Autoradiographic localization of vasopressin and oxytocin binding sites in rat kidney

The presence of vasopressin receptors of the V1 (vascular) type and of oxytocin receptors in the rat kidney was investigated using an autoradiographical approach. Rat kidney sections were incubated with tritiated vasopressin ([3H]vasopressin, 1.5 nM) or oxytocin ([3H]oxytocin, 3 nM). The ligand selectivity of the [3H]vasopressin binding sites detected was deduced from competition experiments using one selective unlabeled ligand for V2 (antidiuretic) vasopressin receptors (1-deamino-[8-D-arginine]-vasopressin, dDAVP) and one selective unlabeled ligand for V1 receptors (des-glycineamide-[1-(beta-mercapto-beta,beta-cyclopentamethylene propionic acid]-arginine vasopressin, des(Gly(NH2)9d(CH2)5-AVP). Specific and dense [3H]vasopressin labeling was observable in the medullopapillary and cortical portions of the kidney. Specific [3H]vasopressin binding in the cortex was insensitive to the V1-selective ligand, des(Gly(NH2)9d(CH2)5-AVP, but was inhibited by dDAVP. Glomerular structures identified as such by microscopical observation of the kidney sections were specifically labeled with [3H]oxytocin and [125I]-SAR1-angiotensin II but not with [3H]vasopressin. It is concluded that V1 receptors which have been evidenced on mesangial cells in culture are not expressed in a detectable quantity on mesangial cells in situ. The specific [3H]oxytocin binding to glomeruli might reflect the presence on glomerular structures of oxytocin receptors involved in the effects of the hormone on renal hemodynamics, and possibly in some of the effects ascribed to vasopressin.     

Vasopressin receptors in human seminal vesicles: identification, pharmacologic characterization, and comparison with the vasopressin receptors present in the human kidney

Because of the presence of a high density of vasopressin receptors in the epithelial cells of porcine seminal vesicles similar to the V2 vasopressin receptors of renal tubules, human seminal vesicles and kidney were investigated using quantitative binding and adenylate cyclase studies. Tissues were obtained at surgery from 17 patients with urologic diseases. A homogeneous class of vasopressin binding sites have been found in both seminal vesicles and renal medulla. However, the vasopressin receptors present in these tissues are different in terms of ligand specificity and adenylate cyclase activation. In seminal vesicles, the V1 vasopressin antagonist d(CH2)5 TyrMeAVP is 36-fold, more potent than the V2 agonist dVDAVP in displacing [3H]AVP binding, while in the medullopapillary portion of kidney dVDAVP is 24-fold, more selective than d(CH2)5 TyrMeAVP for the arginine vasopressin binding site. Furthermore, arginine vasopressin induces a dose-dependent increase in adenylate cyclase activity in renal membranes, while it was ineffective in seminal vesicle membranes. These results indicate that a very high affinity (0.2 nM), low capacity (14 fmoles/mg protein) class of vasopressin receptors is present in human seminal vesicles, having pharmacologic characteristics similar to the V1 subtype of vasopressin receptors. The presence of a high affinity (1.6 nM), high capacity (350 fmoles/mg protein) V2 subtype of vasopressin receptors in human renal membranes is also confirmed. The density of the vasopressin receptors present in human seminal vesicles is inversely correlated with patient age, consistent with a physiologic role for vasopressin in the regulation of accessory sex gland activity.     

Characterization of thromboxane A2 receptors in rat kidney

The renal TXA2 receptors were investigated in preparations derived from rat kidney. Perfused rat kidneys were removed and separated into cortex and medulla. Each subcellular fraction was prepared by a sucrose density gradient method for analyzing the subcellular distribution of TXA2 receptors. The membranes were obtained by 40,000 x g centrifugation for analyzing the renal distribution of TXA2 receptors. Bindings of [3H]SQ 29548, a TXA2 receptor antagonist, to these preparations were carried out using a filtration method. Furthermore, the binding of [3H]SQ 29548 to membranes from rat kidney cortex was compared with its binding to rat washed platelets. The membrane fraction had the highest density of TXA2 receptors among subcellular fractions determined. TXA2 receptors appeared in corticomedullary junction with high density. Scatchard analysis using [3H]SQ 29548 as a ligand revealed a single binding site of TXA2 receptors in membranes from rat kidney cortex, and the dissociation constant (Kd) was remarkably higher than the Kd in rat washed platelets. These findings suggest that TXA2 receptors in membranes from rat kidney cortex are different from those in rat platelets, and TXA2 receptor activation in kidney might play a role in renal function.     

