Positive and negative inotropic effects of carbachol on the embryonic chick atrium.

The effect of carbachol on twitch tension of atrial preparations from chick embryos of different incubation ages (3-14 days) was studied. At every age carbachol evoked negative (at low concentrations) and positive (at higher concentrations) inotropic responses. Maximal response values for both effects increased with age; in 3- and 5-day atria the positive inotropic response prevailed. The muscarinic antagonist pirenzepine inhibited the positive (on 5-day atria) and negative (on strips of 14-day atria) inotropic effects of carbachol with pA2 values of 6.8 and 8.0, respectively, suggesting that muscarinic receptors mediating these effects belong to different receptor subtypes.     

Positive and negative inotropic effects of carbachol on the embryonic chick atrium

The effect of carbachol on twitch tension of atrial preparations from chick embryos of different incubation ages (3–14 days) was studied. At every age carbachol evoked negative (at low concentrations) and positive (at higher concentrations) inotropic responses. Maximal response values for both effects increased with age; in 3- and 5-day atria the positive inotropic response prevailed. The muscarinic antagonist pirenzepine inhibited the positive (on 5-day atria) and negative (on strips of 14-day atria) inotropic effects of carbachol with pA₂ values of 6.8 and 8.0, respectively, suggesting that muscarinic receptors mediating these effects belong to different receptor subtypes.     

Differences in the content and turnover of noradrenaline in rat heart atria and ventricles and circadian-phase-dependency

In light-dark-synchronized male rats the levels of noradrenaline in heart atria were about 3 times that found in heart ventricles. Noradrenaline turnover rate which were about 8-9 fold greater for the atria than for the ventricles displayed a circadian-phase-dependency with increased rates in the dark period in both parts of the rat heart.     

Vitamin D receptors in heart: effects on atrial natriuretic factor.

We report that receptors for vitamin D exist in distinct regions of the heart in female and male mice, predominantly in the right atrium where most of the cardial atrial natriuretic peptide (ANF) is produced. Tritiated 1,25-dihydroxyvitamin D3 (1,25-D3, vitamin D, soltriol) and ANF are colocalized in nuclei and cytoplasm respectively in identical cardiomyocytes. Changes of ANF tissue and blood levels under dietary deficiency and treatment with 1,25-D3 suggest direct genomic actions of vitamin D on myoendocrine cells of the atrium for the regulation of ANF manufacture and secretion. These results were obtained by combining thaw-mount autoradiography with immunocytochemistry using tritiated 1,25-D3 and an antibody against rat ANF. This antibody was also used in a radioimmunoassay to determine atrial natriuretic factor in plasma, atria and ventricles of normal or vitamin D-deficient mice.     

Vitamin D receptors in heart: Effects on atrial natiuretic factor

We report that receptors for vitamin D exist in distinct regions of the heart in female and male mice, predominantly in the right atrium where most of the cardial atrial natriuretic peptide (ANF) is produced. Tritiated 1,25-dihydroxyvitamin D₃ (1,25-D₃, vitamin D, soltriol) and ANF are colocalized in nuclei and cytoplasm respectively in identical cardiomyocytes. Changes of ANF tissue and blood levels under dietary deficiency and treatment with 1,25-D₃ suggest direct genomic actions of vitamin D on myoendocrine cells of the atrium for the regulation of ANF manufacture and secretion. These results were obtained by combining thaw-mount autoradiography with immunocytochemistry using tritiated 1,25-D₃ and an antibody against rat ANF. This antibody was also used in a radioimmunoassay to determine atrial natriuretic factor in plasma, atria and ventricles of normal or vitamin D-deficient mice.     

