Autoradiographic Localization of Dopamine D2-Like Receptors in the Rat Adrenal Gland

The pharmacological profile and the anatomical localization of dopamine D₂-like receptors were studied in sections of the rat adrenal gland using combined radioligand binding and autoradiographic techniques with [³H]-spiroperidol as a ligand. [³H]-Spiroperidol was bound to sections of the rat adrenal gland in a manner consistent with the labelling of dopamine D₂-like receptor sites. The binding was time-, temperature-and concentration-dependent and of high affinity with a dissociation constant (K_s) value of 1.6 ± 0.04 nM and a maximum density of binding sites (B_max) of 60 ± 3.6 fmol/mg tissue. Experiments on the pharmacological specificity of [³H]-spiroperidol binding to sections of the rat adrenal gland suggest the labelling of dopamine D₃ and/or D₄ receptors. The presence of dopamine D₃ and D₄ receptors in the rat adrenal gland was confirmed by the demonstration of a specific binding for the D₃ radioligand [³H]-7-hydroxy-N,N-di-n-propyl-2-aminotetralin (DPAT) and for the D₄ radioligand [³H]-clozapine. Light microscope autoradiography showed the highest accumulation of silver grains which correspond to [3H]-spiroperidol binding sites in the rat adrenal medulla. In the adrenal cortex, where density of silver grains is about 40% lower than in the medulla, the radioligand is accumulated primarily in the zona glomerulosa and to a lesser extent in the zona reticularis. These findings suggest that dopamine D2-like receptor sites in the rat adrenal gland cortex are primarily involved in the modulation of catecholamine secretion from the medulla and of aldosterone secretion from the cortex. The possible relevance of the occurrence of dopamine D3 and D4 receptor subtypes in the adrenal gland is discussed.     

Expression of Dopamine Receptors in Immune Organs and Circulating Immune Cells

The existence of dopamine (DA) D₁- and D₂-like receptors in the rat and pigeon thymus and in human peripheral blood lymphocytes was investigated. The selective D₁-like antagonist [³H]-SCH 23390 was used as a ligand of DA D₁-like receptors (D₁ and D₅ sites). Pharmacological analysis suggests that binding of [³H]-SCH 23390 to sections of thymus and to human peripheral blood lymphocytes belongs mainly to the dopamine D₅ receptor subtype. Light microscope autoradiography, performed in sections of rat and pigeon thymus, revealed that these receptors are located primarily in the cortical layer. DA D₂-like receptors (D₂, D₃ and D₄ sites) were studied in sections of rat thymus and in peripheral blood lymphocytes by using the putative DA D₃ receptor agonist [³H]-7-OH-DPAT as a ligand. Both rat and pigeon thymus and human peripheral blood lymphocytes express a putative DA D₃ receptor. These data are in agreement with recent molecular biology studies performed in human peripheral blood 1ymphocytes. The demonstration of different subtypes of DA receptors in a primary immune organ such as the thymus and in circulating immune cells supports the hypothesis of an involvement of DA in the control of immune function.     

Characterization of the μ and δ Opioid Receptors in the Brain of the C57BL/6 and DBA/2 Mice, Selected for Their Differences in Voluntary Ethanol Consumption

Genetically determined differences in the activity of the hypothalamic β-endorphin system have been demonstrated between the C57BL/6 (alcohol-preferring) and DBA/2 (alcohol-aversive) inbred strains of mice. The present studies examined the distribution and density of the μ and δ receptors in specific brain regions that may mediate the rewarding and reinforcing effects of ethanol, using quantitative auto-radiography and the specific μ agonist FK 33–824 and 6 agonist DPDPE, in their iodinated form. ¹²⁵I-FK 33–824 recognizes a high affinity binding site in brain membrane preparations from both the C57BL/6 (K_d= 1.37 ± 0.22 nM; B_max= 80 ± 12.3 fmol/mg protein) and DBA/2 mice (K_d= 1.02 ± 0.16 nM; B_max= 39.5 ± 9.6 fmol/mg protein), whereas ¹²⁵I-DPDPE binds to a high affinity binding site in brain membranes from both the C57BL/6 (K_d= 1.08 ± 0.34 nM; B_max= 24.4 ± 4.5 fmol/mg protein) and DBA/2 mice (K_d= 0.68 ± 0.24 nM; B_max= 15.3 ± 3.7 fmol/mg protein). The auto-radiographic studies demonstrated differences in the density of the μ opioid receptors between the two strains of mice in brain nuclei that are not directly related to the brain reward system. However, strain-related differences in the density of δ opioid receptors were observed in regions of the limbic system known to mediate the positive reinforcing effects of many drugs of abuse. The density of δ receptors was significantly higher in the ventral tegmental area and nucleus accumbens of the C57BL/6 mice. The results of the present study support the hypothesis that genetically determined differences exist in the density of opioid receptors in distinct regions of the brain between the C57BL/6 and DBA/2 inbred strains of mice, which may play a role in controlling their voluntary ethanol consumption.     

