Increase in [3H]PN 200-110 binding to cardiac muscle membrane in streptozocin-induced diabetic rats

Voltage-sensitive Ca2+ channels in cardiac left ventricular muscle membranes isolated from nondiabetic control and diabetic rats were measured with [3H]PN 200-110, a dihydropyridine derivative, as a ligand. The binding site (Bmax) of [3H]PN 200-110 in cardiac membranes isolated from streptozocin-induced diabetic (STZ-D) rats (128 +/- 10 fmol/mg protein) significantly (P less than 0.01) increased by 64% compared with that of control rats (78 +/- 4 fmol/mg protein) 10 wk after STZ administration without a significant change in Kd. However, the significant increase in Bmax of [3H]PN 200-110 binding in diabetic rats depended on the duration of diabetes such that the increase was not found until 6 wk after STZ injection. An 8-wk intensive insulin treatment, which was initiated 2 wk after STZ injection, normalized the increase in [3H]PN 200-110 binding in STZ-D rats to control levels (85 +/- 4 fmol/mg protein). Furthermore, [3H]PN 200-110 binding to control cardiac membranes was dose-dependently inhibited in the presence of verapamil, a phenylalkylamine Ca2+ antagonist, but that was not the case in cardiac membranes isolated from STZ-D rats. These results indicate that voltage-sensitive Ca2+ channels in cardiac muscle isolated from STZ-D rats are quantitatively and qualitatively altered, because the course of diabetes and the increase in the channels can be prevented by treatment with insulin.     

Changes in the biochemical profiles of mid-cervically located adenosine A1 receptors after repeated theophylline administration in adult rats

BACKGROUND/OBJECTIVE: Adenosine A1 receptors localized in the phrenic motoneurons (PMNs), where the axons of the descending bulbospinal respiratory make synaptic contacts, may be involved in theophylline-induced respiratory-related activity in rats. The objective of this study was to characterize the biochemical profiles of adenosine A1 receptors in 2 groups of rats: (a) na?ve and (b) theophylline-treated (3-day oral administration). METHODS: Biochemical binding characteristics of adenosine A1 receptors in the C3 to C5 (PMN) of adult rats were assessed in na?ve (n = 6) and theophylline-treated animals (n = 6) using [3H]-DPCPX (10 pmol/L to 30 nmol/L), the specific adenosine A1 receptor antagonist in saturation-binding assays. Competition assays used theophylline as the competing ligand (20 mmol/L to 20 pmol/L), and protein concentration was determined with the Bradford assay using a range of standards (0.016-1.0 mg/mL). RESULTS: In saturation-binding assays in na?ve animals, the A1 receptor was characterized by a single binding site with Bmax and Kd values of 256.00 +/- 32.13 fmol/mg protein and 2.89 +/- 0.45 nmol/L, respectively. Analysis of the isotherm in theophylline-treated animals showed 1 site with Bmax and Kd values of 219.00 +/- 26.3 fmol/mg protein and 0.60 +/- 0.21 nmol/L, respectively, and a second site characterized by Bmax and Kd values of 492.6 +/- 3.15 fmol/mg protein and 14.09 +/- 2.06 nmol/L, respectively. CONCLUSIONS: Theophylline administration revealed 2 binding sites on receptors (characterized by the specific adenosine A1 antagonist, [3H]-DPCPX) located in the vicinity of phrenic motoneurons (C3-C5). Alteration of the receptor profiles after theophylline may underlie the respiratory-related actions of the drug.     

D1-like dopamine receptor activation and natriuresis by nitrocatechol COMT inhibitors

