No Correlation of Pineal "Synaptic" Ribbon Numbers and Melatonin Formation in Individual Rat Pineal Glands

As previous circadian studies of pineal "synaptic" ribbon numbers and melatonin formation suggested that a positive correlation of the two variables exists, in the present investigation this problem was examined in individual pineal glands of rats killed at 1200 h and 2400 h, respectively. For this purpose, one half of the gland was processed for electron microscopy and the ribbons were counted in an area of 20,000 micron2 tissue; in the other half serotonin N-acetyltransferase (NAT) activity and melatonin content were determined. No correlation was found to exist between ribbon numbers and pineal NAT activity, pineal melatonin levels and serum melatonin levels, either at day- or at nighttime. It is concluded that the ribbons may perhaps be more closely related to the innervation of the pineal gland than to melatonin formation.     

Effects of Isoproterenol on Synaptic Ribbons in Pinealocytes of the Rat and C57BL/6J Mouse

Synaptic ribbons (SR) in melatonin-deficient pinealocytes of the C57BL/6J mouse were quantitatively compared to SR in pinealocytes of the rat after beta-adrenergic receptor activation by isoproterenol. Two populations of SR comprising synaptic spherules (SRsp) and synaptic rods (SRr) were described in both the mouse and the rat, but species differences existed in the ratio of SRr to SRsp. Isoproterenol caused a significant increase in frequency of SR of the rat but had little or no effect on SR populations in the mouse. It is unlikely that beta-adrenergic receptors are absent on mouse pinealocytes or were not activated since isoproterenol elevated plasma renin concentrations indicating activation of beta-adrenergic receptors. Furthermore the pineal of both species receives heavy sympathetic input. These findings indicate that the role and regulation of pinealocyte SR are complex and are functionally linked to beta-adrenergic receptors as well as other mechanisms related to the production of melatonin.     

Characterization of a Neurohypophyseal Hormone-Like Activity Isolated From Ovine Pineal Glands

The milk-ejecting response of lactating mouse mammary gland tissue to ovine pineal extracts indicated the presence of a neurohormone-like bioactivity in this tissue. After successive fractionation on gel permeation chromatography and reversed-phase liquid chromatography (HPLC) in conjunction with radioimmunoassays (RIA), it was demonstrated that the milk-ejection response to ovine pineal components with an Mr less than 1,000 corresponded to a biologically active peptide sequence that probably differs from that of arginine vasopressin, arginine vasotocin, and oxytocin and from peptides with a COOH-terminal Pro-Arg-Gly-amide ending. Gel permeation chromatography in formic acid appeared also to indicate the presence of a noncovalent interaction of the neurohormone-like bioactivity with proteins (Mr greater than 25,000) of the pineal.     

Daily Profiles of N-Acetyltransferase Measured at a Single Time in Rat Pineal Glands, Retinas, and Harderian Glands

Serotonin N-acetyltransferase activity (NAT) exhibited a daily cycle in light:dark (LD) 14:10 when it was measured in pineal glands taken from rats killed at a sequence of time points. The ratio of peak subjective night NAT to minimum subjective day NAT was 10.9/0.3 nmol per pineal gland per hour. When the rats were placed in constant dark the rhythm persisted (8.2/0.02). When the rats were placed in constant light the rhythm persisted with markedly attenuated amplitude (0.6/0.02).
We also measured NAT profiles in rat pineal glands, Harderian glands, and retinas with alternative methods. We kept rats on six LD 14:10 light-dark cycles with lights-out beginning at midnight, 2 AM, 4 AM, 6 AM, 8 AM, or 10 AM and killing all the rats at one time point, 10 AM. We examined the NAT time profiles 4, 8, and 11 days following placement of the rats in the phase-shifted cycles. In addition, we measured the NAT profile in LD 2:22 and LD 22:2 by keeping the rats on twelve cycles for 11 days and killing all the rats at one time.
Pineal NAT exhibited a rhythm in all the cycles: peak-dark/nadir-light values (nmol product per gland per hour) were 15.6/0.1 in LD 14:10, controls killed at successive time points. The ratios for the profiles obtained using the one time point procedure were 16.7/0.1 in LD 14:10 8.5/0.2 in LD 22.2, and 12.9/0.2 in LD 2.22. Increasing the photoperiod reduced the time to the NAT peak.
In LD 14:10, Harderian NAT was 31–39 nmol per gland per hour but the peak/ nadir radio was only 1.2; retinal NAT was low (0.2–0.7 nmol per retina per hour) and had only a 3.5-fold peak/nadir ratio.     

