 1-42 or GIP 1-31. The existence of functional GIP receptors in human insulinoma substantiates our recent reports demonstrating the presence of GIP binding sites in transplantable hamster insulinoma and indicates that GIP could exert a direct control of the beta-cell function in humans through a purely endocrine pathway.     

Inhibition of murine neuroblastoma growth by dopamine antagonists

C-1300 murine neuroblastoma ( MNB ) contains the catecholamine biosynthetic pathway. This study investigated manipulation of this pathway for effects on cell growth and survival in tumor-bearing mice, and to correlate these findings with specific membrane-bound dopamine-binding activity. The dopamine antagonists domperidone, pimozide, and spiroperidol inhibited macromolecular synthesis in vitro as demonstrated by decreased [3H]TdR and [14C]leu incorporation in a dose-response fashion; 56, 49, and 43% inhibition was noted at 10(-6) M concentration of each drug, respectively, with no loss of cell viability. Dopamine agonists showed no significant inhibition. Scatchard analysis of dopamine binding was consistent with a single class of receptor sites with a mean concentration of 13.2 +/- 2.0 pmole/g wet weight of tissue and mean dissociation constant (Kd) = 0.69 +/- 0.38 nM, compared to a mean receptor concentration of 28.1 +/- 5.2 pmole/g wet weight of tissue and Kd = 0.38 +/- 0.09 nM in receptor-rich dog caudate nucleus, the normal control. A/J mice injected with 1 X 10(6) tumor cells and treated with daily pimozide or domperidone had a significant increase in disease-free survival when compared to controls (15 versus 8.5 days, P less than 0.001) as well as a significant increase in overall survival (35 versus 25 days, P less than 0.001). These data suggest that dopamine antagonists inhibit macromolecular synthesis in the C-1300 MNB . The inhibition of MNB tumor growth in vivo by dopamine antagonists suggests a specific chemotherapeutic approach to neuroblastoma, possibly mediated by dopamine receptors.     

The interaction of neuromuscular relaxants with rabbit lung muscarinic receptors

The interaction of muscle relaxants with airway muscarinic receptors of rabbit lung was investigated in vitro by the [3H]QNB binding technique. Pancuronium, vecuronium, alcuronium and succinyl choline chloride (SCC) inhibited the binding of [3H]QNB to rabbit lung muscarinic receptors in a dose-dependent manner. The values of IC50 (the concentration giving 50% inhibition) of pancuronium, vecuronium, alcuronium and SCC were 1.54 x 10(-5), 2.52 x 10(-5), 8.40 x 10(-5), and 4.00 x 10(-3) mol/l respectively. As the values of Kd increased with minimal change in the value of Bmax, while not influencing the number of receptors, these muscle relaxants had an inhibitory action on the affinity of muscarinic receptors to [3H]QNB in the order: pancuronium greater than or equal to vecuronium greater than alcuronium greater than SCC. Applying IC50 values to human conditions, clinical doses of these muscarinic relaxants are unlikely to exhibit any significant vagolytic action in lung tissue.