BACKGROUND: In recent years, several nitrocatechol derivatives (tolcapone, entacapone, and nitecapone) have been developed and found to be highly selective and potent inhibitors of catechol-O-methyltransferase (COMT). More recently, natriuretic properties were described for two of these compounds (entacapone and nitecapone), although this was not accompanied by enhanced urinary excretion of dopamine. We hypothesized that nitrocatechol derivatives stimulate D1-like dopamine receptors. METHODS: Adult male Wistar rats were treated with a nitrocatechol COMT inhibitor (entacapone, tolcapone, or nitecapone, 30 mg/kg, orally), and the urinary excretion of dopamine and sodium was quantitated. The interaction of nitrocatechol derivatives with D1-like receptors was evaluated by their ability to displace [3H]-Sch23390 binding from membranes of rat renal cortex and cAMP production in opossum kidney (OK) cells. RESULTS: Urinary excretion of sodium (micromol/h) was markedly increased by all three nitrocatechol derivatives: vehicle, 55.0 +/- 5.6; entacapone, 98.4 +/- 9.3; tolcapone, 97.5 +/- 9.3; and nitecapone, 120.5 +/- 12.6. Pretreatment with the selective D1 antagonist Sch 23390 (60 microg/kg) completely prevented their natriuretic effects. Nitecapone and tolcapone were equipotent (IC50s of 48 and 42 micromol/L) and more potent than entacapone and dopamine (IC50s of 107 and 279 micromol/L) in displacing [3H]-Sch23390 binding. In OK cells, all three nitrocatechol derivatives significantly increased cAMP accumulation and reduced Na(+)/H(+) exchange and Na(+),K(+)-ATPase activities, this being prevented by a blockade of D1-like receptors. CONCLUSION: Stimulation of D1-like dopamine receptors and inhibition of Na(+)/H(+) exchange and Na(+),K(+)-ATPase activities by nitrocatechol COMT inhibitors may contribute to natriuresis produced by these compounds.     

Dopaminergic mechanisms controlling urethral function in rats

AIMS: To investigate the role of dopamine receptor subtypes in the control of urethral activity. METHODS: Simultaneous recordings of intravesical and urethral perfusion pressure (UPP) were performed in rats under urethane anesthesia. Changes in coordinated activity of the bladder and urethral sphincter were examined following intravenous (i.v.), intrathecal (i.t.), or intracerebroventricular (i.c.v.) administration of dopamine D1- and D2-like receptor agonists (SKF38393 and quinpirole, respectively) and antagonists (SCH23390 and remoxipride, respectively). RESULTS: Quinpirole (0.03, 0.1, and 0.3 mg/kg i.v.) dose-dependently decreased baseline urethral pressure to 45.33 +/- 5.8, 33.7 +/- 3.3 (P < 0.05, n = 6), and 27.7 +/- 3.3 cm H(2)O (P < 0.05, n = 5) from the control value (46.0 +/- 4.0 cm H(2)O), respectively. i.c.v. injection of quinpirole (1 microg) decreased baseline urethral pressure to 33.6 +/- 5.0 cm H(2)O (P < 0.05, n = 4) from the control value (51.4 +/- 4.9 cm H(2)O) in contrast to the insignificant effects of i.t. administration of the drug (3 microg). The decrement of baseline pressure induced by quinpirole (0.1 mg/kg i.v.) was suppressed by alpha-bungarotoxin (BGT), a neuromuscular blocking agent. SCH23390 (1 and 3 mg/kg, i.v.) dose-dependently decreased the frequency of high frequency oscillation (HFO) of the urethral sphincter. SKF38393 or remoxipride did not have significant effects on any parameters of bladder and urethral activity. CONCLUSIONS: These results indicate that activation of D2-like dopamine receptors at a supraspinal site can suppress activity of the striated muscle urethral sphincter. Thus, decreased urethral resistance induced by D2 dopamine receptor activation might aggravate urge incontinence symptoms often seen in patients with Parkinson's disease (PD).     

Effect of insulin and dietary myoinositol on muscarinic receptor alterations in diabetic rat bladder.

Previous studies from our laboratory demonstrated that there is an up-regulation of muscarinic receptor density in the bladder dome of the 8-wks diabetic rat compared to control. To determine whether the changes observed in receptor density can be corrected by insulin or dietary myoinositol, five groups of rats were maintained for eight weeks: control (C), diabetic (D), diabetic insulin-treated (DI), diabetic myoinositol-treated (DMI), and control myoinositol-treated (CMI). Diabetes was induced by i.v. injection of 65 mg./kg. of streptozotocin. D and DMI animals were smaller, had higher serum glucose and lower serum insulin levels, higher water intakes and urine outputs, and larger bladder domes than the other groups. The density of the muscarinic receptors measured by radioligand receptor binding assays using [3H]quinuclidinyl benzilate were (in fmol./mg. protein): C, 88 +/- 13; D, 176 +/- 19; DI, 94 +/- 5; DMI, 158 +/- 8; CMI, 112 +/- 10. These data indicate that insulin, but not myoinositol treatment normalized diabetes induced abnormalities observed in the general features of streptozotocin-injected rats and prevented the diabetic-induced upregulation of bladder dome muscarinic receptors.     