Longitudinal Observations of Monoamine Oxidase B in Alcoholics: Differentiation of Marker Characteristics

The marker characteristics of monoamine oxidase B (MAO B) in human platelets were investigated in a clinical study of 59 alcoholics (diagnosed according to the criteria of ICD-10) observed over a period of 6 months. Demographic and family history were obtained by a structured interview, including the substance abuse section of CIDI (Composite International Diagnostic Interview). The patient's personality was assessed by Cloninger's Tridimensional Personality Questionnaire (TPQ). Blood samples were first drawn during chronic intoxication (day of admission to the hospital for detoxication), after short-term abstinence (8 days later), medium-term (3 months later), and long-term abstinence (6 months later). A group of 22 matched healthy nonalcoholics served as controls studied under sober conditions and during acute intoxication (4 hr after ingestion of 1 g ethanol/kg body weight). All platelet samples were investigated with 6 kynuramine concentrations as substrate (fluorometric assay) in the absence and presence of 200 itim ethanol (ETOH) in vitro. MAO B activity was significantly reduced in alcoholics during chronic intoxication (V_max: 2.70 ± 0.15 nmol/min/mg protein) compared with sober (V_max: 3.25 ± 0.23 nmol/min/mg protein) and acutely intoxicated controls that turned to normal during abstinence. However, MAO B activity obtained during medium- and long-term abstinence was significantly lowered in patients with high novelty-seeking and impulsiveness scores in the TPQ, a history of suicide attempts, or an alcoholic mother. The affinity of MAO B (K_m values) was unchanged in alcoholics at any time investigated. Addition of ETOH in vitro reduced the affinity. This effect was less pronounced when the blood had been obtained during chronic intoxication and after short-term abstinence, suggesting tolerance toward ETOH. It is demonstrated that reduced MAO B values may serve as state and trait markers of alcoholism and that they can be disentangled in a longitudinally designed study.     

Renal Dopamine and Changes in Dopamine Receptor Ligand Binding During High Sodium Intake

This study was designed to measure changes in plasma levels of atrial natriuretic peptides (ANP), urine volume (UV), urinary excretion of dopamine (U_DAV) and sodium (U_NaV), and dopamine (DA) receptor affinity (K_d) and binding sites (B_max) in kidneys of rats drinking normal saline for a period of 7 days. The saline intake significantly increased UV, U_naV, plasma ANP, U_naV, and its primary metabolite dihydroxyphenyl acetic acid (DOPAC) for the period of 7 days. B_max increased significantly 1 day after the initiation of saline intake, however, the increase appeared to be transient since measurements of B_max made after 7 days of saline intake were not significantly different from the control group. No changes in K_d were observed. These results indicate that renal DA contributes to maintenance of Na balance during increased Na intake and that renal DA receptors undergo transient changes during this period.     