Measurement of Tryptophan Hydroxylase Activity in Rat Pineal Glands and Pinealocytes Using an HPLC Assay With Electrochemical Detection

A method for measuring tryptophan hydroxylase activity by assaying the product 5-HTP using high-performance liquid chromatography (HPLC) with electrochemical detection is described. A nocturnal elevation (80%) in rat pineal gland tryptophan hydroxylase activity was detected. Experiments on isolated rat pinealocytes with the protein synthesis inhibitor cycloheximide indicate that tryptophan hydroxylase turns over rapidly in these cells. This method will be valuable in studies of the adrenergic mechanisms regulating pineal tryptophan hydroxylase activity.     

Immunohistochemical Characterization of Rat Pineal Dopamine β-Hydroxylase-Containing Structures: Use of a Homologous Monoclonal Antibody

Previous immunohistochemical studies using a polyclonal heterologous antibody to dopamine-beta-hydroxylase (DBH) (EC 1.14.17.1) have shown that only a few of the many sympathetic nerve fibers in the rat pineal display DBH-like immunoreactivity. This is in contrast to other sympathetically innervated organs. To ascertain this observation and to rule out antibody- or method-related artifacts, in the present study a well-characterized, monospecific, homologous monoclonal antibody to DBH was used in combination with the biotin-streptavidin-fluorescein-mediated detection system. As in a previous study, immunoreactive fibers were only rarely encountered in the rat pineal gland, with an identical distribution pattern. The present findings indicate that the sympathetic nerve fibers in the rat pineal gland appear to have a low capacity for de novo synthesis of noradrenaline and that most of the noradrenaline present may be taken up from the general circulation. In addition to nerve fibers, perikarya-like cells and endbulb-like structures were seen in the pineal gland similar to those demonstrated by the polyclonal antibody. It is suggested that both structures may be part of an as yet unknown intrapineal regulatory system.     

Effect of Voltage-Dependent Calcium Channel Blockers on Ethanol-Induced β-Endorphin Release From Hypothalamic Neurons in Primary Cultures

The voltage-dependent calcium channel (VDCC) has been shown to mediate calcium entry into neurons that regulates neurotransmission in many neuronal cells. Four major types of VDCCs (three high-voltage-activated L-, N-, and P-types and one low-voltage-activated T-type) have been identified in neurons. Involvement of the VDCC in ethanol-stimulated beta-endorphin (beta-EP) release from hypothalamic neurons has not been studied. In the present study, the role of VDCC on basal and ethanol-induced beta-EP release was determined by using rat fetal hypothalamic cells in primary cultures. Treatments with a 50 mM dose of ethanol for 3 hr increased immunoreactive beta-EP (IR-beta-EP) release from hypothalamic cells maintained in cultures for 9 days. Ethanol-induced IR-beta-EP release was inhibited by a P/Q-type channel blocker omega-agatoxin TK (0.1-1 microM), an N-type channel blocker omega-conotoxin (0.1-1 microM), an L-type blocker nifedipine (1-10 microM), and a T-type blocker flunarizine (1-10 microM). The minimal effective doses of these blockers that blocked the ethanol response produced no significant effects on basal release of IR-beta-EP; neither did these doses of the blockers produce any significant effects on cell viability. These results suggest that ethanol-stimulated IR-beta-EP release is regulated by extracellular calcium involving P-, N-, L- and T-type channels.     

Hamster and Rat Pineal Gland β-Adrenoceptor Characterization With Iodocyanopindolol and the Effect of Decreased Catecholamine Synthesis on the Receptor

Rat and hamster pineal glands were used in binding studies to characterize their beta-adrenoceptors with a new specific antagonist ligand, iodocyanopindolol. The receptors were saturable, and the ligand was selective and demonstrated stereospecificity for both species. The rat pineal had a 20-fold greater density of beta-adrenoceptors, while the affinity was one-third that of the hamster pineal. utilizing this radioligand, we examined the effects of decreased sympathetic input to the pineal on beta-adrenergic receptors in both species. Decreased noradrenergic input to the pineal gland of the hamster was accomplished by superior cervical ganglionectomy, or by exposing the animals to continuous light for 36 hours. Parallel studies were conducted with hamster pineal gland in which catecholamine synthesis was measured. The results indicate that a selective decrease in catecholamine synthesis in the hamster pineal does not change the beta-adrenoceptor density or affinity. In contrast, a concomitant increase in beta-adrenoceptor density but not affinity occurs in the rat pineal gland after similar decreased sympathetic input.     