Effects of insulin treatment on diabetes-induced alterations in endothelin receptors in rat ureter

BACKGROUND: The present investigation was undertaken to examine the effect of insulin treatment on diabetes-induced alterations in endothelin (ET) receptors in rat ureters. METHODS: The biochemical properties of ET receptors were examined in rat ureters from the following groups: 8 weeks diabetic (D8); 8 weeks age-matched control (C8); 16 weeks diabetic (D16); 16 weeks diabetic-insulin treated (insulin started 8 weeks after the onset of diabetes) (DI16); and 16 weeks age-matched control (C16). Diabetes was induced by the i.v. injection of 65 mg/kg streptozotocin (STZ). RESULTS: The densities of ET receptors (Bmax values), as determined by saturation experiments with [125I]-ET-1, in the ureteral plasma membranes of D8, C8, D16, DI16 and C16 were 91.7 +/- 10.1, 42.1 +/- 7.2, 71.1 +/- 2.4, 51.5 +/- 6.3 and 45.1 +/- 3.3 fmol/mg of protein, respectively. [125I]-ET-1 binding to the ET receptors in rat ureteral membrane particulates was inhibited by ET-1 (non-selective), ET-3 (ET(B/C selective), BQ610 (ET(A) selective) and IRL 1620 (ET(B) selective) with the following rank order of Ki values: ET-1 < BQ 610 < ET-3 < IRL 1620. The pharmacological profile of the ET receptors was similar in all groups examined and was consistent with the predominance of the ET(A) receptor subtype in the ureteral membrane particulates. The subtype specificity of ET receptors in the ureteral tissues is confirmed with inhibition data obtained from similar binding studies in cloned human ET(A) and ET(B) receptors. CONCLUSION: The data indicate that diabetes results in an up-regulation of ET receptors in the rat ureter, which is normalized by insulin treatment.     

Presence of dopamine D1 receptors and absence of dopamine D2 receptors in human cerebral meningioma tissue.

Preliminary studies have shown that the dopamine D1 receptor is expressed in cerebral meningioma tissue. The current study presents evidence that the iodinated dopamine D1 antagonist [125I]SCH-23982 bound to dopamine binding sites in 33 of the 45 human cerebral meningiomas examined for this. Saturation curves and the linearity of the Scatchard analysis indicate that [125]SCH-23982 binds to a homogeneous population of binding sites. Competition curves reveal the presence of a dopamine D1 receptor by rank order of various dopaminergic and nondopaminergic antagonists ((+)-SCH-23390 greater than (+/-)-SKF-83566 greater than (cis)-flupentixol greater than (+)-butaclamol greater than chlorpromazine greater than 1-sulpiride greater than mianserin greater than (-)-butaclamol). Stereoselectivity was evaluated by (+)- and (-)-butaclamol. The mean (+/- standard deviation) dissociation rate constant was 369 +/- 196 pM with a density of 31.9 +/- 12.5 fmol/mg membrane protein among 33 meningiomas. The dopamine D2 receptor was not present in the 30 meningiomas examined for this. These findings indicate that the dopamine D1 receptor identified is expressed alone and is therefore regulated independent of a D2 receptor in cerebral meningioma tissue. Although the function of the dopamine D1 receptor in cerebral meningiomas has not so far been defined, previous studies have suggested that the D1 receptor might be involved in the control of proliferative growth of meningiomatous tissue.     