Characteristics of rat kidney dopamine receptors and the effects of renal denervation and dopamine infusion on these receptors

The characteristics of dopamine receptors, as well as the effects of denervation and dopamine infusion on dopamine receptors were studied using the radiolabeled receptor assay of [3H]-spiperone on rat kidney membrane preparations. The rat renal cortex was found to have a single class of [3H]-spiperone binding sites with a dissociation constant (Kd) of 13.5 +/- 2.2 nM. However, neither sulpiride nor serotonin strongly interacted with [3H]-spiperone binding, suggesting that DA1 receptors were predominant in the rat renal cortex. Denervation was performed by surgically stripping the nerves from the renal artery and coating them with 10% phenol. Chronic denervation had no significant effect on the affinity or maximum binding capacity of the renal dopamine receptors, although diuresis and the disappearance of catecholamine fluorophores in the denervated rats were observed. Chronic infusion of dopamine was performed using an osmotic minipump, resulting in a decrease in the number of rat kidney dopamine receptors. These results suggest that the dopamine receptor subtype in the renal cortex was mainly DA1, and that the major source of dopamine which affected the dopamine receptors in the rat kidney was not the nerve ending, but rather the circulation.     

Characterization of nociceptin/orphanin FQ binding sites in dog brain membranes

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ receptor (NOP), whose characteristics in the dog are unknown. We therefore compared [(3)H]N/OFQ binding in dog and rat brain membranes. Radioligand saturation/competition studies with these membranes and leucyl-[(3)H]N/OFQ(1-17)OH or the novel radioligand [(3)H]N/OFQ(1-13)NH(2) were performed to determine receptor density and ligand affinity. The density of classic opioid receptors was determined by using [(3)H]diprenorphine. Leucyl-[(3)H]N/OFQ(1-17)OH binding was concentration dependent and saturable in dog (maximum binding capacity [B(max)], 28.7 +/- 2.8 fmol/mg of protein; equilibrium dissociation constant as negative log [pK(d)], 10.27 +/- 0.11) and rat (B(max), 137.0 +/- 12.9 fmol/mg of protein; pK(d), 10.41 +/- 0.05). In comparison, the B(max) and pK(d) of [(3)H]diprenorphine were, respectively, 77.7 +/- 5.3 fmol/mg of protein and 9.74 +/- 0.09 in dog and 79.1 +/- 18.2 fmol/mg of protein and 9.51 +/- 0.04 in rat. In dog, [(3)H]N/OFQ(1-13)NH(2) binding to NOP receptors was also saturable (B(max), 23.7 +/- 2.0 fmol/mg of protein; pK(d), 10.16 +/- 0.12). In both species, leucyl-[(3)H]N/OFQ(1-17)OH was displaced by various NOP ligands. Dynorphin A, N/OFQ(1-5)NH(2), and nocistatin were essentially inactive. There was a significant positive correlation (r(2) = 0.95; P < 0.0001) between pK(i) values (an estimate of affinity) obtained in displacement studies in rat and dog. We have demonstrated a low density of NOP receptors, measured with two radioligands, in dog, and these receptors display a high degree of pharmacological similarity with those natively expressed in the rat.     

Acute endotoxemia in rats induces down-regulation of V2 vasopressin receptors and aquaporin-2 content in the kidney medulla