A Single, Moderate Ethanol Exposure Alters Extracellular Dopamine Levels and Dopamine D2 Receptor Function in the Nucleus Accumbens of Wistar Rats

Background: The nucleus accumbens (NAc) has been implicated in the neurochemical effects of ethanol (EtOH). Evidence suggests that repeated EtOH exposures and chronic EtOH drinking increase dopamine (DA) neurotransmission in the NAc due, in part, to a reduction in D₂ autoreceptor function. The objectives of the current study were to evaluate the effects of a single EtOH pretreatment and repeated EtOH pretreatments on DA neurotransmission and D₂ autoreceptor function in the NAc of Wistar rats. Methods: Experiment 1 examined D₂ receptor function after a single intraperitoneal (i.p.) injection or repeated i.p. injections of 0.0, 0.5, 1.0, or 2.0 g/kg EtOH to female Wistar rats. Single EtOH pretreatment groups received 1 daily i.p. injection of 0.9% NaCl (saline) for 4 days, followed by 1 day of saline or EtOH administration; repeated EtOH pretreatment groups received 5 days of saline or EtOH injections. Reverse microdialysis experiments were conducted to determine the effects of local perfusion with the D₂‐like receptor antagonist (‐)sulpiride (SUL; 100 uM), on extracellular DA levels in the NAc. Experiment 2 evaluated if pretreatment with a single, moderate (1.0 g/kg) dose of EtOH would alter levels and clearance of extracellular DA in the NAc, as measured by no‐net‐flux (NNF) microdialysis. Subjects were divided into the EtOH‐naïve and the single EtOH pretreated groups from Experiment 1. Results: Experiment 1: Changes in extracellular DA levels induced with SUL perfusion were altered by the EtOH dose (p < 0.001), but not the number of EtOH pretreatments (p > 0.05). Post‐hoc analyses indicated that groups pretreated with single or repeated 1.0 g/kg EtOH showed significantly attenuated DA response to SUL, compared with all other groups (p < 0.001). Experiment 2: Multiple linear regression analyses yielded significantly (p < 0.05) higher extracellular DA concentrations in the NAc of rats receiving EtOH pretreatment, compared with their EtOH‐naïve counterparts (3.96 ± 0.42 nM and 3.25 ± 0.23 nM, respectively). Extraction fractions were not significantly different between the 2 groups. Conclusions: The present results indicate that a single EtOH pretreatment at a moderate dose can increase DA neurotransmission in the NAc due, in part, to reduced D₂ autoreceptor function.     

Inhibitory Effect of Dopamine on Gastric Motility in Rats

Background: There is disagreement with regard to the involvement of dopamine (DA) receptors in gastric motility. The mechanism of the inhibitory effect of DA on rat gastric motility was investigated in association with acetylcholine (Ach) release in the present study.

Methods: In vivo vagotomized, splanchnicectomized rats and control rats were used, and gastric movement was determined as the gastric motility index after DA administration. In vitro study of Ach release from the circular muscle strips of the gastric corpus was investigated after administration of domperidone, SCH23390, phentolamine, or propranolol.

Results: In the in vivo study DA inhibited gastric motility in a dose-dependent manner. Vagotomy and splanchnicectomy had no effect on the inhibitory effect of DA. In the in vitro study DA inhibited [³H]-Ach release in a dose-dependent manner. The inhibitory effect of DA was antagonized by domperidone but not by phentolamine, propranolol, or SCH23390.

Conclusions: Inhibition of gastric motility by dopamine is independent of extrinsic innervation and seems to be mediated by DA₂ receptors in the gastric wall.     

Endothelin-1 receptors in the human myometrium: evidence for different binding properties in post-menopausal as compared to premenopausal and pregnant women

OBJECTIVE We investigated the binding properties of the endothelin receptors in the human myometrium in clinical situations associated with different ovarian steroid levels. SUBJECTS AND METHODS Binding properties of the endothelin receptors were studied in myometrial membranes from post-menopausal women (n= 12), myomatous premenopausal women (n= 14) and pregnant women (n= 14), using ¹²⁵I-labelled endothelin-1. RESULTS The mean (SD) maximal receptor density (B_max) was significantly higher in samples from premenopausal and pregnant women than from post-menopausal women (983 ± 196, 1116 ± 201 and 490 ± 145 pmol/g protein, respectively). Receptor affinity (K_d) did not differ significantly between these groups. Among the pregnant women, mean B_max and K_d values were similar in those who electively underwent Caesarean section prior to the onset of labour and those operated on during the second stage of spontaneous labour. Binding properties of myometrial membranes of either pre or post-menopausal women were unaffected by the presence of high levels of beta-oestradiol or progesterone in the medium. Among samples of premenopausal women, no significant difference was found in binding properties between those operated on either during mid-follicular phase or during mid-luteal phase. CONCLUSIONS In clinical situations associated with relatively high levels of ovarian steroids, the density of endothelin receptors in the myometrium is higher than in situations associated with low ovarian steroid level. Ovarian steroids may exert their influence via the production of other mediators. Changes in density of the endothelin receptor, induced by change in ovarian steroids activity, might play a role in the regulation of myometrial contractility.     