Antinociceptive Effect of Morphine, but not μ Opioid Receptor Number, Is Attenuated in the Spinal Cord of Diabetic Rats

Background: The mechanisms of decreased analgesic potency of [mu] opioids in diabetic neuropathic pain are not fully known. The authors recently found that G protein activation stimulated by the [mu] opioid agonist is significantly reduced in the spinal cord dorsal horn in diabetes. In the current study, they determined potential changes in the number and binding affinity of [mu] opioid receptors in the spinal cord in diabetic rats.

Methods: Rats were rendered diabetic with an intraperitoneal injection of streptozotocin. The nociceptive withdrawal threshold was measured before and after intrathecal injection of morphine by applying a noxious pressure stimulus to the hind paw. The [mu] opioid receptor was determined with immunocytochemistry labeling and a specific [mu] opioid receptor radioligand, [3H]-(d-Ala2,N-Me-Phe4,Gly-ol5)-enkephalin ([3H]-DAMGO), in the dorsal spinal cord obtained from age-matched normal and diabetic rats 4 weeks after streptozotocin treatment.

Results: The antinociceptive effect of intrathecal morphine (2-10 [mu]g) was significantly reduced in diabetic rats, with an ED50 about twofold higher than that in normal rats. However, both the dissociation constant (3.99 +/- 0.22 vs. 4.01 +/- 0.23 nm) and the maximal specific binding (352.78 +/- 37.26 vs. 346.88 +/- 35.23 fmol/mg protein) of [3H]-DAMGO spinal membrane bindings were not significantly different between normal and diabetic rats. The [mu] opioid receptor immunoreactivity in the spinal cord dorsal horn also was similar in normal and diabetic rats.     

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.     

3H]bunazosin, a novel selective radioligand of alpha 1 adrenoceptors in human prostates.

The binding properties of a new radioligand, [3H]bunazosin, were studied in membranes of human prostates with benign prostatic hypertrophy (BPH). Specific binding of [3H]bunazosin was saturable, reversible, and of high affinity (Kd = 0.55 +/- 0.04 nM). The density of [3H]bunazosin binding sites (Bmax) was 676 +/- 33 fmol/mg. protein. [3H]Bunazosin rapidly associated with its binding sites in membranes of human prostates and reached steady state by 20 min. at 25C. The rate constants for association and dissociation of [3H]bunazosin binding were calculated to be 0.11 +/- 0.01/nM/min. and 0.05 +/- 0.02/min. (n = 4), respectively. Seven alpha 1 adrenoceptor antagonists competed with [3H]bunazosin for the binding sites in the rank order: R-(-)-YM-12617 greater than prazosin greater than SGB-1534 greater than bunazosin greater than terazosin greater than naftopidil greater than urapidil. In parallel studies with [3H]bunazosin, the Kd and Bmax values for [3H]prazosin binding in human prostates were slightly lower. There was a similarity in the potency and rank order of seven alpha 1, adrenoceptor antagonists for the inhibition of [3H] bunazosin and [3H]prazosin binding in human prostates. The new [3H]bunazosin binding assay in human prostates is remarkable for its low degree of nonspecific binding as compared to [3H]prazosin, especially at high ligand concentrations. Thus, [3H]bunazosin may become a useful radioligand for the further analysis of the alph 1 adrenoceptor binding sites in human prostates.     

Reduced glomerular thromboxane receptor sites and vasoconstrictor responses in diabetic rats.