Cell Kinetic Study on Histogenesis of Barrett's Esophagus Using Rat Reflux Model

Background: To elucidate the histogenesis of Barrett's esophagus and esophageal adenocarcinoma, we designed a duodeno-gastric reflux model in which normal stomach function and normal nutritional status are retained. Methods: Male Wistar rats were used in the experiment. The esophago-gastric junction was side-to-side anastomosed to a loop of jejunum about 3 &#114 cm distal to Treitz's ligament. The animals were not exposed to any known carcinogens during the experiment. Sequential morphological changes were studied for up to 50 weeks after surgery. Serial sections were made and stained with hematoxylin and eosin (H&E). In addition, immunohistochemical staining for bromodeoxyuridine (BrdU) was performed along with histochemical staining for mucins using paradoxical concanavalin A (ConA), galactose oxidase Schiff (GOS), and high-iron diamine-alcian blue (HID-AB). Results: Severe esophagitis with squamous cell hyperplasia was noted in all animals after surgery. At week 20 after surgery, glandular metaplastic cells positive for ConA first appeared within the basal cell layer of esophageal squamous cell epithelium, and then GOS-positive cells and HID-AB goblet cells appeared. This is a characteristic of the specialized columnar epithelium of Barrett's esophagus. We detected esophageal adenocarcinomas in 1 out of 8 subjects at week 40 and in 3 out of 8 subjects at week 50 after surgery. Conclusions: Reflux of duodenal contents causes specialized columnar epithelium of Barrett's esophagus and esophageal adenocarcinoma. As part of the sequence of events leading to the development of Barrett's esophagus, pyloric-foveolar metaplasia was observed followed by the appearance of intestinal goblet cells. The pyloric-foveolar metaplasia appears to be associated with chronic mucosal damage and regeneration. This multiplastic cell lineage is referred to as 'gut-regenerative cell lineage' (GRCL).     

Effect of Acute Ethanol Administration on the Release of Opioid Peptides From the Midbrain Including the Ventral Tegmental Area

Background:  Experimental evidence suggests that ethanol alters the activity of the endogenous opioid peptide systems in a dose and brain-region dependent manner. These alterations may influence the processes of ethanol reward and reinforcement. Thus, it was the objective of this study to investigate the response of the 3 major opioid peptide systems (endorphins, enkephalins, and dynorphins) to acute ethanol administration, at the level of the midbrain including the ventral tegmental area (midbrain/VTA), a region important for drug, including ethanol reinforcement.
Methods:  Using the in vivo microdialysis technique coupled with specific solid-phase radioimmunoassay for β-endorphin, met-enkephalin, and dynorphin A_1–8, changes in the extracellular concentration of theses peptides at the level of midbrain/VTA were determined at distinct time points following the administration of 0.0 (saline), 0.8, 1.2, 1.6, 2.0, and 2.4 g ethanol/kg B.Wt.
Results:  A biphasic effect of ethanol on β-endorphin release was found, with low to medium (1.2, 1.6, and 2.0 g) but not high (2.4 g) doses of ethanol, inducing a significant increase in the dialysate content of β-endorphin. A late increase in the dialysate content of dynorphin A_1–8 was observed in response to the 1.2 g ethanol dose. However, none of the ethanol doses tested significantly altered the content of met-enkephalin in the dialysate.
Conclusions:  The present findings suggest that the ethanol-induced increase of β-endorphin release at the level of midbrain/VTA may influence alcohol reinforcement.     

Gender specific associations of the Trp64Arg mutation in the beta3-adrenergic receptor gene with obesity-related phenotypes in a Mediterranean population: interaction with a common lipoprotein lipase gene variation