Utilization of hospital resources by alcoholic and nonalcoholic patients

Objective:To measure any difference in the utilization of hospital resources between alcoholic patients and nonalcoholic patients (controls) in a department of internal medicine. Design:Prospective comparative study. Alcoholics were identified as patients with Michigan Alcoholism Screening Test (MAST) scores of ≥8. Controls were defined as patients with MAST scores of ≤4, and matched with alcoholics for sex, age, and time of admission. The length of stay, as well as several indicators of utilization of diagnostic and therapeutic procedures, was used for the comparison of resource utilization. Setting:General wards of internal medicine of a 1,000-bed city and teaching hospital in Lausanne, Switzerland. Participants:One bundred and three alcoholic patients and 103 controls aged 20–75 years, admitted from September 1, 1988, to March 18, 1989. Results:Alcoholics had the same lengths of stay (16 days), durations of intravenous infusions (six days), and durations of bladder catheterization (one day). Statistically nonsignificant differences were found between alcoholics and nonalcoholics regarding the charges for routine laboratory examinations [693 vs. 734 Swiss francs (Sfrs)], antibiotic therapies (218 vs. 145 Sfrs), and x-ray procedures (568 vs. 774 Sfrs; p=0.06). The average number of electrocardiograms (two vs. five; p<0.005) and the duration of intensive care unit (ICU) stay (one vs. two days; p<0.05) were significantly lower for alcoholics than for controls. A total hospital charges index was also lower for alcoholics than for controls (11,900 Sfrs vs. 12,800 Sfrs), but not significantly. Conclusion:The authors’ results suggest that alcoholics do not use more hospital resources per admission than do nonalcoholics. Moreover, alcoholics tend to use less frequently some procedures, such as the ICU, electrocardiography, and x-ray examinations. Several hypotheses are developed to explain these results in relation to those of previous studies, which showed more use of medical care by alcoholics than by nonalcoholics. Support by a grant from the Swiss National Research Foundation (no 3200-009282) and by a grant from the “Fondation du 450^eme Anniversaire de l’Université de Lausanne.”     

Family history of alcoholism is associated with lower 5-HT2A receptor binding in the prefrontal cortex

Background: 5‐Hydroxytryptophan (5‐HT_2A) receptor involvement in alcoholism is suggested by less 5‐HT_2A binding in alcohol preferring rats, association of a 5‐HT_2A receptor gene polymorphism with alcohol dependence and reduced alcohol intake with 5‐HT_2A antagonists. We sought to determine postmortem whether 5‐HT_2A receptors are altered in the prefrontal cortex (PFC) of alcoholics. Methods: Brain tissue from 25 alcoholics and 19 controls was collected at autopsy. Diagnosis of DSM‐IV alcoholism/abuse and other psychiatric disorders and the determination of family history of alcoholism were made by psychological autopsy. Specific binding to 5‐HT_2A (³H‐ketanserin) receptors in the PFC was measured by quantitative autoradiography. Results: 5‐HT_2A binding decreased with age [Brodmann areas (BA) 9, 46 gyrus; r = ?0.381, ?0.334, p < 0.05]. No differences in receptor binding between alcoholics and controls were detected in the gyrus or sulcus of any PFC area examined. Cases (controls or alcoholics) with a family history of alcoholism (n = 23) had less 5‐HT_2A binding throughout PFC than subjects without (n = 21) a family history of alcoholism (p < 0.05). 5‐HT_2A receptor binding in alcoholics without a family history of alcoholism (n = 7) did not differ from controls without a family history of alcoholism (n = 14). There was no association between alcoholism or alcohol rating and genotype. There was an association between genotype and the total amount of ³H‐ketanserin binding in BA46 with the TT genotype having more binding (TT>TC≈CC). Conclusions: Lower 5‐HT_2A receptor binding in the PFC of cases with a family history of alcoholism suggests a genetic predisposition to alcoholism. Alcohol abuse by itself did not have a significant effect on PFC 5‐HT_2A binding and as 5‐HT_2A binding in alcoholics is not different from controls and antagonists may be therapeutic, fewer receptors may result in downstream developmental effects on the brain resulting in a predisposition to alcoholism.     