Glomerular thromboxane production and urinary thromboxane excretion are increased in early diabetes, but in spite of this renal blood flow and glomerular filtration rate are significantly higher than in control animals. To study the possibility of a defect in thromboxane actions in the kidney, we have measured glomerular thromboxane receptors and the renal hemodynamic response to the administration of a stable thromboxane analog in diabetic rats. Glomerular thromboxane receptors were studied in hyperglycemic diabetic rats 7 to 10 days after injection of streptozotocin (65 mg/kg, i.v.) and in normal controls. Scatchard analysis of equilibrium binding using the thromboxane antagonist, [3H]-SQ29548, demonstrated one class of high affinity thromboxane receptor sites in control (Kd = 19.9 +/- 2.6 nM, N = 16) and diabetic rats (Kd = 19.8 +/- 2.1 nM, N = 8, P = NS). The number of thromboxane receptors was reduced by 44% in diabetic rats (control, 374 +/- 20 vs. diabetic, 210 +/- 21 fmol/mg, P less than 0.01). Thromboxane binding in diabetic rats was not restored to normal levels by thromboxane synthetase inhibition with OKY046. Diabetic rats had higher renal blood flow (diabetic, 7.03 +/- 0.18 vs. control, 6.33 +/- 0.13 ml/min, P less than 0.05) and glomerular filtration rate (2.42 +/- 0.10 vs. 1.96 +/- 0.07 ml/min, P less than 0.05). Infusion of the stable thromboxane agonist, U46619 (0.1 micrograms/kg/min), reduced renal blood flow and glomerular filtration rate in all animals, but the constrictor responses were blunted by 50% in hyperglycemic diabetic rats compared with normal controls or euglycemic diabetic rats (P less than 0.05). Control of blood glucose with insulin normalized the number of glomerular thromboxane receptor sites, reversed hyperfiltration and restored glomerular responses to thromboxane agonist. The abnormalities of glomerular thromboxane receptors are similar to changes in angiotensin II receptors, and suggest a generalized defect in vasoconstrictor receptors in the diabetic kidney.     

Role of dopamine D1 and D2 receptors in the micturition reflex in conscious rats

To clarify the role of dopamine D1 and D2 receptors in the volume-induced micturition reflex, conscious, female rats were investigated cystometrically before and after intravenous administration of SKF 38393 (a selective D1 receptor agonist), SCH 23390 (a selective D1 receptor antagonist), quinpirole (a selective D2 receptor agonist), and remoxipride (a selective D2 receptor antagonist). The effect of quinpirole was also investigated in the presence of remoxipride. Intravenous administration of SKF 38393 (0.01-3.0 mg/kg) did not affect any cystometric parameters investigated. On the other hand, SCH 23390 (0.1-1.0 mg/kg i.v.) reduced bladder capacity and micturition volumes and increased the micturition pressure in a dose-dependent manner. Quinpirole (0.01-0.1 mg/kg) given intravenously, dose-dependently decreased bladder capacity and micturition volumes. Pre-treatment with remoxipride (1.0 mg/kg i.v.) significantly attenuated the effect of quinpirole (0.1 mg/kg i.v.). Remoxipride (0.1-1.0 mg i.v.) itself did not cause any significant changes in the cystometric parameters. These results suggest that in conscious rats, D1 receptors tonically inhibit the micturition reflex and that D2 receptors are involved in facilitation of the micturition reflex. It may be speculated that detrusor hyperreflexia associated with Parkinson's disease results from activation failure of D1 receptors and that administration of D2 receptor agonists might worsen the condition.     

Mononuclear leukocyte beta 2-adrenergic receptors and adenylate cyclase sensitivity in insulin-dependent diabetes mellitus

Patients with insulin-dependent diabetes mellitus (IDDM) have been found to have a heightened hyperglycemic response to epinephrine. To determine if patients with IDDM have increased sensitivity of cellular beta 2-adrenergic receptor-effector systems, we assessed beta 2-adrenergic receptors and adenylate cyclase sensitivities to isoproterenol in partially purified mononuclear leukocyte (MNL) plasma membranes from 10 patients with IDDM (without adrenergic neuropathy) and 10 matched nondiabetic controls. MNL beta 2-adrenergic receptor densities (Bmax = 48 +/- 8 fmol [3H] DHA/mg protein in IDDM, 44 +/- 3 fmol [3H] DHA/mg protein in controls) and binding affinities (apparent KD = 0.3 +/- 0.07 nM in IDDM, 0.3 +/- 0.04 nM in controls) did not differ. Further, MNL adenylate cyclase activities were not significantly different either at baseline (325 +/- 86 pmol/mg protein/15 min in IDDM, 275 +/- 49 pmol/mg protein/15 min in controls) or in response to isoproterenol (842 +/- 229 pmol/mg protein/15 min in IDDM, 608 +/- 86 pmol/mg protein/15 min in controls). Thus, the data do not support the presence of a generalized alteration of beta-adrenergic receptors or adenylate cyclase sensitivity in IDDM. To the extent that MNL beta 2-adrenergic receptors and adenylate cyclase activities reflect those of extravascular catecholamine target cells, these findings suggest that the heightened hyperglycemic response to epinephrine exhibited by patients with IDDM is not due to increased sensitivity of cellular beta 2-adrenergic receptor-effector systems and is best attributed to the altered hormonal milieu of the insulin-deficient state.     