OBJECTIVE: To investigate the association between the Trp64Arg beta3-adrenergic receptor (ADRB3) mutation and obesity-related phenotypes in a Mediterranean Spanish population considering the effect of other genetic and environmental factors. DESIGN AND SUBJECT: Cross-sectional study in 1063 (476 men and 587 women) randomly selected from this population (aged: 18-68 years). MEASUREMENTS: Anthropometric (weight, height and waist-to-hip ratio), blood pressure, biochemical (lipids, fasting glucose, and uric acid), life-style variables, and the Trp64Arg, HindIII-Lipoprotein lipase (LPL) and apolipoprotein E polymorphism. RESULTS: Frequency of the Arg64 allele was low (0.051; 95% CI: 0.042-0.060). We found gender-specific associations between the Trp64Arg mutation and obesity related phenotypes. In men, carriers of the Arg64 variant had higher body mass index (BMI) (27.63 +/- 3.81 vs. 26.34 +/- 3.57 kg m-2, P=0.049) and total cholesterol (5.85 +/- 1.45 vs. 5.28 +/- 1.06 mmol L-1; P=0.011) compared with wild-type individuals. Logistic regression analysis, revealed that the risk of overweight was two times higher in male carriers of the Arg64 allele. In women, the Arg64 variant was only associated with higher fasting glucose (P=0.031). These genotype effects persisted after adjustment for age, genetic and life-style variables. For the LPL polymorphism, the H-/H- genotype was associated with lower BMI and with lower risk of overweight (OR: 0.49; 95% CI: 0.30-0.81) in both men and women. However, after adjustment for covariates, these associations only remained statistically significant (P < 0.02) in women. Moreover, in women, a statistically significant interaction (P=0.026) between the LPL and the ADRB3 gene loci in determining BMI was found. Thus, the Arg64 allele was associated with a higher BMI only in H+/H+ women. CONCLUSIONS: The Trp64Arg mutation was associated with BMI and lipids in men. In women, an additional gene-gene interaction with the LPL-HindIII polymorphism may explain the results.     

TRP64ARG polymorphism of the beta 3-adrenergic receptor gene and obesity risk: effect modification by a sedentary lifestyle

Aim: We performed a case–control study to assess the association between obesity risk and the Trp64Arg polymorphism of the β₃-adrenergic receptor gene.
Methods: Obese subjects [n = 159; body mass index (BMI) > 30 kg/m²] and controls (n = 154; BMI < 25 kg/m²) were compared using multivariable logistic regression to control for potential confounders.
Results: A higher obesity risk (adjusted OR: 2.98; 95% CI: 1.00–8.56; p = 0.05) was associated with the Trp64Arg polymorphism among sedentary, but not among more active people.
Conclusions: Our results suggest that the TRP64ARG polymorphism of the ADRB3 seems to be a risk factor for obesity that is dependent on a sedentary lifestyle.     

Hyperkinetic contraction of a nonischemic segment of ischemic left ventricle in anesthetized dogs

Summary Regional myocardial function during acute coronary artery occlusion was studied with ultrasonic dimension gauges in 20 open-chest anesthetized dogs. Two pairs of ultrasonic crystals were implanted in the left ventricular free wall near the epicardium in an ischemic segment and in a control nonischemic segment, and the segment length (SL) and maximum velocity of systolic shortening (max dL/dt) were measured. In six dogs, the wall thickness (WT) was measured simultaneously in the same regions with sonomicrometry. Left ventricular pressure (LVP), aortic pressure (AoP), and plasma norepinephrine concentration in the coronary sinus (NE_CS) were also measured. The heart rate was kept constant (180 beats/min) with atrial pacing. The left anterior descending coronary artery was occluded at its distal portion without propranolol in 12 dogs (group 1) and 30 min after propranolol in eight dogs (group 2). In the ischemic region, coronary artery occlusion resulted in an increase in end-diastolic SL (50% at 3 min after occlusion in group 1,P<0.005), and a decrease in max dL/dt in systole (36% at 5 min after occlusion in group 1,P<0.02). In the nonischemic region, end-diastolic SL did not change significantly, but an increase in max dL/dt (29% at 10 min after occlusion in group 1,P<0.005) was observed in systole. Under propranolol (group 2), the results were similar to those of group 1. There were no significant changes in LVP, AoP, and NE_CS during occlusion. We conclude that: (1) SL and WT in the same region present a mirror image, suggesting that WT is a useful index for evaluating regional myocardial function; (2) after coronary artery occlusion, while the ischemic region showed hypokinesis, the nonischemic region presented significant hyperkinesis without an increase in preload (end-diastolic SL) or a decrease in ventricular afterload (AoP); and (3) since these results did not change significantly after propranolol and were not accompanied by an increase in NE_CS, the hyperkinesis in the nonischemic region does not seem to be related to-adrenergic receptors and is not due to the Frank-Starling mechanism.     