Influence of α-Methyldopa Treatment on Adrenergic Receptor Binding in Rat Forebrain

We studied the influence of 6 days treatment with α₃-methyldopa (50 mg/kg s.c.) twice daily on radioligand binding to α₁ ((³H)prazosin), α₂ ((³H)clonidine) and β((³H)dihydroalprenolol (D₃HA)) receptors in rat forebrain. A 27% rise (p lt; 0.05) in the B_max for (³H)prazosin without change in Kd was found. Following α-methyldopa, the K_d for (³H)clonidine was increased from 1.78 ± 19 to 3.03 ± 0.30 nM and B_max fell from 197 ± 20 to 167 ± 19 fmoles/mg protein (p < 0.05). These findings suggest that chronic α-methyldopa therapy induces changes in α₁ and α₂ receptors which may modify the antihypertensive effect of α-methyldopa.     

The presence of corticosteroid receptor activity in the gills of the brook trout,Salvelinus fontinalis

Gill tissue from brook trout was examined for the presence of cortisol receptors. Both cytosolic and nuclear preparations from the gills manifested high equilibrium association constants (K_a) and low maximum binding capacities (N_max) indicative of high-affinity and low-capacity receptor activity (cytosol: K_a = 0.31 ± 0.02 × 10⁹/M, N_max = 223.9 ± 22.8 fmol/mg protein; nuclear extract: K_a = 0.02 ± 0.003 × 10⁹/M, N_max = 424.6 ± 96.3 fmol/mg protein). Gel permeation (Sephacryl S-300) column chromatography gave two incompletely separated peaks at 326,000 and 189,000 Da and Stokes radii of 5.96 and 4.81 nm using [³H]triamcinolone acetonide and only one peak at 219,000 Da and 5.4 nm using [³H]cortisol. The binding of the synthetic compounds, triamcinolone acetonide and dexamethasone, appears to be different from that of the natural steroid, cortisol. The receptor activity appears to be highly specific for cortisol since cortisone and 11β,17α,21-trihydroxy-4-pregnen-3,20-dione-2l-phosphate bind with much lower affinity. The gill tissue cytosol fractions had the highest cortisol-binding activity, followed by liver, intestine, and muscle. The association constants for the liver, intestine, and muscle were the same order of magnitude as that for the gill. These results are consistent with the concept of nonmembrane steroid receptors of target organs.     

Temperature Dependence of Macroscopic L-Type Calcium Channel Currents in Single Guinea Pig Ventricular Myocytes