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.     

Antinociceptive effect of morphine, but not mu opioid receptor number, is attenuated in the spinal cord of diabetic rats

BACKGROUND: The mechanisms of decreased analgesic potency of mu opioids in diabetic neuropathic pain are not fully known. The authors recently found that G protein activation stimulated by the mu opioid agonist is significantly reduced in the spinal cord dorsal horn in diabetes. In the current study, they determined potential changes in the number and binding affinity of mu opioid receptors in the spinal cord in diabetic rats. METHODS: Rats were rendered diabetic with an intraperitoneal injection of streptozotocin. The nociceptive withdrawal threshold was measured before and after intrathecal injection of morphine by applying a noxious pressure stimulus to the hind paw. The mu opioid receptor was determined with immunocytochemistry labeling and a specific mu opioid receptor radioligand, [3H]-(D-Ala2,N-Me-Phe4,Gly-ol5)-enkephalin ([3H]-DAMGO), in the dorsal spinal cord obtained from age-matched normal and diabetic rats 4 weeks after streptozotocin treatment. RESULTS: The antinociceptive effect of intrathecal morphine (2-10 microg) was significantly reduced in diabetic rats, with an ED50 about twofold higher than that in normal rats. However, both the dissociation constant (3.99 +/- 0.22 vs. 4.01 +/- 0.23 nm) and the maximal specific binding (352.78 +/- 37.26 vs. 346.88 +/- 35.23 fmol/mg protein) of [3H]-DAMGO spinal membrane bindings were not significantly different between normal and diabetic rats. The mu opioid receptor immunoreactivity in the spinal cord dorsal horn also was similar in normal and diabetic rats. CONCLUSIONS: The reduced analgesic effect of intrathecal morphine in diabetes is probably due to impairment of mu opioid receptor-G protein coupling rather than reduction in mu opioid receptor number in the spinal cord dorsal horn.     

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.     

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.     

Androgen receptor binding activity in meningiomas

Analyses of androgen receptor binding activity in 54 intracranial, intraspinal, and metastatic meningiomas were performed with a specific radioligand binding technique using [3H]R 1881 as radioligand. [3H]R 5020 was used for the concurrent determination of progesterone receptor binding activity. Moderate concentrations of androgen receptors (33.4 +/- 5.4 fmol/mg protein) were detected in 35 (65%), whereas high levels of progesterone binding components (236 +/- 35 fmol/mg protein) were demonstrated in 48 (89%) tumors. The androgen receptor binding activity was positively correlated with the progesterone receptor binding activity (rs = 0.38, p less than 0.05). This relationship is suggestive of an androgen regulation of the progesterone receptor via the androgen receptor system. The presence of androgen and progesterone receptors in a large proportion of meningiomas, and the tendency for a dependence of androgen receptor and progesterone receptor binding activity on the histological subtype could have implications for tumor therapy.     

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.     

Differential effects of ketamine and pentobarbitone on acetylcholine release from the rat hippocampus and striatum

Using microdialysis, we examined the effects of ketamine and pentobarbitone on acetylcholine (ACh) release from the rat hippocampus and striatum. Ketamine 25 and 50 mg kg-1 increased ACh release from the hippocampus to 295% and 353% of basal release, respectively, but not from the striatum. SCH 23390 1 microgramsmol litre-1, a D1 antagonist, significantly inhibited the facilitatory effect of ketamine 50 mg kg-1 on hippocampal ACh release (to 241% of basal level). In contrast, pentobarbitone 20 and 40 microgramsmg kg-1 decreased basal ACh release from both the hippocampus by 41% and 69%, respectively, and the striatum by 37% and 58%, respectively. The results suggest that ketamine and pentobarbitone exert opposite effects on ACh release from the rat hippocampus and that the stimulating effect of ketamine may involve dopamine D1 receptors.