BACKGROUND: Endotoxemia can lead to fluid metabolism alterations despite unchanged or elevated plasma vasopressin (VP) levels, suggesting a refractoriness of the kidney to the effect of the peptide. To test this hypothesis, we examined the effect of lipopolysaccharide (LPS) injection on the expression of V2 receptors and aquaporin-2 in the kidney. METHODS: Plasma VP and urine osmolality, and binding of [3H]VP to kidney membranes, Western blot, and immunohistochemical analysis of aquaporin-2, in situ hybridization for V2 VP receptors and cytokines mRNAs were measured in the kidney 3 to 24 hours after LPS injection, 250 microg/100 g, intraperitoneally. RESULTS: LPS injection caused prolonged decreases in urine osmolality (up to 24 hours) without significant changes in plasma levels of sodium or VP. This was associated with marked decreases in V2 VP receptor mRNA and VP receptor number in the kidney, which were evident for up to 12 hours after LPS injection. Aquaporin-2 in kidney inner medulla was also reduced by about 50%. LPS induced interleukin (IL)-1beta in the kidney medulla by 3 hours, reached maximum at 6 hours, and started to decline by 12 hours, while it increased IL-6 mRNA significantly only at 3 hours. Interleukin mRNA expression was absent in kidneys of control rats. In vitro incubation of kidney medulla slices with IL-1beta reduced VP binding. CONCLUSION: The inflammatory response to acute endotoxemia down regulates V2 VP receptors and aquaporin-2 of the kidney inner medulla resulting in prolonged impairment of the renal capacity to concentrate urine.     

Polarized expression of the vasopressin V2 receptor in Madin-Darby canine kidney cells.

BACKGROUND: The vasopressin V2 receptor is expressed in the polarized principal cell of the renal collecting duct. Inactivating mutations of the vasopressin V2 receptor gene cause X-linked nephrogenic diabetes insipidus (NDI). Most of the mutant V2 receptors show transport defects, as analyzed in non-polarized cells, but data pertaining to polarized cells have not previously been presented. METHODS: Madin-Darby canine kidney cell (MDCK) II clones stably expressing c-myc-tagged human V2 receptors were characterized for [3H]-arginine vasopressin (AVP)-binding and AVP-sensitive adenylyl cyclase activity. The V2 receptors were immunocytochemically localized using the tyramide signal amplification technique in conjunction with an anti-c-myc antibody. RESULTS: The introduction of the c-myc epitope at the N- or C-terminus did not affect the functional properties of the V2 receptor expressed in MDCK II clones. However, the use of standard immunofluorescence methodology for these MDCK II clones yielded only weak signals. With the tyramide signal amplification technique, strong signals were obtained, showing the V2 receptor to be mainly localized within the lateral and, to a minor extent, apical membrane. In MDCK II clones stably expressing the c-myc-tagged V2 receptor NDI mutant L44P, fluorescent signals were found exclusively within the cell. CONCLUSION: The wild-type V2 receptor is expressed mainly in the lateral membrane, whereas the L44P mutant is completely retained within the cell. In conjunction with tyramide signal amplification, MDCK II cells constitute a suitable model for the analysis of transport-defective mutants of the V2 receptor.     

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.     

Historadioautographic localization of oxytocin and V1a vasopressin binding sites in the kidney of developing and adult rabbit,mouse and merione and of adult human

The localization of oxytocin (OT) binding sites and vasopressin (VP) binding sites of the V1a subtype was investigated by radioautography in kidneys of rabbits, mice and meriones during postnatal development and in the adult, and in the human kidney. Kidney sections were incubated in the presence of selective radioiodinated OT and V1a antagonists, respectively. The localizations were compared with those previously described in the rat. The main finding of the study was the almost constant presence in the cortex of V1a binding sites in the connecting tubule, the cortical collecting duct and in the juxtaglomerular apparatus (on the intra- and extraglomerular mesangium and the afferent arteriole). This distribution suggests an interaction of VP via V1a receptors and the kallikrein-kinin system in the kidney. OT binding sites, in comparison with V1a binding sites, were fewer and less constantly detectable in the kidney of the different species. In the mouse, their presence on the limbs of Henle's loop in the medulla points to the possibility of their involvement in the medullary concentrating process. In the kidneys of the various species, OT and V1a binding sites occurred always in differential structures. In contrast, in the human kidney cortex, a colocalization of OT and V1a binding sites was almost constantly observed. This raises the question as to the specificity of the neurohypophysial hormone receptors in the human kidney.     

Laudanosine does not displace receptor-specific ligands from the benzodiazepinergic or muscarinic receptors

The present study was designed to investigate whether D,L-laudanosine (a breakdown product of the neuromuscular relaxant atracurium besylate) interacts with benzodiazepinergic receptors or muscarinic receptors, both of which are involved in epilepsy and other types of seizures. The ability of D,L-laudanosine (10(-10) to 5 X 10(-5) M) to displace ligands specific for these receptors from their binding sites was tested. D,L-Laudanosine failed to inhibit the binding of [3H]flunitrazepam to central benzodiazepine receptors in the cerebral cortex, the binding of [3H]PK 11195 to peripheral benzodiazepine binding sites in the cerebral cortex and kidney, the binding of [3H]Ro 5-4864 to peripheral benzodiazepine binding sites in the kidney, or the binding of [3H]quinuclidinyl benzilate to muscarinic receptors in the cerebral cortex. These results suggest that laudanosine does not exert its convulsive effect via interaction with benzodiazepinergic or muscarinic receptors.     