Caffeine intake induces an alteration in human neutrophil A2A adenosine receptors

Caffeine is the most widely used drug in the world and acts mainly through antagonism of the effects mediated by the adenosine receptor subtypes A1, A2A, A2B and A3. We determined whether repeated caffeine administration at different doses and for different periods of time (400 or 600 mg/day for 1 week and 400 mg/day for 2 weeks) alters human neutrophil A2A adenosine receptor density and function. Saturation binding assays showed an increase in affinity (K(D)) and density (B(max)) of A2A adenosine receptors after caffeine intake. These changes were accompanied by increases in cAMP accumulation and decreases in superoxide anion production after stimulation of the A2A receptor subtype using the agonist 5'-N-ethylcarboxamidoadenosine (NECA). Binding and functional changes of A2A receptors returned to baseline after 48 h of caffeine withdrawal. The findings are consistent with a potential anti-inflammatory effect of caffeine mediated by neutrophil A2A receptors.     

The enteric neural receptor for 5-hydroxytryptamine

An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using 3H-5-HT as a radioligand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of 3H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these 3H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of 3H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of 3H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of 3H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.     

The enteric neural receptor for 5-hydroxytryptamine

Summary An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using³H-5-HT as a radiologand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of³H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these³H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of³H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of³H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of³H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.     

Uptake of noradrenaline in high altitude native's heart

Summary Uptake of³H-noradrenaline by the heart was studied with sections of isolated atria obtained from high or lowlanders. In native highlanders, affinity for³H-noradrenaline by human atria is more significant than in lowlanders. Furthermore, the Michaelis Menten constant is lower in high altitude native's heart.     

Serotonergic and cholinergic interaction in the regulation of pituitary-adrenal function in rats

It has been proposed that the central serotonergic inputs which modulate pituitary-adrenal secretion are mediated by cholinergic neurons. We have tested this hypothesis in intact rats. Male Sprague-Dawley rats were injected with cholinergic and serotonergic agents which enhanced transmitter function and with receptor blocking agents. Agents were injected, singly and in combination, into both unstressed and stressed animals. Since the response to cholinergic agents might be due to changes to vasopressin release, Brattleboro (vasopressin deficient) rats were also injected with cholinergic agents. The level of plasma corticosterone at 1-h post-injection was determined. Results indicate that the serotonin receptor blockade decreased the stimulatory, cholinergic effect of physostigmine. Cholinergic receptor blockers did not significantly reduce the corticosterone rise induced by 5-hydroxytryptophan. These results do not support the hypothesis of cholinergic mediation of serotonergic input. Nicotinic and muscarinic receptors appeared to exert opposing influences on the system. The nicotinic receptor antagonist was able to block the stimulatory effect of physostigmine. The muscarinic receptor antagonist significantly elevated plasma corticosterone levels. No differences were found in the effect of physostigmine on Brattleboro rats as compared to controls. These data are interpreted as suggesting that 1) the acetylcholine-induced stimulation of pituitary-adrenal function is mediated, in part, by serotonergic neurons; and 2) stimulation of nicotinic receptors is facilitatory whereas stimulation of muscarinic receptors is inhibitory to pituitary-adrenal function.     

Tbx2 and Tbx3 induce atrioventricular myocardial development and endocardial cushion formation

A key step in heart development is the coordinated development of the atrioventricular canal (AVC), the constriction between the atria and ventricles that electrically and physically separates the chambers, and the development of the atrioventricular valves that ensure unidirectional blood flow. Using knock-out and inducible overexpression mouse models, we provide evidence that the developmentally important T-box factors Tbx2 and Tbx3, in a functionally redundant manner, maintain the AVC myocardium phenotype during the process of chamber differentiation. Expression profiling and ChIP-sequencing analysis of Tbx3 revealed that it directly interacts with and represses chamber myocardial genes, and induces the atrioventricular pacemaker-like phenotype by activating relevant genes. Moreover, mutant mice lacking 3 or 4 functional alleles of Tbx2 and Tbx3 failed to form atrioventricular cushions, precursors of the valves and septa. Tbx2 and Tbx3 trigger development of the cushions through a regulatory feed-forward loop with Bmp2, thus providing a mechanism for the co-localization and coordination of these important processes in heart development.     