Calcium Channels and Temperature. Introduction: Lowering temperature greatly reduces calcium influx through calcium channels. Studies on a number of tissues demonstrate that the peak inward current, I_Ca exhibits Q₁₀ values ranging from 1.8 to 3.5; however, it remains unclear which component(s) of calcium channel gating may give rise to this large temperature sensitivity. Components of gating that may affect channel availability include phosphorylation and changes in [Ca²⁺)_i, processes that vary in pertinence depending on the channel examined. This study addresses this problem by examining the temperature sensitivity (from 34° to 14°C) of cardiac I_Ca under control conditions, during attenuation or activation of protein kinase A (PKA) activity, and when intracellular [Ca²⁺] has been elevated. Methods and Results: I_Ca was studied using the whole cell configuration of the patch clamp technique. In control, lowering temperature from 34° to 24°C resulted in a shift in the potential for maximum slope (V_a and the peak current (Y_max) toward more positive membrane potentials. The Q₁₀, values for the decrease in Y_max and the macroscopic slope conductance (G_max), which reflects the number of available channels, were 3.15 ± 0.19 and 2.57 ± 0.13, respectively. At 0 mV the Ca²⁺ current decayed biexponentially, and the two time constants (T₁ and T₂) showed Q₁₀ values of 1.79 ± 0.21 and 2.06 ± 038, while their contribution to the total current (I₁ and I₂) showed a Q₁₀ of 5.99 ± 0.83 and 1.61 ± 0.22. In myocytes loaded with inhibitors of the PKA cycle sufficient to inhibit the increase of I_Ca to 1 μM isoprenaline, the Q₁₀ values for some of the kinetic parameters were increased with the Q₁₀ for I₁ increasing to 17.06 ± 3.48. Stimulation of I_Ca by exposing myocytes to 1 μM isoprenaline reduced the temperature sensitivity of Y_max, G_max, and I₁, yielding respective values of 2.00 ± 0.18, 1.85 ± 0.07, and 2.04 ± 0.15. Raising [Ca²⁺], to enhance Ca²⁺_i-dependent inactivation, while affecting inactivation and activation kinetics, affected temperature sensitivity little compared to control. The Q₁₀ for time to peak changed little under experimental conditions (2.3 to 2.4) Conclusions: Increasing the phosphorylated states of calcium channels, but not Ca²⁺_i-dependent inactivation, reduces temperature sensitivity of certain gating parameters. The data suggest that the rate of the transitions between the unavailable and also between the various closed states are changed in the opposite direction to that induced by PKA-dependent phosphorylation. Processes, e.g., inhibitory mechanisms, may be involved to maintain channels in unavailable or “unphosphorylated” states, and it may he these that contribute to the high Q₁₀ of macroscopic channel currents.     

Effects of Chronic Alcohol Consumption and Aging on Dopamine D2 Receptors in Fischer 344 Rats

Aging and chronic alcohol consumption are each accompanied by significant changes in dopamine and dopamine receptors. This study extended previous work by investigating the combined effects of chronic alcoholism and aging on total dopamine D₂ receptors in brain areas associated with the nigrostriatal and mesocorticolimbic systems. In addition, the effects of chronic alcohol consumption and aging on the high-affinity state of D₂ receptors and their conversion to the low-affinity form is included. Quantitative autoradiography was used to assess [³H]spiperone-labeled D₂ receptors in tissue sections from 5- to 14- and 24-month Fischer 344 rats that were pair-fed a control or 6.6% (v/v) ethanol-containing liquid diet for 6 weeks. In addition, D₂ receptors were determined in rats given the control liquid diet ad libitum. The results of these experiments demonstrated age-related changes in the nigrostriatal system. There was an age-related loss of total dopamine D₂ receptors in the rostral and caudal striatum (～25% decrease in B_max). This decline in D₂ receptors may be associated with changes in motor function. Despite the age-related decline in D₂ receptors, there were no significant differences in the proportion of striatal receptors in the high-affinity form or in their conversion to the low-affinity state. Both aging and chronic alcohol consumption produced significant changes in the concentration of D₂ receptors in brain areas associated with the mesocorticolimbic system. That is, the specific binding of [³H]spiperone was decreased in the frontal cortex of aged rats. In addition, chronic alcoholism was associated with a significant increase (～20%) in the B_max for D₂ receptors in the nucleus accumbens. Nonetheless, neither age nor chronic alcohol consumption altered the proportion of high-affinity D₂ receptors in the nucleus accumbens or their conversion to the lower affinity state. The observed changes in D₂ receptors in the frontal cortex and nucleus accumbens are of interest because of the involvement of the mesocorticolimbic dopamine areas in the rewarding properties of alcohol and other drugs of abuse. Although aging and chronic alcoholism both produced significant changes in dopamine D₂ receptor concentrations, alcohol did not accentuate the age-related loss of D₂ receptors. We cannot eliminate the possibility that a more prolonged exposure or higher ethanol dose may potentiate age-related changes in the dopaminergic system.     