Sodium excretion in response to vasopressin and selective vasopressin receptor antagonists

The mechanisms by which arginine vasopressin (AVP) exerts its antidiuretic and pressor effects, via activation of V2 and V1a receptors, respectively, are relatively well understood, but the possible associated effects on sodium handling are a matter of controversy. In this study, normal conscious Wistar rats were acutely administered various doses of AVP, dDAVP (V2 agonist), furosemide, or the following selective non-peptide receptor antagonists SR121463A (V2 antagonist) or SR49059 (V1a antagonist). Urine flow and sodium excretion rates in the next 6 h were compared with basal values obtained on the previous day, after vehicle treatment, using each rat as its own control. The rate of sodium excretion decreased with V2 agonism and increased with V2 antagonism in a dose-dependent manner. However,for comparable increases in urine flow rate, the V2 antagonist induced a natriuresis 7-fold smaller than did furosemide. Vasopressin reduced sodium excretion at 1 mug/kg but increased it at doses >5 umg/kg,an effect that was abolished by the V1a antagonist. Combined V2 and V1a effects of endogenous vasopressin can be predicted to vary largely according to the respective levels of vasopressin in plasma,renal medulla (acting on interstitial cells), and urine (acting on V1a luminal receptors). In the usual range of regulation, antidiuretic effects of vasopressin may be associated with variable sodium retention. Although V2 antagonists are predominantly aquaretic, their possible effects on sodium excretion should not be neglected. In view of their proposed use in several human disorders, the respective influence of selective (V2) or mixed (V1a/V2) receptor antagonists on sodium handling in humans needs reevaluation.     

Vasopressin antagonists in polycystic kidney disease

Increased cell proliferation and fluid secretion, probably driven by alterations in intracellular calcium homeostasis and cyclic adenosine 3,5-phosphate, play an important role in the development and progression of polycystic kidney disease. Hormone receptors that affect cyclic adenosine monophosphate and are preferentially expressed in affected tissues are logical treatment targets. There is a sound rationale for considering the arginine vasopressin V2 receptor as a target. The arginine vasopressin V2 receptor antagonists OPC-31260 and tolvaptan inhibit the development of polycystic kidney disease in cpk mice and in three animal orthologs to human autosomal recessive polycystic kidney disease (PCK rat), autosomal dominant polycystic kidney disease (Pkd2/WS25 mice), and nephronophthisis (pcy mouse). PCK rats that are homozygous for an arginine vasopressin mutation and lack circulating vasopressin are markedly protected. Administration of V2 receptor agonist 1-deamino-8-D-arginine vasopressin to these animals completely recovers the cystic phenotype. Administration of 1-deamino-8-D-arginine vasopressin to PCK rats with normal arginine vasopressin aggravates the disease. Suppression of arginine vasopressin release by high water intake is protective. V2 receptor antagonists may have additional beneficial effects on hypertension and chronic kidney disease progression. A number of clinical studies in polycystic kidney disease have been performed or are currently active. The results of phase 2 and phase 2-3 clinical trials suggest that tolvaptan is safe and well tolerated in autosomal dominant polycystic kidney disease. A phase 3, placebo-controlled, double-blind study in 18- to 50-yr-old patients with autosomal dominant polycystic kidney disease and preserved renal function but relatively rapid progression, as indicated by a total kidney volume >750 ml, has been initiated and will determine whether tolvaptan is effective in slowing down the progression of this disease.     