Galanin – 25 years with a multitalented neuropeptide

The pathophysiology of depression remains unclear, but involves disturbances in brain monoaminergic transmission. Current antidepressant drugs, which act by enhancing this type of transmission, have limited therapeutic efficacy in a number of patients, and not rarely serious side-effects. Increasing evidence suggests that neuropeptides, including galanin, can be of relevance in mood disorders. Galanin is coexpressed with and modulates noradrenaline and serotonin systems, both implicated in depression. Pharmacological and genetic studies have suggested a role for galanin in depression-like behaviour in rodents, whereby the receptor subtype involved appears to play an important role. Thus, stimulation of GalR1 and/or GalR3 receptors results in depression-like phenotype, while activation of the GalR2 receptor attenuates depression-like behaviour. These findings suggest that galanin receptor subtypes represent targets for development of novel antidepressant drugs.     

Galanin – 25 years with a multitalented neuropeptide

Galanin, a neuropeptide widely expressed in the central and peripheral nervous systems and in the endocrine system, has been shown to regulate numerous physiological and pathological processes through interactions with three G-protein-coupled receptors, GalR1 through GalR3. Over the past decade, some of the receptor subtype-specific effects have been elucidated through pharmacological studies using subtype selective ligands, as well as through molecular approaches involving knockout animals. In the present review, we summarize the current data which constitute the basis of targeting GalR1, GalR2 and GalR3 for the treatment of various human diseases and pathological conditions, including seizure, Alzheimer's disease, mood disorders, anxiety, alcohol intake in addiction, metabolic diseases, pain and solid tumors.     

Role of adenosine A1 and A2 receptors in the regulation of aldosterone production in rat adrenal glands

To investigate the roles of adenosine A1 and A2 receptors in the regulation of aldosterone production, we examined the effects of adenosine and adenosine agonists (N6-cyclohexyl adenosine; selective adenosine A1 receptor agonist and 5'-N-ethylcarboxamine adenosine; selective adenosine A2 receptor agonist) on aldosterone and cyclic AMP production in rat adrenal capsular cells. Neither adenosine nor 5'-N-ethylcarboxamine adenosine caused significant effects on basal aldosterone or cyclic AMP production. Also, adenosine (10(-3) M) showed no consistent effects on aldosterone and cyclic AMP production induced by ACTH. On the other hand, N6-cyclohexyl adenosine exhibited a significant inhibition of basal aldosterone and cyclic AMP production at doses of 10(-4) M and 10(-3) M; furthermore, 10(-3) M N6-cyclohexyl adenosine inhibited aldosterone and cyclic AMP production stimulated by ACTH. These results suggest that adenosine A1 receptors are coupled to and inhibit adenylate cyclase and may be involved in the inhibition of aldosterone production.     

Genetic and pharmacological models of cholinergic supersensitivity and affective disorders

Summary Increased muscarinic sensitivity has been associated with altered hormonal states (hypothyroidism and hyperadrenocorticism), chronic administration of muscarinic antagonists or antidepressants with muscarinic actions, selective breeding for anticholinesterase sensitivity, and certain inbred strains of rats and mice. Thus, both genetic and environmental factors may influence muscarinic receptor sensitivity. The reasonably detailed studies on the selectively-bred rats have revealed that the Flinders Sensitive Line (FSL) rats weigh less, are less active, are more sensitive to muscarinic agonists and to stressors, and have higher concentrations of hippocampal and striatal muscarinic receptors than normal, or the selectively-bred, Flinders Resistant Line (FRL) rats. Thus, there are a number of parallels between FSL rats and depressed humans. The FSL rats may be the first animal model of depression to mimic the actual trait of depression, and not just the state.     

Genetic and pharmacological models of cholinergic supersensitivity and affective disorders

Increased muscarinic sensitivity has been associated with altered hormonal states (hypothyroidism and hyperadrenocorticism), chronic administration of muscarinic antagonists or antidepressants with muscarinic actions, selective breeding for anticholinesterase sensitivity, and certain inbred strains of rats and mice. Thus, both genetic and environmental factors may influence muscarinic receptor sensitivity. The reasonably detailed studies on the selectively-bred rats have revealed that the Flinders Sensitive Line (FSL) rats weigh less, are less active, are more sensitive to muscarinic agonists and to stressors, and have higher concentrations of hippocampal and striatal muscarinic receptors than 'normal', or the selectively-bred, Flinders Resistant Line (FRL) rats. Thus, there are a number of parallels between FSL rats and depressed humans. The FSL rats may be the first animal model of depression to mimic the actual trait of depression, and not just the state.     