Dopamine receptors and hypertension

Dopamine plays an important role in regulating renal function and blood pressure. Dopamine synthesis and dopamine receptor subtypes have been shown in the kidney. Dopamine acts via cell surface receptors coupled to G proteins; the receptors are classified via pharmacologic and molecular cloning studies into two families, D₁-like and D₂-like. Two D₁-like receptors cloned in mammals, the D1 and D5 receptors (D1A and D1B in rodents), are linked to adenylyl cyclase stimulation. Three D₂-like receptors (D2, D3, and D4) have been cloned and are linked mainly to adenylyl cyclase inhibition. Activation of D₁-like receptors on the proximal tubules inhibits tubular sodium reabsorption by inhibiting Na/H-exchanger and Na/K-adenosine triphosphatase activity. Reports exist of defective renal dopamine production and/or dopamine receptor function in human primary hypertension and in genetic models of animal hypertension. In humans with essential hypertension, renal dopamine production in response to sodium loading is often impaired and may contribute to hypertension. A primary defect in D₁-like receptors and an altered signaling system in proximal tubules may reduce dopamine-mediated effects on renal sodium excretion. The molecular basis for dopamine receptor dysfunction in hypertension is being investigated, and may involve an abnormal posttranslational modification of the dopamine receptor.     

Specific 125I Neuropeptide Y Binding to Intact Cultured Bovine Adrenal Medulla Capillary Endothelial Cells

Objective: This study involved the pharmacological detection and characterization of binding sites for the neuromodulator neuropeptide Y (NPY) in an in vitro preparation of capillary endothelial cells derived from bovine adrenal medulla. Methods: Equilibrium binding assays were conducted on intact cells with ¹²⁵I Bolton-Hunter labeled NPY (¹²⁵I-BH-NPY). The specificity of the high-affinity binding site was evaluated in competition experiments with cold NPY, (Leu³¹, Pro³⁴)NPY (a Y₁ receptor ligand, Y₁RL), NPY_13–36 (a Y₂ receptor ligand, Y₂RL), and two other members of the pancreatic polypeptide-fold (PP-fold) family: peptide YY (PYY) and avian pancreatic polypeptide (APP). Forskolin-stimulated adenylate cyclase activity was assessed to detect the participation of this second messenger pathway in the neuromodulator action at the studied cell preparation. Results: Nonlinear regression analysis of the binding data indicated the existence of high-affinity binding sites with an equilibrium dissociation constant (K_d) value of 39.00 ± 12.84 nM and a maximal binding (B_max) of 489.89 ± 155.49 fmol/10⁶ cells (mean ± SE, n = 6). NPY, Y₁RL, and PYY displayed a concentration that inhibits the specific binding by 50% IC₅₀ (nM) values of 4.06 ± 1.66 (n = 4), 2.94 ± 0.75 (n = 5), and 18.36 ± 10.36 (n = 3), respectively. APP and Y₂RL were unable to compete with ¹²⁵I-NPY in the concentration range 0.001–1 μM. Further evaluation of second messenger pathways suggested that NPY binding sites in this model are coupled to the inhibition of adenylate cyclase. NPY significantly inhibited the forskolin-stimulated adenosine cyclic 3′,5′-(hydrogen phosphate) (cAMP) accumulation with a maximal effect of 37.03 ± 6.28%, n = 5 and an IC₅₀ of 5.96 ± 1.87 nM. The Y₁RL produced a comparable response (IC₅₀ = 5.35 ± 1.39 nM, n = 4; maximal inhibition of 61.05 ± 13.03%) and Y₂LR had no detectable effect at a similar concentration range. Conclusions: The results demonstrate the existence of a Y₁ receptor in the adrenal medulla capillary endothelial cells, which may be relevant to the postjunctional effect of NPY on this gland.     

Intramuscular versus subcutaneous injection of soluble and lispro insulin: comparison of metabolic effects in healthy subjects