Alpha-2A is the predominant alpha-2 adrenergic receptor subtype in human spinal cord.

alpha-2 Adrenergic receptors can be subdivided into four subtypes based on their pharmacologic properties. The subtype of alpha-2 adrenergic receptor present in human spinal cord has not been reported previously. The affinities of nine alpha-2 subtype-selective drugs for the alpha-2 adrenergic receptor in human spinal cord homogenates were determined using [3H]rauwolscine and [3H]RX821002. These drug affinities (pKi values) were highly correlated with those obtained in a tissue or cell line containing only the alpha-2A adrenergic subtype (correlation coefficient of 0.99 and 0.98 for human platelet and HT29 cells, respectively). In contrast, the correlation of pKi values for the human spinal cord with tissues or cell lines containing other adrenergic receptor subtypes was poor. The correlation coefficients for alpha-2B (neonatal rat lung), alpha-2C (OK cell), and alpha-2D (bovine pineal gland) were 0.15, 0.68, and 0.81, respectively. These data suggest that the predominant alpha-2 adrenergic subtype present in human spinal cord is the alpha-2A subtype. Both [3H]rauwolscine and [3H]RX821002 appeared to label a single class of binding sites. The alpha-2 adrenergic receptor density was significantly greater in the sacral region of the cord as compared to either the lumbar or thoracic regions.     

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.     

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.     

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.     

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.     

Effects of nitrous oxide and halothane on mu and kappa opioid receptors in guinea-pig brain

The effects of two general anesthetics, nitrous oxide and halothane, and oxygen on mu and kappa opioid receptor subtypes from guinea-pig brain were investigated. mu receptor binding was defined using [3H]dihydromorphine as the ligand. Nitrous oxide (100%) and halothane (2%) decreased the [3H]dihydromorphine binding affinity (Kdair = 0.87 nM, KdN2O = 1.45 nM, Kdhalothane = 2.30 nM) without affecting the density of binding sites. A decrease in the [3H]dihydromorphine binding affinity without influence on the density of binding sites was also observed in the presence of 100% oxygen (KdO2 = 1.40 nM). kappa receptor binding was defined using [3H](-)ethylketocyclazocine as the ligand, in the presence of 100 nM D-ala2-D-leu5-enkephalin and 30 nM morphine. While 100% nitrous oxide caused a slight decrease in [3H](-)ethylketocyclazocine binding affinity (Kdair = 0.24 nM, KdN2O = 0.31 nM) and a substantial decrease in the density of binding sites (Bmaxair = 115 fmol/mg protein, BmaxN2O = 84 fmol/mg protein), halothane dramatically affected both the affinity (Kdhalothane = 0.70 nM) and density (Bmaxhalothane = 38 fmol/mg protein). Oxygen (100%) reduced [3H]dihydromorphine binding affinity. Differential effects of two anesthetics on the same receptor or distinct actions of the same anesthetic on different receptors could indicate the presence of specific targets for anesthetics at the membrane level. Conversely, effects of volatile anesthetics on opioid receptors could reflect a non-specific perturbation of the lipidic and proteinaceous components of the membranes.     

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.     

Characterization of muscarinic acetylcholine receptors in human penile corpus cavernosum: studies on whole tissue and cultured endothelium

The binding characteristics of the muscarinic acetylcholine receptor antagonist, [3H]quinuclidinyl benzilate, to isolated membranes of human corpus cavernosum and endothelial cells, cultured from this tissue, were investigated. [3H]quinuclidinyl benzilate bound to membranes of human corpus cavernosum and endothelial cells with high affinity and limited capacity. Analysis of the binding data by Scatchard plot revealed the presence of one class of binding sites. The ligand binding specificity was determined by competitive binding assay. The data obtained show that [3H]quinuclidinyl benzilate was displaced by unlabeled competitors in the following order of efficacy in both membrane preparations: quinuclidinyl benzilate greater than scopolamine greater than atropine greater than 4-diphenylacetoxy-N-methyl-piperidine methiodide, M3 antagonist greater than pirenzepine, M1 antagonist greater than oxotremorine greater than (4-hydroxy-2-butynyl)trimethylammonium chloride m-chlorocarbanilate, M1 agonist greater than carbachol greater than hexamethonium. Solubilization of the muscarinic acetylcholine receptors from human corpus cavernosum and endothelial cells, with 1% digitonin and 0.02% cholate and subsequent analysis on sucrose density gradients, demonstrated the presence of a macromolecule specifically bound to [3H]quinuclidinyl benzilate sedimenting at the 6.2 S region of the gradient. These results demonstrate the presence of muscarinic acetylcholine receptors in human corpus cavernosum and in endothelial cells cultured from this tissue.