The action of β-adrenergic blocking and stimulating agents on insulin secretion. Characterization of the type of β receptor

Summary As a result of our experiments designed to studyin vivo in the anaesthetized dog, the role of the beta-adrenergic receptors involved in insulin secretion, we have found: 1. that isoprenaline (global stimulator of the beta-adrenergic receptors) provoked a considerable increase in the secretion of insulin. — 2. that propranolol (blocking agent of the beta-adrenergic receptors) partially and temporarily inhibited the secretion of insulin. — 3. that isoprenaline, after blockage of the beta-adrenergic receptors by propranolol, provoked a strong, long-lasting inhibition of insulin secretion. — 4. that practolol (selectively ₁ blocking agent) did not counteract the stimulating effects of isoprenaline ( ₁ and ₂ stimulating agent). This suggests that the beta-adrenergic receptor involved in insulin secretion is of the type ₂. — 5. that salbutamol (selective ₂ stimulating agent) provoked an abundant secretion of insulin, an effect which was found to be blocked by propranolol. This last fact confirms that the beta-adrenergic receptor involved in the insulin secretion provoked by isoprenaline is of type ₂. — All these findings underline the importance of the ₂ adrenergic receptors of the beta cell of the islets of Langerhans, in the process of insulin secretion.

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Die Wirkung von blockierenden und stimulierenden, -adrenergischen Substanzen auf die Insulinsekretion. Charakterisierung des -Rezeptortyps

Zusammenfassung Wir fanden in unseren Experimenten, die dazu angelegt waren, am anästhesierten Hundin vivo die Rolle der-adrenergischen Rezeptoren für die Insulinsekretion zu erforschen: 1. daß Isoprenaline (welches die-adrenergischen Rezeptoren stimuliert), eine starke Erhöhung der Insulinsekretion hervorruft. — 2. daß Propranolol (ein Blocker der-adrenergischen Rezeptoren) die Insulinsekretion hemmt. — 3. daß Isoprenaline nach der Blockierung der-adrenergischen Rezeptoren durch Propranolol die Insulinsekretion stark, und dauerhaft hemmt. — 4. daß Practolol (welches mehr die Rezeptoren ₁ hemmt) nicht die Stimulation des Isoprenaline aufhebt, welches sowohl auf die ₁ und ₂ Rezeptoren wirkt. Dieses deutet darauf hin, daß die-adrenergischen Rezeptoren, die bei der Insulinsekretion mitwirken, dem Typ ₂ angehören. — 5. daß Salbutamol (welches mehr die ₂-Rezeptoren anregt) eine übermäßige Insulinsekretion bewirkt, die wiederum durch Propranolol zu hemmen ist. Diese Tatsache bestätigt, daß die-adrenerischen Rezeptoren, welche an der durch Isoprenaline hervorgerufenen Insulinsekretion beteiligt sind, dem Typ ₂ angehören. — Alle diese Tatsachen unterstreichen die Bedeutung der ₂ adrenergischen Rezeptoren der-Zelle der Langerhansschen Inseln für den Prozess der Insulinsekretion.

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Action sur l'insulino-sécrétion des substances bloquant et stimulant les récepteurs adrénergiques. Caractérisation du type de récepteur

Résumé Il résulte de nos expériences destinées à étudierin vivo, chez le chien anesthésié, le rôle des récepteurs-adrénergiques impliqués dans l'insulino-sécrétion: 1. que l'isoprénaline (stimulant des récepteurs bêta-adrénergiques) provoque une augmentation importante de la sécrétion d'insuline. — 2. que le propranolol (bloquant des récepteurs bêta-adrénergiques) freine temporairement la sécrétion d'insuline. — 3. quel'isoprénaline, après blocage des récepteurs bêta-adrénergiques par le propranolol, inhibe l'insulino-sécrétion d'une manière puissante et durable. — 4. que le practolol (bloquant plus sélectif des récepteurs ₁) ne s'oppose pas aux effets stimulants de l'isoprénaline qui agit sur les récepteurs ₁ et ₂, ce qui suggère que le récepteur bêta-adrénergique impliqué dans l'insulinosécrétion est de type ₂. — 5. que le salbutamol (stimulant plus sélectif des récepteurs ₂) provoque une abondante sécrétion d'insuline, effet qui se trouve bloqué par le propranolol. Ce fait confirme que le récepteur adrénergique impliqué dans l'insulino-sécrétion provoquée par l'isoprénaline est de type ₂. -Tous ces faits soulignent l'importance des récepteurs adrénergiques ₂ de la cellule bêta des îlots de Langerhans dans le processus d'insulinosécrétion.