The aim of this study was to compare the glucodynamic effects of soluble insulin and the rapid acting insulin analogue insulin lispro after subcutaneous (s.c.) and intramuscular (i.m.) injection. Twelve healthy male volunteers (age 26.8 ± 1.7 years, BMI 23.2 ± 2.3 kg m⁻²; mean ± SD) participated in this single-centre, open-labelled, euglycaemic glucose clamp study on four different days. Soluble insulin or insulin lispro (0.2 U kg⁻¹) were injected s.c. or i.m. into the thigh by syringe. The glucodynamic effects were assessed by registering the glucose infusion rates necessary to maintain blood glucose at 5.0 mmol l⁻¹ for the subsequent 420 min. Intramuscular injection of soluble insulin led to an earlier peak of metabolic action when compared to s.c. administered soluble insulin (t_max 138 ± 29 vs 179 ± 34 min; p < 0.05). The maximal metabolic effect and metabolic activity during the first 2 h after i.m. and s.c. injection of soluble insulin were comparable (GIR_max 9.7 ± 2.3 vs 7.8 ± 2.3 mg kg⁻¹ min⁻¹; n.s., AUC_{0–120 min} 0.60 ± 0.18 vs 0.50 ± 0.15 g kg⁻¹ 120 min; n.s.). Subcutaneous administration of insulin lispro led to a metabolic effect resembling that induced by i.m. application of soluble insulin (t_max 116 ± 26 vs 138 ± 29 min; n.s., GIR_max 11.1 ± 2.3 mg vs 9.7 ± mg kg⁻¹ min⁻¹; n.s.). However, the overall metabolic response during the first 2 h after injection was higher with s.c. insulin lispro (AUC_{0–120 min} 0.81 ± 0.26 vs 0.60 ± 0.18 g kg⁻¹ 120 min; p < 0.05). The glucodynamic activity of i.m. applied insulin lispro was comparable to that of lispro s.c.. Following i.m. injection of soluble insulin, the metabolic activity peaked more rapidly than with s.c. administration. In contrast, the metabolic effect of insulin lispro was similar with either route. The time–action profile of i.m. injected soluble insulin lies between that of s.c. applied soluble insulin and insulin lispro. © 1998 John Wiley & Sons, Ltd.     

Dopamine D2 Receptor Polymorphisms in Inflammatory Bowel Disease and the Refractory Response to Treatment

Dopamine and its receptors may be involved in inflammatory reaction. The availability of this molecule depends on its receptors. The DRD2 gene, which codifies for the D2 dopamine receptor, has several polymorphisms. In this study, the DRD2 TaqIA polymorphism, which confers a decreased receptor density, was evaluated in 313 individuals including 220 inflammatory bowel disease patients (143 patients with Crohn’s disease and 77 with ulcerative colitis) and in 93 healthy blood donors. The analysis was carried out by PCR-RFLP techniques. The frequencies of A ₁ A ₁ and A ₂ A ₂ genotypes were similar among Crohn’s disease, ulcerative colitis patients, and health controls. Also, the genotype frequency was similar in different groups of disease localization, behavior, and age of disease onset. However, the Crohn’s disease patients carriers of A ₂ A ₂ genotype showed a lower risk for development refractory Crohn’s disease (37 out 65) than A ₁ A ₁ and A ₁ A ₂ carriers (28 out of 65) [(OR=0.4, 95% CI 0.21–0.87; p=0.02)]. Our results support an involvement of the dopamine receptor in inflammatory bowel disease and suggest a new potential target for therapy in refractory Crohn’s disease patients.     

Echocardiographic abnormalities in chronic asymptomatic alcoholics

Alcohol abuse is a frequent contributor to elevated blood pressure, but the literature is ambiguous about the role of hypertension in producing left ventricular dysfunction. Fifty asymptomatic male alcoholics admitted for detoxification were studied using echocardiograms and systolic time intervals. Alcoholics were separated into Group I (28 with hypertension) and Group II (22 without hypertension). Forty-four patients had analyzable echocardiograms and were compared to 29 nonalcoholics. Group III consisted of 14 nonalcoholics with hypertension. Group IV consisted of 15 normotensive nonalcoholics (controls). The ejection fraction and shortening fraction were reduced in Group I (p less than 0.05). Hypertensive alcoholics had increased left ventricular mass indices but less than hypertensive nonalcoholics. Left ventricular wall stress was compared to mass as an index of ventricular compensation. The wall stress to mass index for hypertensive alcoholics was 1.65 as compared to 1.43 for the controls. Alcoholics without hypertension had a wall stress to mass ratio of 1.54. Hypertensive patients had a reduced wall stress to mass ratio of 1.38 when compared to controls. These data suggest an inappropriate compensatory response to afterload. Alcohol and hypertension combined may be more harmful to left ventricular function than either disease alone.