Use of3H-QNB in the isolation of plasma membrane from smooth muscle of the urinary bladder: Effect of oxalate on calcium uptake by the membrane fractions

Summary Specific binding of tritiated quinuclidinyl benzilate (³H-QNB) to surface membrane muscarinic receptors was utilized to identify plasma membrane (PM) fractions from smooth muscle of the rabbit urinary bladder. Accumulation of³H-QNB in the PM fraction was 4–5-fold higher than that in fractions of endoplasmic reticulum (EM) or mitochondria (M). A similar pattern of     

Morphine 6 glucuronide stimulates nitric oxide release in mussel neural tissues: evidence for a morphine 6 glucuronide opiate receptor subtype

We have previously demonstrated that Mytilus edulis pedal ganglia contain opiate alkaloids, i.e., morphine and morphine 6 glucuronide (M6G), as well as mu opiate receptor subtype fragments exhibiting high sequence similarity to those found in mammals. Now we demonstrate that M6G stimulates pedal ganglia constitutive nitric oxide (NO) synthase (cNOS)-derived NO release at identical concentrations and to similar peak levels as morphine. However, the classic opiate antagonist, naloxone, only blocked the ability of morphine to stimulate cNOS-derived NO release and not that of M6G. CTOP, a mu-specific antagonist, blocked the ability of M6G to induce cNOS-derived NO release as well as that of morphine, suggesting that a novel mu opiate receptor was present and selective toward M6G. In examining a receptor displacement analysis, both opiate alkaloids displaced [³H]-dihydromorphine binding to the mu opiate receptor subtype. However, morphine exhibited a twofold higher affinity, again suggesting that a novel mu opiate receptor may be present. Received 1 November 2001; received after revision 1 February 2002; accepted 1 February 2002     

Dual effect of cannabinoid CB<Subscript>1</Subscript> receptor stimulation on a vanilloid VR1 receptor-mediated response

Cannabinoid CB1 receptors and vanilloid VR1 receptors are co-localized to some extent in sensory neurons of the spinal cord and dorsal root ganglia. In this study, we over-expressed both receptor types in human embryonic kidney (HEK)-293 cells and investigated the effect of the CB1 agonist HU-210 on the VR1-mediated increase in intracellular Ca2+ ([Ca2+]i), a well-known response of the prototypical VR1 agonist capsaicin. After a 5-min pre-treatment, HU-210 (0.1 microM) significantly enhanced the effect of several concentrations of capsaicin on [Ca2+]i in HEK-293 cells over-expressing both rat CB1 and human VR1 (CB1-VR1-HEK cells), but not in cells over-expressing only human VR1 (VR1-HEK cells). This effect was blocked by the CB1 receptor antagonist SR141716A (0.5 microM), and by phosphoinositide-3-kinase and phospholipase C inhibitors. The endogenous agonist of CB1 and VR1 receptors, anandamide, was more efficacious in inducing a VR1-mediated stimulation of [Ca2+]i in CB1-VR1-HEK cells than in VR1-HEK cells, and part of its effect on the former cells was blocked by SR141716A (0.5 microM). Pre-treatment of CB1-VR1-HEK cells with forskolin, an adenylate cyclase activator, enhanced the capsaicin effect on [Ca2+]i. HU-210, which in the same cells inhibits forskolin-induced enhancement of cAMP levels, blocked the stimulatory effect of forskolin on capsaicin. Our data suggest that in cells co-expressing both CB1 and VR1 receptors, pre-treatment with CB1 agonists inhibits or stimulates VR1 gating by capsaicin depending on whether or not cAMP-mediated signalling has been concomitantly activated.